©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Copper Binding to Mouse Liver S-Adenosylhomocysteine Hydrolase and the Effects of Copper on Its Levels (*)

(Received for publication, July 15, 1994; and in revised form, June 26, 1995)

Kathleen E. Bethin Thomas R. Cimato Murray J. Ettinger (§)

From the Department of Biochemistry, State University of New York at Buffalo, Buffalo, New York 14214

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The dissociation constant and stoichiometry of copper binding to mouse liver S-adenosylhomocysteine hydrolase (SAHH) was determined as part of characterizing the possible roles of SAHH in copper metabolism. Copper (Cu(II)) binding was measured by an ultrafiltration method in the presence of EDTA as a competing ligand. The K was 3.9 ± 0.7 10M, and the stoichiometry was one g atom of copper per 48-kDa subunit. Western blots indicated that the liver contains approx12 times more SAHH than the kidney, which in turn contains approx5 times more SAHH than the brain. The high concentration and copper affinity of SAHH in the liver may contribute to the liver's ability to preferentially accumulate copper, and the low levels of SAHH in the brain may contribute to the sensitivity of the brain to copper deficiency. The effects of genetic defects of copper metabolism and copper deficiency on SAHH were also determined. Normal SAHH levels were detected in brindled mouse liver, kidney, and brain. However, SAHH from brindled mouse liver eluted abnormally from phenyl Superose columns implying an effect of the brindled mouse defect on SAHH protein structure. Hepatic cytosols from the toxic milk mouse contained approx42% the amount of SAHH detected in controls, and hepatic levels of SAHH were also decreased by approx45% in copper-deficient mice. The binding properties of SAHH and the effects of abnormal states of copper metabolism on its levels are consistent with significant roles for SAHH in normal and abnormal copper metabolism. SAHH may have roles in regulating tissue copper levels and the distribution of intracellular copper.


INTRODUCTION

A cDNA was recently cloned, which encodes a major copper binding protein (CuBP) (^1)in mouse liver(1, 2) . The deduced amino acid sequence showed 98 and 96% identities to rat (3) and human (4, 5) S-adenosylhomocysteine hydrolase (SAHH), respectively. Comparisons of the structure-function properties of mouse liver CuBP and human placental SAHH confirmed that CuBP was SAHH(6) . SAHH was proposed to be a bifunctional protein with roles in methionine/cysteine metabolism and copper metabolism(6) . Since homocysteine is required for cysteine biosynthesis, SAHH enzymatic activity may be required for copper metabolism because high cysteine levels are necessary for metallothionein synthesis(7) , and cysteine is also used to synthesize glutathione, which may have a role in copper metabolism(8, 9, 10) . Moreover, the copper binding activity of SAHH may play a role in regulating the intracellular distribution of copper(2, 6) .

It was reasoned that determining the binding properties of SAHH was essential for examining its possible functions in copper metabolism and that sensitivity to abnormal copper states of copper metabolism would further imply a significant role for SAHH in copper metabolism. Here, we report the dissociation constant and stoichiometry of copper binding to mouse liver SAHH and its isotopic exchange properties. The effects of copper deficiency and genetic defects of copper metabolism on SAHH levels or properties were also determined. Since the liver, kidney, and brain are particularly sensitive to abnormalities of copper metabolism (11, 12) , the levels of SAHH in these organs were also determined.

The brindled mouse and toxic milk mouse have inborn errors of copper metabolism. The brindled mouse (13) is an animal model of Menkes disease(11, 12, 14) , which is a fatal, X-linked disease characterized by decreased activities of several copper enzymes, defective brain development, and connective tissue abnormalities(11, 12, 14) . The toxic milk mouse defect is lethal to all offspring of homozygous dams because the copper deficiency that develops during fetal development is exacerbated by the copper-deficient milk of the affected dams(15, 16) . However, all offspring survive if fed normal amounts of copper(15) , and adult toxic milk mice have elevated hepatic copper and metallothionein (MT). While the defect in the toxic milk mouse is unknown, a candidate gene (cDNA) for Menkes disease was recently cloned(17, 18, 19) . The deduced amino acid sequence is homologous to P-type ATPases, which are Cd(II) or Cu(II) transporters in bacteria (20, 21) . Based on the properties of cells from Menkes patients and the brindled mice, the Menkes gene product was proposed to be an intracellular membrane copper transporter(17) . Interestingly, the candidate gene (cDNA) for Wilson's disease, the other well characterized inherited disease of copper metabolism in humans, is homologous to the candidate gene for Menkes disease(22, 23, 24, 25) .

The copper binding properties of SAHH are consistent with significant roles for SAHH in regulating tissue copper levels and the distribution of intracellular copper. Moreover, the fact that each of the three abnormal states of copper metabolism that were examined here affect the levels or properties of SAHH is consistent with significant roles of SAHH in normal and abnormal copper metabolism.


EXPERIMENTAL PROCEDURES

Materials

All HPLC columns (Superose-12 HR 10/30, Mono Q HR 5/5, Hi Load 16/10 Q Sepharose HP (Hi Q), chelating Superose HR 10/2, and phenyl Superose 5/5), the HPLC pump (model 2150), the HPLC controller (model 2152), and all other HPLC accessories including a titanium prefilter were from Pharmacia Biotech Inc. The ultrafiltration units (Ultrafree-MC, 10,000 daltons) for the copper binding studies were from Millipore. Bovine serum albumin was from Calbiochem or Intergen (Purchase, NY). Siliconized 0.65-ml microcentrifuge tubes were from Marsh (Rochester, NY). Cu(NO(3))(2) was obtained from Buffalo Materials Research Center (Buffalo, NY) with a specific activity of approx10 mCi/mg. The protein assay reagents, bicinchoninic acid (BCA) and micro-BCA, were from Pierce. Immobilon-P membranes for immunoblotting were from Millipore. I-Protein A was from ICN (Irvine, CA). Antirabbit IgG alkaline phosphatase conjugate, p-nitrophenyl phosphate, phenylmethylsulfonyl fluoride, leupeptin, MOPS, MES, and HEPES were obtained from Sigma. Silver nitrate (99.9+%) was from Alfa (Ward Hill, MA). X-ray film (XAR-5) was from Eastman Kodak Co. Nitroblue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate were from Bio-Rad. All other chemicals, unless otherwise specified, were from Fisher.

Purification of Mouse Liver SAHH

SAHH was purified in milligram quantities by modifying the previously used procedure (2) as follows. All solutions for HPLC were made with distilled, deionized (mixed bed resin, D8922, Barnstead, Dubuque, IA) water. The copper content was 0.0005 ppm by flameless atomic absorption. All solutions were filtered through 0.2-µm nylon filters (Nalgene, Rochester, NY) and then stored at 4 °C. Hi Load Q buffers were used within 3 weeks of preparation. All other column buffers were prepared freshly 1 day prior to use. All column buffers were degassed for 30 min just prior to use. All samples (cytosols, column fractions, or other samples) were filtered through 0.22-µl Millex GV syringe filters (Millipore) before use. Protein absorbance was monitored at 280 nm by a Single Path Monitor UV-1 (Pharmacia) using 10-mm, 7.9-µl flow cells. An estimated dead volume of 1.5 ml (the volume between the gradient mixing valve and the end of the prefilter) was accounted for while programming the gradients. All HPLC steps were at 4 °C. In the previously published protocol, samples after each purification step were applied to Superose columns, and SAHH was detected by its elution volume, SDS-PAGE, and the Cu binding activity of each Superose fraction(2) . In the modified procedure, SAHH was monitored by SDS-PAGE and Western blots.

