©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of a Major Hepatic Copper Binding Protein as S-Adenosylhomocysteine Hydrolase (*)

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

Kathleen E. Bethin Nenad Petrovic 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 properties of a mouse liver copper binding protein (CuBP) and human placental S-adenosylhomocysteine hydrolase (SAHH) were compared to test the hypothesis that CuBP is SAHH. CuBP and SAHH migrated identically on SDS-polyacrylamide gel electrophoresis gels, and their 48-kDa monomers both self-associate to tetramers. Human placental SAHH cross-reacted with polyclonal antibodies to mouse liver CuBP, and CuBP from mouse liver cross-reacted with two monoclonal antibodies to human placental SAHH. A third monoclonal antibody to human placenta SAHH reacted weakly with the mouse liver protein but well with CuBP from human lymphoblasts. NAD-activated CuBP has high SAHH enzymatic activity. Moreover, human placental SAHH, like mouse liver CuBP, has a single high affinity copper binding site per 48-kDa subunit. Thus, the data confirm that CuBP is SAHH, and SAHH is proposed to be a bifunctional protein with roles in sulfur-amino acid metabolism and copper metabolism. The copper binding activity of SAHH is proposed to play a significant role in the intracellular distribution of copper, and SAHH enzymatic activity may influence copper metabolism through its role in cysteine biosynthesis from methionine.


INTRODUCTION

A copper binding protein (CuBP) (^1)was recently purified from mouse liver(1) . The 48-kDa subunit of CuBP self-associates to a dimer and tetramer(1) . CuBP has a single, high affinity copper-binding site per 48-kDa monomer(2) . CuBP was proposed to be a major copper binding protein in mouse liver cytosols because it coeluted with high copper binding fractions from HPLC columns and had a higher affinity for copper than other proteins in those fractions(1) . A possible role for CuBP in intracellular copper metabolism was inferred from the abnormal properties of CuBP that were isolated from the livers of brindled mice(3) . The brindled mouse is an animal model (4) for Menkes disease that is a fatal X-linked disease of copper metabolism(5, 6, 7) . Candidate genes (cDNAs) for the defects in Menkes disease and Wilson's disease were recently cloned(8, 9, 10, 11, 12, 13, 14) , which are homologous to bacterial membrane copper and cadmium transporters(15, 16) . Based on these homologies and the characteristics of these diseases, the proteins encoded by these genes may be intracellular membrane copper transport proteins(8) . Our working hypothesis is that CuBP interacts with these proteins in copper metabolism.

The cDNA for CuBP was recently cloned from a mouse liver cDNA library(17) . The deduced amino acid sequence of CuBP (17) showed greater than 95% identities to the deduced amino acid sequences of rat (18) and human (19, 20) S-adenosylhomocysteine hydrolase (SAHH). Human placental and rat liver SAHH are 48-kDa proteins that self-associate to tetramers(21, 22) . SAHH catalyzes the reversible hydrolysis of S-adenosylhomocysteine (SAH) to adenosine and homocysteine(23) . SAHH activity may be required in copper metabolism for the synthesis of cysteine from homocysteine. Cysteine is required in large amounts for the synthesis of metallothionein (24) and glutathione, which, in turn, have postulated roles in copper metabolism.

The cDNA encoding CuBP was cloned by polymerase chain reaction and library hybridization screening based on the partial sequence of a cyanogen bromide peptide from CuBP(17) . While the sequence of the cDNA that was cloned clearly indicated that it was the cDNA encoding SAHH, that alone did not prove that CuBP was SAHH. The molecular and functional properties of the mouse liver CuBP and human placental SAHH are compared here to further test the hypothesis that CuBP is SAHH. Since the results are consistent with this hypothesis, SAHH is proposed to be a bifunctional protein with roles in methionine/cysteine metabolism and copper metabolism.