The Hi Q column was equilibrated with the starting buffer (0.05 M triethanolamine, pH 7.9) and the high salt buffer (0.05 M triethanolamine, 1 M NaCl, pH 7.9) at a flow rate of 1.5 ml/min by the following protocol: 0-18 min, high salt buffer; 18-37 min, starting buffer. These steps were repeated twice, and the column was then equilibrated in the starting buffer for 33 min. The cytosol samples (2, 26) containing approx200 mg of protein were diluted 1:2 in the starting buffer and loaded onto the column. The flow rate for sample loading was 1.0 ml/min. After washing the column with 30-60 ml of starting buffer, the proteins, which were bound to the column matrix, were eluted at a flow rate of 0.6 ml/min with the following gradient: 0-16.7 min, 0.1 M NaCl; 16.7-166.7 min, 0.1-0.26 M NaCl; 166.7-166.8 min, 0.26-1.0 M NaCl. 47 min after the start of the gradient, 60 drops (1.65 ml) were collected into fractions containing 20 µl of (0.05 mg/ml) leupeptin. Fractions containing SAHH were identified as described above and pooled for the next step. After completion of the gradient, high salt buffer was run through the column for 30 min, and the column was treated with 0.1% pepsin in 0.1 M HCl (3 ml). The column was stored in pepsin at 4 °C.

In the immobilized metal affinity chromatography step, chelating Superose HR 10/2 was charged with Zn(II). Three buffers were used: starting buffer (50 mM NaOAc, 50 mM MOPS, 50 mM MES, 250 mM NaCl, pH 7.9), a 1 M NH(4)Cl buffer (50 mM NaOAc, 50 mM MOPS, 50 mM MES, 250 mM NaCl, 1 M NH(4)Cl, pH 6.4), and a 2 M NH(4)Cl buffer (50 mM NaOAc, 50 mM MOPS, 50 mM MES, 250 mM NaCl, 2 M NH(4)Cl, pH 4.9). 7-9 ml (4-5 column volumes) of 0.1 M ZnSO(4) were passed through the column. The charged column was then rinsed with 2-3 volumes of water, 4-6 column volumes of the 1 M NH(4)Cl buffer, and then 4-6 column volumes of starting buffer. The charged column was used approx10 times before recharging. Hi Q fractions containing SAHH were pooled, diluted 1:2 in starting buffer, and loaded onto the column at a flow rate of 0.5 ml/min. The column was washed with starting buffer until the A profile returned to the base line (approx30 min), and the remaining proteins were eluted by a stepwise gradient of 0-6 min, 0.7 M NH(4)Cl (pH 6.4); 6-16 min, 1 M NH(4)Cl (pH 6.4); 16-32 min, 1.4 M NH(4)Cl (pH 4.9); 32-39 min, 1.85 M NH(4)Cl (pH 4.9). 32 drops (880 µl) were collected into test tubes containing leupeptin. After the gradient was completed, the column was rinsed in at least 10 ml of water and re-equilibrated in starting buffer. Fractions containing SAHH were identified as described above.

Hydrophobic interaction chromatography was used in the third purification step. Phenyl Superose HR 5/5 was equilibrated with at least 15 ml of the starting buffer (0.05 M potassium phosphate, 1.5 M (NH(4))(2)SO(4), pH 6.9). Pooled fractions from the chelating Superose step were diluted 1:2 in starting buffer and then applied to the column. After the A returned to the base line, the remaining proteins were eluted at a flow rate of 0.5 ml/min with the following gradient: 0-10 min, 0.3 M (NH(4))(2)SO(4); 10-17.5 min, 0.15 M(NH(4))(2)SO(4); 17.5-38 min, 0 M(NH(4))(2)SO(4), 50 mM potassium phosphate, pH 6.9. 32 drops (880 µl) per fraction were collected into test tubes. After the buffer gradient was completed (at 38 min), water was passed through the column at a flow rate of 0.5 ml/min, and 100 drops (2.75 ml) were collected into each fraction to elute SAHH(2) . After the protein was eluted, at least 15 ml of water were passed through the column, and the column was treated with 2 ml of 0.1% NaN(3). Before use, the column was washed with 15 ml of water and then at least 15 ml of starting buffer.

Fractions containing pure SAHH were identified as described above, and the pooled fractions were concentrated in an Amicon (Beverly, MA) concentrator, C-30. Concentrated SAHH was diluted and reconcentrated in 100 mM NaCl, 50 mM MOPS, pH 7.4, for the binding studies. The amount of protein purified was determined by a micro-BCA assay, using BSA as the standard(27) . SAHH was estimated to be approx0.5% of the total hepatic cytosol protein. The copper contents of each preparation were determined by flameless atomic absorption using a Perkin-Elmer HGA 700 graphite furnace with a 1100 B atomic absorption spectrophotometer.

Determination of the Dissociation Constant for Copper Binding to SAHH

A form of the binding equation was used in which copper was held constant while the protein concentration was varied (see ``Results''). EDTA was used as competing ligand to facilitate the binding measurements. Protein was incubated with EDTA and Cu(II) in a final volume of 200 µl. The concentrations of EDTA and copper were kept constant at 0.2 and 0.25 µM, respectively. The concentration of protein (BSA or SAHH) was varied from 0.3 to 2.1 µM. Five or six replicates were used for each binding measurement. EDTA was first added to the protein, and then the Cu was added. The Cu, protein, and EDTA were incubated overnight (approx17 h) at 22 °C in siliconized tubes with gentle shaking. The next day, 190 µl from each sample were ultrafiltered and centrifuged for 15 min at 9,000 RPM in a microfuge to separate protein-bound Cu(II) from EDTA-Cu(II), free EDTA, and free Cu in the ultrafiltrate. The filters and ultrafiltrates were then counted in a LKB counter (model 1282) correcting for decay. The Cu(II) in the ultrafiltrate was used to correct for nonspecific Cu bound as described below. Total protein was determined by a micro-BCA assay(27) . Protein-bound Cu was subtracted from the total amount of protein to calculate the amount of free protein. The measured Cu-protein concentration (±S.D.) and the calculated free protein were then used to calculate the K, apparent by a non-linear least squares fit (28) to (see ``Results''). The K for copper binding to the protein was then calculated from the apparent K, using the reported value for the K of EDTA, 1.4 10M, corrected for pH 7.4(29) , and the known concentration of EDTA, i.e.K, apparent = K (1 + [I]/K). The program provides the S.D. of the parameters and a bias measure of how well the data fit the equation used. A bias value higher than 2.0 indicates a poor fit to the equation used(28) .

Determination of Nonspecific Cu Binding to the Filter

0.2 µM EDTA was incubated with 0.05-0.2 µMCu(II) and ultrafiltered. The nonspecifically bound Cu on the filter was plotted versus the Cu in the ultrafiltrate. The linear plot obtained was used to determine how much Cu was nonspecifically bound to the filter from the measured Cu in the ultrafiltrate. The nonspecifically bound Cu was then subtracted from the measured Cu bound to the filter to obtain the corrected Cu bound to the protein.

To determine the nonspecifically bound copper for the stoichiometry experiments (described in the ``Results''), 5 µg of lysozyme were incubated with 0.105-0.525 µMCu overnight at 22 °C. The next day, the solution was transferred to the ultrafilters and centrifuged. Since lysozyme does not bind copper, any Cu on the filters indicated nonspecifically bound copper. The Cu in the ultrafiltrate was plotted versus the Cu bound to the filter, and the linear plot obtained was used to determine the nonspecifically bound Cu. The nonspecifically bound Cu on the filters was subtracted from the measured Cu bound to the filters to determine the corrected Cu bound to SAHH protein.

Isotope Exchange

SAHH (0.625 µM) was incubated for 20 h at 22 °C in 200 µl of 100 mM NaCl, 50 mM MOPS, pH 7.4, with or without 0.44 µM Cu(NO(3))(2). The next day, 0.25 µMCu(II) and 0.20 µM EDTA were added. Also, 0.44 µM Cu(NO(3))(2) was added to the samples containing SAHH that had not been preincubated with stable copper. All of the samples were then incubated at 22 °C for 17 h. The next day, 190 µl of each solution were transferred to an ultrafiltration unit and centrifuged for 15 min at 9,000 rpm. The filters and ultrafiltrates were then counted as described above.

Animals

Normal mice (C57 BL/6) were from Harlan Sprague-Dawley. The brindled mice were bred as previously described(30, 31) . Brindled males were treated with a single subcutaneous injection of 10 µl of 0.5% CuCl(2) in polypropylene glycol at 7-9 days after birth(30, 31, 32) .

Four pairs of C57BL/6 mice were mated for the copper deficiency studies. 7 days after parturition, mouse dams with their litters were divided into two dietary groups. Two dams were maintained on standard laboratory chow and tap water, ad libitum, and two dams were maintained on a copper-deficient diet (ICN) and deionized water, ad libitum. 23 days after birth, the male mice were weaned and maintained on the same diet as their respective mothers for an additional 4 or 7 weeks. The diets were ground in a mortar and suspended in 1% nitric acid (0.0002 ppm Cu) for trace metal analysis. The copper contents were determined by flameless atomic absorption. The standard diet and copper-deficient diets contained 25.5 and 0.15 ppm, respectively. The tap water and deionized water contained 0.06 and 0.0005 ppm copper, respectively.

Preparation of Cytosols for Copper Deficiency Studies

After anesthetizing the mice, blood was drawn from the inferior vena cava and used for the hemoglobin and ceruloplasmin assays. Liver cytosols were prepared as previously described (2) with the following modifications. The mice were perfused through the heart. 1 gram of liver was homogenized in 3 volumes of 0.25 M sucrose, 50 mM HEPES, pH 7.4, buffer containing 0.05 mg/ml phenylmethylsulfonyl fluoride and 0.5 µg/ml leupeptin to inhibit proteolysis. Homogenates were centrifuged for 1.5 h at 105,000 g at 4 °C. Protein concentrations were determined using BCA(27) .

Hemoglobin Concentrations

Blood samples were drawn from the inferior cava. Samples (100 µl) were added to tubes containing 10 µl of EDTA (15 mg/ml, pH 7.4) to prevent coagulation. Hemoglobin levels were determined spectrophotometrically by a cyanomethemoglobin method(33) . Five replicates were used for each assay.

Ceruloplasmin Activities

An aliquot of blood was obtained from the inferior vena cava and allowed to coagulate. The serum was obtained by centrifugation for 15 min in a microcentrifuge at 4 °C. The resultant serum was frozen at -20 °C until it was assayed for ceruloplasmin oxidase activity. Ceruloplasmin activity was measured by the o-dianisidine method of Schosinsky et al.(34) and Prohaska(35) .

Hepatic Copper Levels

Aliquots of whole liver homogenates were dissolved and diluted in 1% nitric acid (0.0002 ppm copper), and the tissue copper levels were determined by flameless atomic absorption.

Superose Chromatography of Hepatic Cytosols

Column buffer was prepared as described above and used within 3 weeks. The Superose column was equilibrated with at least two column volumes (48 ml) of HEPES buffer (0.05 M HEPES, 0.1 M NaCl, pH 7.4). Samples were thawed, filtered, and stored on ice until used. Liver cytosol protein concentrations were adjusted to 14 mg/ml with the sucrose homogenization buffer. Radiolabeled copper (0.65 µMCu(II)) was added, and the sample was incubated at room temperature for 15 min. Approximately 2.8 mg in 200 µl were injected onto the Superose-12 column through a titanium loop. Fractions were collected at a flow rate of 0.4 ml/min beginning at 16 min, and 8-drop (approx220 µl) fractions were collected in test tubes containing 10 µl of a phenylmethylsulfonyl fluoride (1.6 mg/ml) and leupeptin (10 µg/ml) solution to inhibit proteolysis. The amount of Cu in each fraction was determined by an LKB model 1282 counter, correcting for decay. Fractions were stored at -20 °C for SDS-PAGE and Western blot analyses. The following molecular mass markers were used: ferritin (445 kDa), aldolase (161 kDa), ovalbumin (45 kDa), and myoglobin (17 kDa).

Electrophoresis and Immunoblotting

The Laemmli (36) method of SDS-PAGE was used with a mini-gel apparatus (Bio-Rad) as previously described(6) . Immunoblots were done as previously described using polyclonal antibodies to mouse liver SAHH (6) . Immunoreactivity was either detected by I-protein A, quantitating the blots by laser densitometry or by an antirabbit IgG alkaline phosphatase method as previously described(6) . The linearity of the densitometric data was confirmed by varying the amount of hepatic cytosol applied to SDS-PAGE gels.

Statistics

A two-tailed student's t test was used to test the statistical significance of the data.


RESULTS

Rationale of the Method Used to Determine the Dissociation Constant for Copper Binding to SAHH

Superose chromatography of SAHH with copper and EDTA (1) suggested that the affinity of mouse liver SAHH for copper was similar to that of EDTA (K approx1.4 10M at pH 7.4)(29) . A micro-ultrafiltration method was developed to determine the exact dissociation constant and stoichiometry of copper binding to SAHH. To circumvent errors in measuring low concentrations of free copper, copper binding was measured in the presence of a competitive chelator, EDTA, and an alternative form of the binding equation was used. is a standard binding equation,

where P is the concentration of the ligand bound to protein, B(max) is the maximum concentration of ligand bound, K is the dissociation constant, and L is the concentration of free ligand. is an alternative form of .

Here, P is the concentration of protein bound with copper, Cu is the total concentration of Cu, P is the concentration of free protein, K is the dissociation constant for binding of Cu to the protein, [I] is the concentration of competitive inhibitor, and K is the dissociation constant for Cu binding to the inhibitor. This equation allows the total concentration of the ligand and competitive inhibitor to be held constant while varying the concentration of protein. An analogous equation has been used by enzymologists for bisubstrate reactions to measure the affinity of one of the substrates while varying the concentration of enzyme. Non-linear least squares fits of the binding data to gave the best fit parameters of , i.e.K and Cu. A comparison of the calculated Cu to the actual total copper used provided an independent measure of the quality of the fits.