EXPERIMENTAL PROCEDURES

Materials

Immobilon-P membranes for immunoblotting were from Millipore. I-Protein A was from ICN (Irvine, CA). The protein assay (28) reagents bicinchoninic acid and microbicinchoninic acid were from Pierce. Homocysteine thiolactone, NAD, adenosine, inosine, S-adenosylhomocysteine, antirabbit IgG alkaline phosphatase conjugate, antimouse polyvalent immunoglobulins/alkaline phosphatase conjugate, phenylmethylsulfonyl fluoride, leupeptin, MOPS, and HEPES were obtained from Sigma. Silver nitrate (99.9+%) was from Alfa (Ward Hill, MA), nitroblue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate were from Bio-Rad, and [8-^14C]adenosine (approx60 mCi/mmol) was from DuPont NEN.

Animals

Normal mice (C57 BL/6) were from Harlan SpragueDawley. The brindled mice were bred as previously described (25, 26) . 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, which prolongs their life span(25, 26, 27) . Control and brindled mice were fed normal diets and tap water ad libitum.

CuBP and SAHH Proteins

CuBP was purified from mouse liver as recently described(1, 2) . Human placental SAHH and recombinant SAHH from human placental SAHH cDNA expressed in Escherichia coli, which does not normally contain SAHH, were kind gifts from Dr. Michael Hershfield (Duke University, Durham, NC). Since the human proteins were isolated in 1 mM dithiothreitol (DTT) and 1 mM EDTA, they were diluted in 100 mM NaCl, 50 mM MOPS, pH 7.4, and then concentrated by centrifugation in a Centricon-30 concentrator (Amicon). The protein was diluted and reconcentrated 10-15 times until no EDTA or DTT remained.

Antibodies

Polyclonal antibodies to CuBP were generated in two female (approx12 weeks old) New Zealand White rabbits (Becker Farms, Lockport, NY). 3 weeks prior to immunization, approx15 ml of preimmune sera were obtained from each rabbit. Three monoclonal antibodies raised to human placental SAHH were obtained from Dr. Michael Hershfield (Duke University, Durham, NC).

Electrophoresis and Immunoblotting

SDS-PAGE was performed by the Laemmli method (29) using a mini-gel apparatus (Bio-Rad). Immunoblots were done as described by Towbin et al.(30) with a mini-blot system (Bio-Rad). Gels were transferred to Immobilon-P for 45-50 min at 100 V in a cold room. The transfer buffer was precooled and kept cool during transfer by an ice block. A 3% gelatin solution was used to block nonspecific binding, and 1% gelatin was included in all antibody incubations. The membranes were blocked for 2 h and then incubated with antibody overnight. Immune or preimmune sera was used at a 1:300 dilution; monoclonal antibodies were used at a 1:10,000 dilution. I-Protein A or antirabbit IgG conjugated to alkaline phosphatase (for polyclonal antibodies to CuBP) or antimouse IgG conjugated to alkaline phosphatase (for monoclonal antibodies to SAHH) was used to detect antibody bound to the membrane. When [I]-protein A was used, 4 µCi of I-protein A were incubated with the blot for 2 h. When the alkaline phosphatase method of detection was used, the anti-immunoglobulin alkaline phosphatase conjugate at a 1:10,000 to 1:15,000 dilution was incubated with the blot for 1 h. Nitroblue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate were used to visualize the alkaline phosphatase-linked antibody. After washing and drying the blot, the blot was wrapped in plastic wrap and then exposed to x-ray film with an intensifying screen at -70 °C. Band intensities were quantitated by laser densitometry (Ultrascan XL, Life Technologies, Inc.).

Copper Binding to Recombinant Human SAHH

The dissociation constant for copper binding to recombinant human placental SAHH was determined by a recently developed ultrafiltration method(2) . EDTA was included as a competing ligand, and the protein concentration was varied instead of the copper concentration to reduce the error of the binding measurements(2) . The binding data were corrected for nonspecific binding of Cu(II) to the filters as described in the accompanying paper(2) .