Validation of the Ultrafiltration Method Using Albumin

Copper binding to BSA was used to establish the optimal conditions and validity of the method. Pilot experiments indicated that Cu-EDTA and/or free Cu bound nonspecifically to the filters. Therefore, a correction curve for nonspecific binding was established as described under ``Experimental Procedures.'' Since pilot experiments also showed that excess copper bound to the sample tubes, the total concentration of protein and EDTA always exceeded the concentration of Cu. The correction for nonspecific binding was always less than 10% of the total copper retained by the filter. The K determined for BSA by the ultrafiltration method and was 10.7 ± 1.6 10M, which is within experimental error of the literature value of 6.7 10M(37) .

Determination of K for SAHH

The copper dissociation constant for mouse liver SAHH was determined by the ultrafiltration method. As with albumin (data not shown), the corrected experimental data fit very well to (Fig. 1). The data in Fig. 1represent the average from two separate experiments, and each data point is the average of five or six data points. The K was 3.9 ± 0.7 10M. The bias was 0.03 (see ``Experimental Procedures''), and the calculated [Cu] was 0.23 ± 0.016 µM, which was in excellent agreement with the actual 0.25 µM copper used. Thus, the dissociation constant determined for SAHH was close to the K for copper binding to EDTA as previously estimated from Superose Cu binding data(2, 38) .


Figure 1: Cu binding to SAHH in the presence of EDTA. SAHH (0.3-2.1 µM) was incubated with 0.2 µM EDTA and 0.25 µMCu. Cu-SAHH was separated from Cu-EDTA and Cu by ultrafiltration. The amount of Cu-SAHH was measured and subtracted from the total SAHH added to calculate the free SAHH. The bound Cu was corrected for nonspecific binding to the filter using the data as described in the text. The opencircles represent the means ± S.D. of five replicates of the corrected observed data. The closedcircles represent the calculated amount bound from the best fit of the data to . The best fit parameters were K = 3.9 ± 0.7 10M and [Cu] = 0.23 ± 0.016 µM.



The Stoichiometry for Copper Binding to CuBP

The form of the binding equation that was used to determine the K does not allow n, the g atom of copper bound per 48-kDa subunit, to be determined unambiguously. Therefore, n was determined directly by varying the equivalents of copper per 48-kDa subunit and measuring the Cu bound per 48-kDa subunit. At less than one equivalent of copper added, a correction factor was required for the amount of Cu that passed through the filter to the ultrafiltrate. The validity of this correction factor was confirmed with BSA. Corrections for nonspecific Cu binding to the filters were made by determining the amount of Cu retained by filters in the presence of lysozyme as described under ``Experimental Procedures.'' The correction for nonspecific binding was always less than 10% of the total copper bound.

The µmol of Cu bound per µmol of SAHH were plotted versus the equivalents of Cu added per 48-kDa subunit (Fig. 2, A and B). The curve was linear until approx1.0 equivalent of copper was added and then reached a plateau at approx1.1 equivalents of Cu added. The slope of the linear portion of the curve is n, the µmol of Cu bound per µmol 48-kDa subunit per µmol equivalents of copper added. The calculated n from the least squares slope of Fig. 2B was 0.93 ± 0.05 g atom of copper per 48-kDa subunit of SAHH.


Figure 2: A, the stoichiometry of Cu binding to SAHH. SAHH (0.525 µM) was incubated with 0.105-1.05 µMCu. Bound and free Cu were separated by ultrafiltration. The bound Cu was corrected for nonspecific binding using the data in Fig. 4as explained in the text. The abscissa is the micromolar equivalents of copper added per micromole of 48-kDa subunit. The ordinate is the micromoles of copper bound per micromole of 48-kDa subunit. The data shown are the means ± S.D. of five replicates. All of the S.D. were equal to or less than the size of the data point circles shown. B, the least squares best fit line for the linear portion of A.




Figure 4: Western blot of control and brindled mouse liver, kidney, and brain cytosols. The Western blot was incubated with a 1:300 dilution of rabbit antiserum to mouse liver SAHH. Bound antibodies were detected with goat antirabbit alkaline phosphatase conjugate and the substrates nitroblue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate. Lane1, 19.1 µg of control mouse liver cytosol; lane2, 19.5 µg of brindled mouse liver cytosol; lane3, 59 µg of control mouse kidney cytosol; lane4, 56 µg of brindled mouse kidney cytosol; lane5, 66 µg of control mouse brain cytosol; lane6, 55 µg of brindled mouse brain cytosol. The lanes marked N are from control mice, and the lanes marked B are from brindled mice. In the topline, Liv are liver cytosols, K are kidney cytosols, and B are brain cytosols. The lane marked S indicates prestained molecular weight markers.



Isotope Exchange

Mouse liver SAHH, as isolated, was found to have 0.213 ± 0.117 g atom of copper bound per 48-kDa subunit. The stoichiometry of approx1.0 g atom of copper bound per 48-kDa subunit suggested that stable copper and Cu readily exchange. However, to test directly for exchange, SAHH was incubated with or without 0.7 equivalents of stable copper per 48-kDa subunit overnight at 22 °C. Copper, 0.25 µMCu (0.4 equivalents), and 0.2 µM EDTA (the concentrations used in the binding assays), with or without 0.7 equivalents of stable copper, were added to each sample and incubated overnight as above. No significant difference was detected between the amount of Cu bound to SAHH preincubated with cold copper and SAHH preincubated with only buffer (Table 1). Using the K of 3.9 10M, it was calculated that only 0.029 µMCu would have been bound to SAHH if exchange did not occur, and if exchange occurred, 0.093 µMCu would be bound to SAHH. The measured concentration was approx0.12 µMCu bound. Thus, complete isotopic exchange occurs over the time period that was used to determine the binding parameters and stoichiometry.



Determination of K for Brindled Mouse Liver SAHH

The copper dissociation constant for SAHH from the brindled mouse liver was also determined. The corrected experimental data again fit very well to (data not shown); the bias was 0.05, and the calculated [Cu] was 0.20 ± 0.007 µM. The K calculated from the fit to the equation was 1.8 ± 0.32 10M, which is similar to the Kdetermined for control mouse liver CuBP. It had been estimated from Superose chromatography binding data that the differences detected in copper binding by purified SAHH from control and brindled mice could be accounted for if the brindled mouse SAHH had approx2-fold lower binding constant(38) . The method used here cannot distinguish a 2-fold difference in binding constants.

SAHH Levels in the Liver and Kidney

The levels of SAHH in hepatic and renal cytosols were determined by immunoblots using polyclonal antibody to mouse liver SAHH(6) . Fig. 3shows a Western blot of mouse liver and kidney cytosols. The preimmune serum (left) did not react with SAHH, and the immune serum only reacted with SAHH in liver cytosols. Two additional components were detected in some kidney cytosol samples, which probably represented artifacts since these bands were not detected reproducibly. The autoradiographs were quantitated by densitometry as described under ``Experimental Procedures.'' Hepatic cytosols contained 12 times the amount of SAHH in renal cytosols.