SAHH Activity

SAHH activity was measured in the synthesis direction by a TLC method described by Hershfield(31) . Just prior to use, L-homocysteine thiolactone was converted to homocysteine by incubation at 37 °C with 0.05 N NaOH for 30 min, followed by neutralization with HCl. Assay samples were mixed in a 50-µL final volume of 50 mM K-phosphate buffer, pH 7.4; 1 mM DTT, 5 mM homocysteine, and 50 µM [8-^14C]adenosine was added last to initiate the reaction. When NAD was included, the protein was incubated with 5 molar equivalents of NAD for 15 min at 22 °C prior to initiating the reaction. The reaction mixture was incubated at 37 °C, and 1-µl samples were spotted on TLC plates at 5-min intervals for 30 min. The chromatograms were developed in butanol-1/glacial acetic acid/water (12:3:5, v/v) and dried. Adenosine, inosine, and SAH standards were used to identify the substrate and product under ultraviolet light. The complete spots containing [^14C]adenosine and [^14C]SAH were then detected by exposing x-ray film to the TLC plate. The radiolabeled substrate and product were then cut from the chromatograms and counted by liquid scintillation spectrometry. The amount of product formed at each time point was corrected for the total yield of [^14C]adenosine and ^14C-labeled S-adenosylhomocysteine. Specific activities were determined from the linear least squares slopes of the product versus time plots and the milligrams of protein in the sample.


RESULTS

The Subunit Properties of CuBP and SAHH

As predicted from the literature(1, 21, 22, 32) , mouse liver CuBP, human placental SAHH, and human recombinant SAHH migrated identically on SDS-PAGE at 48 kDa (Fig. 1). Also noteworthy, doublets were sometimes detected on SDS-PAGE with both CuBP and SAHH(1, 19, 33) . The higher molecular weight form of CuBP converted to the lower molecular weight form after storage at low ionic strength or repetitive Superose chromatography(1) , indicating that the two components detected by SDS-PAGE represented two forms of CuBP.


Figure 1: Silver-stained SDS-PAGE analysis of mouse liver CuBP/SAHH and human placental and recombinant CuBP/SAHH. Lanes1 and 8 are 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. Lanes2 and 7, 300 ng of mouse liver CuBP/SAHH (labeled M); lanes3 and 4, 300 ng of human recombinant placental CuBP/SAHH (labeled R); and lanes5 and 6, 250 ng of human placental CuBP/SAHH (labeled P).



Antibody Cross-reactivities of SAHH and CuBP

Recombinant SAHH and human placental SAHH cross-reacted with polyclonal antibodies to mouse liver CuBP (Fig. 2). There was less placental SAHH than recombinant SAHH or mouse liver CuBP loaded onto the gel that was used for Fig. 2. Three monoclonal antibodies to human SAHH (Dr. M. Hershfield, Duke University, Durham, NC) were tested for their cross-reactivities to mouse liver CuBP from brindled and control mice (Fig. 3, A-C). Two of the three monoclonal antibodies reacted with both the control and brindled mouse liver CuBP as well as with human SAHH. However, the third monoclonal antibody showed only faint reactivity to mouse liver CuBP. Human lymphoblast CuBP (which was partially purified and concentrated by Mono Q chromatography(2) ), human recombinant SAHH, and human placental SAHH were tested with the third monoclonal antibody (Fig. 3D). Parallel SDS-PAGE gels stained with silver indicated similar amounts of 48-kDa protein in the three samples. While this monoclonal antibody reacted only weakly with mouse liver CuBP (Fig. 3C), it reacted well with human lymphoblast CuBP (Fig. 3D). Thus, this monoclonal antibody apparently recognizes a species-specific epitope on SAHH, which is noteworthy considering the >95% sequence identities between mouse CuBP and human SAHH(17) .