Figure 3: Autoradiograph of mouse liver and kidney cytosol Western blot. Proteins were separated by SDS-PAGE. The proteins were then transferred to Immobilon-P. Lanes1 and 2 were incubated with preimmune sera in a 1:300 dilution. Lanes3-8 were incubated with rabbit antiserum to mouse liver SAHH in a 1:300 dilution. Bound antibodies were detected with I-protein A. Lanes1 and 3-6 are 19 µg of mouse liver cytosol protein, and lanes2, 7, and 8 are 59 µg of mouse kidney cytosol protein. The lanes marked A-D represent liver cytosols from four different mice, and the lanes marked 1 and 2 represent kidney cytosols from two different mice. The lane marked L is mouse liver cytosol, and the lane marked K is mouse kidney cytosol.



SAHH Levels from the Brindled Mouse Liver, Kidney, and Brain

SAHH levels in the brindled mouse liver, kidney, and brain were determined by immunoblots using alkaline phosphatase to detect SAHH, which was more sensitive than the I-protein A method used for Fig. 3. The protein components that were detected in addition to SAHH by the alkaline phosphatase method also reacted with preimmune sera (Fig. 4). More total kidney and brain cytosolic protein was applied to the gels than liver cytosol protein (Fig. 4). As obtained by the I-protein A method (Fig. 3), the liver contained much more SAHH than the kidney. The level of SAHH in normal mouse brain was approx5-fold lower than in the kidney and approx60-fold less than in the liver.

The brindled mouse liver, kidney, and brain each contained approximately the same amount of SAHH as in the corresponding control mouse tissues (Fig. 4). The most striking abnormality of SAHH isolated from the livers of brindled mice was a markedly abnormal elution from the last HPLC step used in its purification. SAHH prepared from brindled mouse liver by the modified procedure described under ``Experimental Procedures'' exhibited the identical abnormal elution characteristics as previously reported(38) . SAHH from brindled mice eluted approx7 column volumes sooner than SAHH from control mice. The striking reproducibility of this result with the modified purification protocol confirms that SAHH from brindled mouse liver has abnormal properties.

SAHH Levels in Normal and Menkes Lymphoblasts

Since pilot studies showed that lymphoblasts contained low levels of SAHH, SAHH from normal and Menkes lymphoblasts was partially purified and concentrated by Mono Q chromatography. Immunoblots of the lymphoblast samples and partially purified mouse liver cytosol are shown in Fig. 5. Parallel silver-stained SDS-PAGE gels indicated that the normal and Menkes lymphoblast cytosols contained about the same amount of 48-kDa protein. The preimmune serum did not react with SAHH in any sample. Menkes lymphoblasts appeared to contain similar levels of SAHH as normal lymphoblasts.


Figure 5: Western blot of normal and Menkes human lymphoblast cytosols and mouse liver cytosol. Lymphoblast cytosols partially purified and concentrated on a Hi Q column were immunoblotted. The immunoblot was incubated with a 1:300 dilution of rabbit antiserum to mouse liver SAHH. Bound antibodies were detected with goat antirabbit alkaline phosphatase conjugate and the substrates nitroblue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate. Lane1, Menkes lymphoblast (labeled M); lane2, normal lymphoblast (labeled N); lane3, mouse liver cytosol (labeled Liv).



The Level of SAHH in Toxic Milk Mouse Liver

SAHH levels were determined in hepatic cytosols from control (parent strain) mice, toxic milk mice, and a C57 BL/6 mouse, which is the control mouse from which SAHH was purified. A decreased level of SAHH was detected in each of the toxic milk mouse cytosols (Fig. 6), and densitometry of autoradiographs of Western blots indicated that the toxic milk mouse liver contained 42 ± 19% of the level of SAHH detected in the controls (p < 0.05).


Figure 6: Autoradiograph of Western blot of toxic milk and control mouse liver cytosol. The Western blot was incubated with a 1:300 dilution of rabbit antiserum to mouse liver SAHH. Bound antibodies were detected with I-protein A. Lanes1-3, 24 µg of control strain mouse liver cytosol (labeled C); lanes4-6, 24 µg of toxic milk mouse liver cytosol (labeled T); lane7, 24 µg of liver cytosol from the C57 BL/6 control strain of mouse (labeled C57).



Assessment of Copper Deficiency

Neonatal mice were fed a copper-deficient diet for 4 or 7 weeks postweaning (see ``Experimental Procedures''). The degree of copper deficiency was estimated by determining hemoglobin levels, ceruloplasmin activities, and liver copper levels. As shown in Table 2, two levels of copper deficiency were detected based on hemoglobin levels. The moderately copper-deficient mice had approx57% of control mean hemoglobin levels while the severely copper deficient mice, which were on the diet for 7 weeks, had approx26% of control levels (the terms moderate and severe are used only to distinguish the two levels of copper deficiency as determined by hemoglobin levels and not the severity of clinical symptoms from copper deficiency). The hemoglobin differences between each copper-deficient group and the control group were statistically significant as was the difference between the moderately and severely copper-deficient mice (Table 2). Also, both the moderately and severely copper-deficient groups showed little ceruloplasmin activity (Table 2) which was not significantly different from each other (Table 2). The severely copper-deficient mice had significantly lower copper levels than the control mice (43%). Although the moderately deficient group had a liver copper level that was intermediate between the control and severely deficient group, this was not statistically different from the control mice (Table 2). The variability in hepatic copper levels was much higher than the hemoglobin levels. Therefore, the hemoglobin levels were taken as the most reliable quantitative measure of the level of copper deficiency.



Hepatic SAHH Levels in Copper-deficient Mice

Copper deficiency led to decreased levels of SAHH in the liver cytosols from both moderately and severely copper-deficient mice. A approx45% decrease in SAHH levels was detected in both moderately and severely deficient mice (p < 0.0001) (Table 3). SAHH levels were not significantly different between the two groups of copper-deficient mice (Table 3). SDS-PAGE gels of mouse hepatic cytosols were silver stained to determine whether copper deficiency affected the levels of other proteins. While most proteins were unaffected by copper deficiency, a decrease in a 23-kDa protein and increases in a 55-kDa and a 80-kDa protein were detected in the copper-deficient mice samples (Fig. 7).




Figure 7: Silver-stained SDS-PAGE of control and copper-deficient mouse liver cytosols. Mouse liver cytosols (14 µg) were separated on a 10% SDS-PAGE and silver stained. Lane1 contains the following molecular weight markers; beta-galactosidase, 116 kDa; phosphorylase b, 97.4 kDa; bovine serum albumin, 66 kDa; ovalbumin, 45 kDa; glyceraldehyde-3-phosphate dehydrogenase, 36 kDa; carbonic anhydrase, 29 kDa; and trypsinogen, 24 kDa. Lanes1, 2, 5, and 6 are liver cytosols from copper-deficient mice (labeled D). Lanes3, 4, 7, and 8 are liver cytosols from control mice (labeled C).