Figure 2: Autoradiograph of Western blot of mouse liver and human placental and recombinant CuBP/SAHH using polyclonal antibodies raised to mouse liver CuBP/SAHH. The Western blot was incubated with a 1:300 dilution of rabbit antisera to mouse liver CuBP. Bound antibodies were detected with I-protein A. Lane1, 300 ng of mouse liver CuBP/SAHH (labeled M); lane2, 300 ng of human recombinant placental CuBP/SAHH(labeled R); and lane3, 250 ng of human placental CuBP/SAHH (labeled P).




Figure 3: Western blot analysis, using monoclonal antibodies raised to human placental CuBP/SAHH, of mouse liver CuBP/SAHH, human lymphoblast CuBP/SAHH, and human placental and recombinant CuBP/SAHH. Bound antibody was detected with antimouse antibody alkaline phosphatase conjugate (diluted 1:8,000) and the substrates nitroblue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate as described under ``Experimental Procedures.'' A, the immunoblot was incubated with a 1:10,000 dilution of monoclonal antibody 1. Lane1, 220 ng of mouse liver CuBP/SAHH (labeled M); lanes2 and 3, 220 ng of human recombinant CuBP/SAHH (labeled R); and lanes4 and 5, 190 ng of human placental CuBP/SAHH (labeled P). B, the immunoblot was incubated with a 1:10,000 dilution of monoclonal antibody 2. Lane1, 220 ng of mouse liver CuBP/SAHH (labeled M); lane2, 220 ng of brindled mouse liver CuBP/SAHH (labeled Br); lane3, 220 ng of human recombinant CuBP/SAHH (labeled R); and lane4, 190 ng of human placental CuBP/SAHH (labeled P). C, the immunoblot was incubated with a 1:10,000 dilution of monoclonal antibody 3. Lane1, 220 ng of mouse liver CuBP/SAHH (labeled M); lane2, 220 ng of brindled mouse liver CuBP/SAHH (labeled Br); lane3, 220 ng of human recombinant CuBP/SAHH (labeled R); and lane4, 190 ng of human placental CuBP/SAHH (labeled P). D, the immunoblot was incubated with a 1:10,000 dilution of monoclonal antibody 3. Lanes1 and 2, partially purified and concentrated human lymphoblast CuBP/SAHH (labeled L); lane3, 220 ng of human recombinant CuBP/SAHH (labeled R).



The Catalytic Activity of Mouse Liver CuBP

SAHH enzymatic activity was measured in the synthesis direction by TLC analysis of the rate of formation of SAH from [^14C]adenosine and homocysteine (see ``Experimental Procedures''). The specific activity of mouse liver CuBP was low (Fig. 4). However, it was known from the literature that SAHH frequently has low activity when first isolated after purification (34) . In some cases, SAHH was activated by simply adding NAD(35, 36) , which is required for enzymatic activity. In other cases, NADH needed to be removed from SAHH and then replaced with NAD(35, 37) . When CuBP was incubated with 5 molar equivalents of NAD, its specific activity increased 33-fold to 0.039 µmol/min/mg (Fig. 4). The addition of NAD to human placental SAHH, which was purified differently than the mouse protein, had no effect on its catalytic activity. Although CuBP had high SAHH activity after NAD was added, human placental SAHH was approx15 times more active than NAD-activated mouse liver CuBP. The specific activity of CuBP isolated from brindled mouse liver was as low as from control mice, and the brindled mouse liver enyzme was activated 29-fold by 5 molar equivalents of NAD.


Figure 4: SAHH activity of mouse liver CuBP/SAHH. SAHH activity was measured in the synthesis direction in 50 mM potassium phosphate, pH 7.0, 1 mM DTT. 1.36 µg of mouse liver CuBP/SAHH was incubated at 37 °C with 5 mM homocysteine and 50 µM [8-^14C]adenosine for the times indicated. The opencircles represent the reaction without any additions. The closedcircles represent the reaction with five equivalents of NAD. The opensquares represent the reaction with five equivalents NAD and two equivalents CuSO(4).