Superose Chromatography of Copper-deficient and Control Mouse Hepatic Cytosols

Radiolabeled copper (0.65 µMCu(II)) was added to the liver cytosols, and the Cu-labeled cytosols were chromatographed on Superose-12 to determine how copper deficiency and decreased SAHH affected copper binding in hepatic cytosols from the moderately deficient mice. The amounts of total liver cytosolic protein were equalized in each sample. The Cu binding profiles from four control and five moderately deficient mouse liver cytosols were averaged to obtain the profiles shown in Fig. 8. As previously reported(2) , two major Cu binding protein fractions were detected. These are labeled I(S) and II(S) in Fig. 8. Decreases in both fractions were detected. The 26% decrease in the I(S)Cu binding protein fraction was significant, but the 16% decrease in II(S) binding was not statistically significant (Table 4). SAHH was detected by immunoblots of individual fractions from a Superose column (Fig. 9), which also showed decreased SAHH levels in the column fractions from the copper-deficient mice (Fig. 9). Thus, a approx45% decrease in SAHH was associated with approx25% decrease in copper binding in the I(S) fraction, which is consistent with a significant contribution of copper binding to SAHH in the I(S) fraction (Fig. 9). The amount of SAHH monomer in the II(S) fraction was too low to quantitate. Cu binding was also detected where MT and GSH elute from Superose (Fig. 8)(2) . It should be noted that any Cu that binds less in the I(S) and II(S) fractions from copper-deficient cytosols would be expected to bind to MT when Cu was added to the cytosols. Thus, the increase in MT-bound Cu that was detected (Fig. 8) was most likely due to secondary decreases in Cu binding in the other two fractions rather than an effect on MT levels.


Figure 8: Superose Cu binding profiles of liver cytosol from moderately copper-deficient and control mice. Liver cytosols were incubated with 0.65 µMCu and then fractionated on Superose 12 using 0.1 M NaCl, 0.05 M HEPES, pH 7.4, at a flow rate of 0.4 ml/min and 0.22-ml fractions. The Cu (pg) bound to individual fractions was determined in a counter. The opencircles represent the moderately copper-deficient mice, and the closedcircles represent the control mice. Fraction I(S) is where the tetramer and dimer of SAHH elute, and the II(S) fraction is where the monomer of SAHH elutes. The data shown are the means of four control and five copper-deficient replicates.






Figure 9: Autoradiograph of Western blot of individual fractions from Superose chromatography from a severely copper-deficient and a control mouse liver cytosol. Liver cytosols were incubated with 0.65 µMCu and then fractionated on Superose 12 as described in Fig. 8. The indicated fractions from the I(S)Cu binding region were immunoblotted. The immunoblot was incubated with a 1:300 dilution of rabbit antiserum to mouse liver SAHH. Bound antibodies were detected with I-protein. Lanes1-6 correspond to fraction numbers 27-32 from the control mouse, and lanes7-12 correspond to fraction numbers 27-32 from a severely copper-deficient mouse.



The Superose Cu binding profiles from severely copper-deficient cytosols were variable (data not shown). However, the severely copper-deficient cytosols consistently showed a significant, approx40% decrease in II(S)Cu binding (Table 4). Cytosols from two mice with the lowest hemoglobin concentrations showed increased I(S)Cu binding (see ``Discussion''), while other severely copper-deficient cytosols showed decreases in I(S)Cu binding.

Analysis of Renal Cytosols

Radiolabeled copper (0.65 µMCu) was added to the renal cytosols, and the Cu-labeled cytosols were chromatographed on Superose-12. The total amounts of renal cytosolic protein were equalized in each sample. -There were no significant differences between the control and copper-deficient kidney cytosol Cu binding profiles (data not shown). Immunoblot analysis showed no significant differences between SAHH levels from control and copper-deficient kidney cytosols (data not shown). Thus, copper deficiency had a greater affect on SAHH levels in the liver than in the kidney.


DISCUSSION

A micro-ultrafiltration method for the determination of the binding constant at a high affinity site on a protein was established here, which should be useful with other systems. The method was validated by obtaining a copper dissociation constant for BSA, which was close to the value from the literature. This method requires a centrifugation step that requires approximately 15 min to complete. This time is probably not long enough to significantly disturb the binding equilibrium attained with a high affinity protein, as demonstrated by the determination of the correct binding constant for BSA. The holdup volume in the ultrafilters was 2-3% of the total volume applied to the ultrafilters. Calculations done with and without correcting for the holdup volume indicated that this volume would not significantly alter the data.

A single, high affinity copper binding site (K = 3.9 10M) per 48-kD(a) subunit was determined for mouse liver SAHH. Although mouse liver SAHH exists as a monomer-dimer-tetramer equilibrium(2) , only one dissociation constant for SAHH was detected by this method. The excellent fit of the experimental data to with the best fit parameters and the agreement of the calculated total copper concentration with the actual concentration used strongly substantiate the single, specific high affinity site interpretation. Also, the fact that SAHH has copper bound to it after purification from mouse liver indicates that SAHH binds copper in vivo. That less than one equivalent per 48-kDa subunit was detected most likely reflects losses during purification; Mono Q and chelating Superose bind copper. Moreover, SAHH may not be saturated with copper in vivo.

The immunoblot data clearly indicate that hepatic cytosols contain much higher levels of SAHH than the renal cytosols, and renal cytosols contain much more SAHH than brain cytosols. These results are consistent with the relative enzymatic activities of SAHH in these organs from the rat(39, 40) . High levels of CuBP/SAHH in the liver may play a role in the liver's ability to accumulate a high percentage of copper that enters the circulation after an oral or intravenous dose (41, 42) . Copper uptake data in cell cultures suggested that a high affinity intracellular copper binding protein was present at higher concentrations in the liver than other tissues and that this protein(s) played a role in preferential accumulation of copper by the liver(43) . CuBP/SAHH has the requisite properties to significantly contribute to this function of the liver. The kidney often acts as a second line of defense against potential toxicity from excess copper in copper toxicity and Wilson's disease(11, 44) , and under normal conditions, copper uptake by the kidney is second only to the liver(42) . The fact that SAHH levels in renal cytosols is intermediate between hepatic and brain cytosols is consistent with SAHH contributing to the relatively high copper uptake by the kidney. The brain has relatively high levels of copper, which may be due to high copper retention and low rates of efflux through the blood brain barrier. However, copper uptake and efflux through the blood brain barrier have not been well studied.

Although copper is tightly bound to SAHH, stable copper bound to SAHH freely exchanges with radioactive copper. This is not the case for typical enzymes that use copper at their active sites. The copper binding properties of SAHH are similar to those of albumin, which is known to transport and deliver copper to the liver and other tissues (45, 46) . Both proteins exchange copper and have similar dissociation constants of 6.7 10 (albumin) (37) and 3.9 10M (SAHH). Thus, the binding properties of SAHH are consistent with a possible role as an intracellular trafficking factor, which affects the intracellular distribution of copper by either equilibrating with various copper pools or by being part of a sequential series of donor/acceptor steps along a specific copper delivery pathway.

The immunoblot data clearly indicate that copper deficiency leads to decreased levels of SAHH in mouse liver. At least for the moderately copper-deficient mice, the decreases in SAHH levels correlated with decreased copper binding in the Superose fractions containing SAHH. While decreases in other proteins may have contributed to the decreases in Cu binding detected, the effect of decreased SAHH levels is likely to be a significant factor in the decreased Cu binding detected. The increase in the I(S)Cu binding fraction from some of the severely copper-deficient hepatic cytosols may be due to increases in specific proteins, especially an 80-kDa component, a putative heat shock protein, that elutes in the I(S) fraction(26) .