Since SAHH has a high affinity for copper(2) , the effect of copper on the catalytic activity of SAHH was examined in vitro. The addition of 2 molar equivalents of copper had no significant effect on the catalytic activities of either mouse liver CuBP (Fig. 4) or human placental SAHH (data not shown) under the assay conditions used.

The Affinity of Human SAHH for Copper

The dissociation constant for copper binding to recombinant human SAHH was determined by an ultrafiltration method(2) . Fig. 5shows the calculated and observed copper binding data with human SAHH. The corrected experimental data fit very well to the binding equation. The K determined for recombinant SAHH was 5.8 ± 0.4 10M, which is approx7-fold lower than the K for copper binding to mouse liver SAHH(2) . Thus, human placental SAHH has a slightly higher affinity for copper than mouse liver CuBP, which may be due to species differences or the different purification methods that were used to purify the proteins used for these studies.


Figure 5: Cu binding to human recombinant 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 opencircles represent the means ± S.D. of triplicates of the corrected observed data. The closedcircles represent the calculated amount bound from the best fit of the data to the binding equation. The best fit K was 5.8 ± 0.4 10M.




DISCUSSION

The deduced >95% amino acid sequence identities of mouse liver CuBP, rat liver SAHH, and human placental SAHH (17) clearly indicated that the cDNA cloned was the cDNA for mouse liver SAHH. However, the sequence identity alone did not unequivocally establish that CuBP is SAHH. The possibilities were that 1) CuBP and SAHH were copurified, and the amino acid sequence of the cyanogen bromide peptide that was used for cloning the cDNA was from SAHH rather than CuBP; 2) CuBP and SAHH shared at least one homologous domain and also contained non-homologous domains; or 3) that mouse CuBP was indeed a homologue of rat and human SAHH.

SAHH as a contaminant of CuBP purification seemed unlikely because the cyanogen bromide peptide used was not a minor peptide. Moreover, CuBP purification required three different types of purification steps(1, 2) , i.e. Mono Q, chelating Superose, and phenyl Superose chromatography. Though possible, it seems improbable that both SAHH and CuBP would have similar isoelectric points, similar amounts and distributions of surface histidines, and similar surface hydrophobicities that these HPLC steps require and also have the same monomeric molecular weights and yet be two different proteins. Also, the K for copper binding to mouse liver CuBP/SAHH is similar to the K reported here for human SAHH (2) . If copper binding by CuBP were only due to contaminating SAHH, their measured K values would be very different, especially because the protein concentration was varied rather than copper concentration in the binding method used.

Because CuBP and SAHH comigrated on SDS-PAGE and both oligomerized to a tetramer of the same molecular weight, it seemed highly unlikely that they only shared a homologous domain. Also, if CuBP and SAHH shared only one homologous domain, then at least two cDNAs in the cDNA library should have hybridized to SAHH-specific probes, but only one was detected(17) . However, it was possible that the CuBP cDNA was in much lower abundance than the SAHH cDNA in the library used for cloning. Therefore, it was possible that although the cDNA cloned was clearly the cDNA encoding SAHH, it was not the gene for mouse liver CuBP.

Since none of the above possibilities could be unequivocally excluded, it was imperative to compare the physical and functional properties of the purified protein that was first identified as a CuBP with the protein identified as SAHH. Polyclonal antibodies raised to mouse liver CuBP cross-reacted with human placental and recombinant SAHH. Since polyclonal antibodies react with numerous epitopes, cross-reactivity to proteins that share a domain is expected. Also, if SAHH had copurified with CuBP, the antibodies would react with SAHH. Therefore, cross-reactivity with polyclonal antibodies did not unambiguously indicate that CuBP was SAHH. However, cross-reactivities of similar magnitudes with three different monoclonal antibodies is less likely with two similar but non-identical proteins. Because it is not known which epitopes on SAHH the monoclonals react with, it is possible, though unlikely, that all three monoclonals reacted with the same epitopes on a putative homologous domain. However, one of the monoclonal antibodies recognized one of the few epitopes that was unique to the human protein, indicating that this monoclonal antibody recognized a different epitope than the other two monoclonal antibodies.