Ceruloplasmin activity is known to be very sensitive to copper deficiency(44) . Inactive, apo-ceruloplasmin is still synthesized and secreted in copper deficiency(47, 48) . At both levels of copper deficiency obtained, ceruloplasmin activity was barely detectable, and at both degrees of copper deficiency, hepatic levels of SAHH decreased to almost half of normal. Thus, both levels of copper deficiency studied had a maximal effect on SAHH levels and delivery of copper to apo-ceruloplasmin. That is the expected result if either SAHH copper was a precursor of ceruloplasmin copper or SAHH and ceruloplasmin had a common precursor for their copper. In any event, the leveling off of SAHH levels at 55% of normal may help the liver conserve copper once a critical level of copper deficiency is reached. It is interesting to note that SAHH may thus fulfill a copper metabolic function at these concentrations without adversely affecting its enzymatic role because a approx50% decrease in SAHH activity may only lead to a small increase in the steady-state levels of its substrate, SAH(49) .

Although investigators have searched for possible regulators of SAHH protein or mRNA levels, none were detected. Thus, copper is the first known regulator of SAHH levels. Preliminary Northern blot data (^2)indicate that SAHH mRNA levels are decreased by approx30% under conditions that decrease SAHH protein levels by approx40%. This suggests that effects of copper deficiency on transcription or mRNA half-life contribute significantly to the effect detected on protein levels. However, the data thus far do not exclude some effect on mRNA expression or SAHH protein half-life.

The sensitivity of SAHH to copper status that was detected here is consistent with a significant role for SAHH in intracellular copper metabolism. Thus far, every abnormal copper state tested affects either the levels or properties of SAHH. SAHH may affect copper metabolism by two mechanisms. SAHH enzymatic activity may influence the levels of MT and GSH(6) , which, in turn, may affect the distribution and utilization of copper, and copper binding by SAHH may influence the distribution of intracellular copper. SAHH-bound copper could influence copper distribution by equilibrating with various copper pools or by direct transfer of SAHH copper to copper enzymes or other proteins involved in copper metabolism, such as the proteins encoded by the candidate genes for Menkes (17, 18, 19) and Wilson's disease(22, 23, 24, 25) .

The abnormal properties of SAHH from the brindled mouse most likely reflect a secondary consequence of the primary defect. Mutations in the mouse X-chromosome homologue of the candidate gene for Menkes disease have been reported for three of the five mutant alleles of the mottled locus(50, 51, 52) , and a mutation in this gene was also detected in the brindled mouse. (^3)The secondary effects of the brindled mouse defect on SAHH are consistent with a metabolic interaction between SAHH and the mouse homologue of the Menkes protein. Abnormal properties of SAHH, may contribute to the abnormal phenotypic characteristics of Menkes disease and the brindled mouse defect. Similarly, decreased levels of SAHH in the toxic milk mouse may be due to a secondary effect of an abnormal distribution of intracellular copper in this defect. The gene for SAHH maps to human chromosome 20 (53) . As expected by the loci of closely linked genes, SAHH mapped to mouse chromosome 2(54) . However, the rat cDNA for SAHH also hybridized to mouse genes on chromosomes X and 8(54) . These may be pseudogenes or homologous genes that are expressed at some stage of development.


FOOTNOTES

*
This work was supported by U. S. Dept. of Agriculture Grant NYR-9101242. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Dept. of Biochemistry, State University of New York at Buffalo, 140 Farber Hall, Buffalo, NY 14214. Tel.: 716-829-3257; Fax: 716-829-2725; cammje{at}ubvms.cc.buffalo.edu.

(^1)
The abbreviations used are: CuBP, copper binding protein; SAHH, S-adenosylhomocysteine hydrolase; MT, metallothionein; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography; MOPS, 4-morpholinepropanesulfonic acid; MES, 4-morpholineethanesulfonic acid; BSA, bovine serum albumin.

(^2)
X. Zhou and M. J. Ettinger, unpublished results.

(^3)
B. Levinson (University of California, San Francisco) and J. Mercer (University of Melbourne), personal communications.


ACKNOWLEDGEMENTS

We thank Dr. Harold Rauch (University of Massachusetts, Amherst) for generously supplying toxic milk mice and parent strain breeding pairs and detailed instructions on how to breed and care for these animals.


REFERENCES

  1. Petrovic, N. (1993) Copper Incorporation Into Copper Zinc Superoxide Dismutase in Normal and Menkes Lymphoblasts and Cloning the cDNA for a Copper Binding Protein Involved in Copper Metabolism , Ph.D. dissertation, State University of New York at Buffalo
  2. Seo, H. C., and Ettinger, M. J. (1993) J. Biol. Chem. 268,1151-1159 [Abstract/Free Full Text]
  3. Ogawa, H., Gomi, T., Mueckler, M. M., Fujioka, M., Backlund, P. S., Jr., Aksamit, R. R., Unson, C. G., and Cantoni, G. L. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,719-723 [Abstract]
  4. Arredondo-Vega, F. X., Charlton, J. A., Edwards, Y. H., Hopkinson, D. A., and Whitehouse, D. B. (1989) Ann. Hum. Genet. 53,157-167 [Medline] [Order article via Infotrieve]
  5. Coulter-Karis, D. E., and Hershfield, M. S. (1989) Ann. Hum. Genet. 53,169-175 [Medline] [Order article via Infotrieve]
  6. Bethin, K. E., and Ettinger, M. J. (1995) J. Biol. Chem. 270,20698-20702 [Abstract/Free Full Text]
  7. Squibb, K. S., Cousins, R. J., and Feldman, S. L. (1977) Biochem. J. 164,223-228 [Medline] [Order article via Infotrieve]
  8. Ciriolo, M. R., Desideri, A., Paci, M., and Rotilio, G. (1990) J. Biol. Chem. 265,11030-11034 [Abstract/Free Full Text]
  9. Freedman, J. H., Ciriolo, M. R., and Peisach, J. (1989) J. Biol. Chem. 264,5598-5605 [Abstract/Free Full Text]
  10. Steinkuhler, C., Sapora, O., Carri, M. T., Nagel, W., Marocci, L., Ciriolo, M. R., Weser, U., and Rotilio, G. (1991) J. Biol. Chem. 266,24580-24587 [Abstract/Free Full Text]
  11. Danks, D. M. (1989) in The Metabolic Basis of Inherited Disease (Scriver, C., Beaudet, A., Sly, W., and Valle, D., eds) pp. 1411-1432, McGraw-Hill, New York
  12. Ettinger, M. J. (1984) Life Chem. Rep. 5,169-186
  13. Hunt, D. M. (1974) Nature 249,852-853 [Medline] [Order article via Infotrieve]
  14. Menkes, J. H., Alter, M., Steigleder, G. K., Weakley, D. R., and Sung, J. H. (1962) Pediatrics 29,764-779 [Abstract]
  15. Rauch, H. J. (1983) J. Hered. 74,141-144 [Medline] [Order article via Infotrieve]
  16. Rauch, H., Stockert, R. J., and Sternlieb, I. (1984) Hepatology 4,1072
  17. Vulpe, C., Levinson, B., Whitney, S., Packman, S., and Gitschier, J. (1993) Nat. Genet. 3,7-13 [Medline] [Order article via Infotrieve]
  18. Chelly, J., Tümer, Z., T&ncjs1134;nesen, T., Petterson, A., Ishikawa-Brush, Y., Tommerup, N., Horn, N., and Monaco, A. P. (1993) Nat. Genet. 3,14-19 [Medline] [Order article via Infotrieve]
  19. Mercer, J. F. B., Livingston, J., Hall, B., Paynter, J. A., Begy, C., Chandrasekharappa, S., Lockhart, P., Grimes, A., Bhave, M., Siemieniak, D., and Glover, T. W. (1993) Nat. Genet. 3,20-25 [Medline] [Order article via Infotrieve]
  20. Nucifora, G., Chu, L., Misra, T. K., and Silver, S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86,3544-3548 [Abstract]
  21. Odermatt, A., Suter, H., Krapf, R., and Solioz, M. (1993) J. Biol. Chem. 268,12775-12779 [Abstract/Free Full Text]
  22. Yamaguchi, Y., Heiny, M. E., and Gitlin, J. D. (1993) Biochem. Biophys. Res. Commun. 197,271-277 [CrossRef][Medline] [Order article via Infotrieve]
  23. Bull, P. C., Thomas, G. R., Rommens, J. M., Forbes, J. R., and Cox, D. W. (1993) Nat. Genet. 5,327-337 [Medline] [Order article via Infotrieve]
  24. Petrukhin, K., Fischer, S. G., Pirastu, M., Tanzi, R. E., Chernov, I., Devoto, M., Drzustowicz, L. M., Cayanis, E., Vitale, E., Russo, J. J., Matseoane, D., Boukhgalter, B., Wasco, W., Figus, A. L., Loudianos, J., Cao, A., Sternlieb, I., Evgrafov, O., Parano, E., Pavone, L., Warburton, D., Ott, J., Penchaszadeh, G. K., Scheinberg, I. H., and Gilliam, T. C. (1993) Nat. Genet. 5, 338-343 [Medline] [Order article via Infotrieve]
  25. Tanzi, R. E., Petrukhin, K., Chernov, I., Pelleques, J. L., Wasco, W., Ross, B., Romano, D. M., Parano, E., Pavone, L., Brzustowicz, L. M., Devoto, M., Peppercorn, J., Bush, A. T., Sternlieb, I., Pirastu, M., Gusella, J. F., Evgrafov, O., Penchaszadeh, G. K., Honig, B., Edelman, I. S., Soares, M. B., Scheinberg, I. H., and Gilliam, T. C. (1993) Nat. Genet. 5, 344-350 [Medline] [Order article via Infotrieve]
  26. Palida, F. A., and Ettinger, M. J. (1991) J. Biol. Chem. 266,4586-4592 [Abstract/Free Full Text]
  27. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeka, N. M., Olson, B. J., and Klenk, D. C. (1985) Anal. Biochem. 150,76-85 [Medline] [Order article via Infotrieve]
  28. Michael, C. K., Menten, L. E., and Garfinkel, D. (1979) Comp. Biomed. Res. 12,461-469 [Medline] [Order article via Infotrieve]
  29. Dawson, R. M. C. (1986) in Data for Biochemical Research (Dawson, R. M. C., Elliott, D. C., Elliott, W. H., and Jones, K. M., eds) pp. 399-405, Clarendon Press, Oxford
  30. Darwish, H. M., Hoke, J. E., and Ettinger, M. J. (1983) J. Biol. Chem. 258,13621-13626 [Abstract/Free Full Text]
  31. Waldrop, G. L., and Ettinger, M. J. (1990) Biochem. J. 267,417-422 [Medline] [Order article via Infotrieve]
  32. Mann, J. R., Camakaris, J., Danks, D. M., and Walliczek, E. G. (1979) Biochem. J. 180,605-612 [Medline] [Order article via Infotrieve]
  33. Koepke, J. A. (1984) in Laboratory Hematology (Koepke, J. D., ed) Vol. 2, pp. 868-869, Churchill Livingston, New York
  34. Schosinsky, K. H., Lehmann, H. P., and Beeler, M. R. (1974) Clin. Chem. 20,1556-1563 [Abstract/Free Full Text]
  35. Prohaska, J. R. (1991) J. Nutr. 121,355-363 [Medline] [Order article via Infotrieve]
  36. Laemmli, U. K. (1970) Nature 227,680-685 [Medline] [Order article via Infotrieve]
  37. Lau, S. J., and Sarkar, B. (1971) J. Biol. Chem. 246,5938-5943 [Abstract/Free Full Text]
  38. Seo, H. C., and Ettinger, M. J. (1993) J. Biol. Chem. 268,1160-1165 [Abstract/Free Full Text]
  39. Finkelstein, J. D., and Harris, B. (1973) Arch. Biochem. Biophys. 159,160-165 [Medline] [Order article via Infotrieve]
  40. Finkelstein, J. D. (1974) Metabolism 23,387-398 [Medline] [Order article via Infotrieve]
  41. Owen, C. A., Jr. (1965) Am. J. Physiol. 209,900-904 [Medline] [Order article via Infotrieve]
  42. Sass-Kortsak, A. (1965) Adv. Clin. Chem. 8,1-67 [Medline] [Order article via Infotrieve]
  43. Waldrop, G. L., Palida, F. A., Hadi, M., Lonergan, P. A., and Ettinger, M. J. (1990) Am. J. Physiol. 259,G219-G225
  44. Owen, C. A., Jr. (1981) Copper Deficiency and Toxicity, Noyes, Park Ridge, NY
  45. Bearn, A. G., and Kunkel, H. G. (1954) Proc. Soc. Exp. Biol. Med. 85,44-48
  46. Marceau, N., Aspin, N., and Sass-Kortsak, A. (1970) Am. J. Physiol. 218,377-383 [Medline] [Order article via Infotrieve]
  47. Matsuda, I., Pearson, T., and Holtzman, N. A. (1974) Pediatr. Res. 8,821-824 [Medline] [Order article via Infotrieve]
  48. Sato, M., and Gitlin, J. D. (1991) J. Biol. Chem. 266,5128-5134 [Abstract/Free Full Text]
  49. Hasobe, M., McKee, J. G., Ishii, H., Cools, M., Borchardt, R. T., and DeClerca, E. (1989) Mol. Pharmacol. 36,490-496 [Abstract]
  50. Levinson, B., Vulpe, C., Elder, B., Martin, C., Verley, F., Packman, S., and Gitschier, J. (1994) Nat. Genet. 6,369-373 [Medline] [Order article via Infotrieve]
  51. Mercer, J. F. B., Grimes, A., Ambrosini, L., Lockhart, P., Paynter, J. A., Dierick, H., and Glover, T. W. (1994) Nat. Genet. 6,374-378 [Medline] [Order article via Infotrieve]
  52. Das, S., Levinson, B., Vulpe, C., Whitney, S., Gitschier, J., and Packman, S. (1995) Am. J. Hum. Genet. 56,570-576 [Medline] [Order article via Infotrieve]
  53. Hershfield, M. S., and Francke, U. (1982) Science 216,739-742 [Medline] [Order article via Infotrieve]
  54. Pilz, A., Letissier, P., Moseley, H., Peters, J., and Abbott, C. (1992) Mamm. Genome 3,633-636 [Medline] [Order article via Infotrieve]

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