Most important, CuBP and SAHH share the same functional properties, i.e. both proteins have a high affinity for copper and catalyze the synthesis of S-adenosylhomocysteine from adenosine and homocysteine. The fact that human placental SAHH has a single binding site for copper per 48-kDa subunit that has a K 5.8 10M is clearly consistent with SAHH being CuBP, i.e. human SAHH has a copper binding activity similar to that of CuBP(2) . Finally, the fact that mouse liver CuBP has SAHH catalytic activity confirms that CuBP is SAHH. Incubation of mouse liver CuBP with 5 molar equivalents of NAD activated its hydrolase activity 33-fold. Although the mouse liver enzyme was still 15-fold less active than the human placental SAHH, CuBP has high SAHH activity. The hydrolysis reaction catalyzed by SAHH is unusual in that it requires an NAD-dependent oxidation step prior to the hydrolysis step(38) . The reverse reaction, SAH synthesis, also begins with an oxidation step that requires NAD(38) . Thus, this reaction could not be catalyzed by a nonspecific hydrolase that is not homologous to SAHH. The fact that CuBP, as isolated, was inactive was not surprising in view of the SAHH literature. SAHH, as isolated, was often either inactive or partially active(34) . The chelating Superose step in the purification of CuBP involves a pH 4.9 buffer at high ionic strength, and the phenyl Superose step involves isolating CuBP at very low ionic strength(1, 2) . Either of these steps may have partially stripped NAD or NADH from CuBP, thereby accounting for the partial activation that was detected when NAD was added. Further, it was not surprising that CuBP was not fully activated because the NAD in SAHH can become irreversibly reduced to NADH(36, 39) . Since NAD/NADH is very tightly bound to SAHH, the only method to fully activate SAHH is to acid precipitate the protein and replace the NADH that is released at low pH with NAD(35, 37) .

A significant role for CuBP/SAHH in cellular copper metabolism is indicated by its high affinity for copper, the fact that SAHH levels are affected by copper levels(2) , and the effects that genetic defects of copper metabolism and copper deficiency have on its properties or levels(2) . The role of CuBP/SAHH in copper metabolism could involve both its enzymatic and copper binding activities. The catalytic activity of SAHH may be related to copper metabolism through its role in cysteine synthesis. SAH is the product of all methyl transferase reactions involving S-adenosylmethionine. SAHH catalyzes the reversible hydrolysis of SAH to adenosine and homocysteine(23) . In vivo, the reaction is driven in the hydrolysis direction by the deamination of adenosine to inosine by adenosine deaminase. In vitro, the synthesis reaction is favored thermodynamically. Homocysteine is a pivotal intermediate for either salvaging methionine or for the synthesis of cysteine. While prokaryotes and plants make methionine from cysteine, the pathway is unidirectional in animals, i.e. animals can only synthesize cysteine from methionine(40, 41) . This is the only pathway for synthesizing cysteine in animals, and cysteine is an essential amino acid in newborns(42) . MT is a cysteine-rich, metal binding (including copper) protein(43) . It was reported that when MT is induced, about 30 percent of the cysteine in a cell is used to synthesize MT(24) . Thus, MT synthesis may link SAHH catalytic activity with copper metabolism. Another possibility involves glutathione (GSH) synthesis. Cysteine is used to synthesize GSH. A direct role for GSH in copper metabolism as a copper trafficking factor has been proposed(44, 45, 46) . Also, the level of reduced GSH may be critical to the formation of Cu(I), which may be essential for copper incorporation into some copper enzymes. More generally, interactions between sulfur metabolism and copper metabolism have not been thoroughly studied and need to be explored further.

Copper is apparently not required for the catalytic activity of CuBP/SAHH; both mouse liver and human placental CuBP/SAHH are active as isolated with less than 1 g atom of copper/mol of 48-kDa subunit(2) . Moreover, copper had no significant effect on CuBP/SAHH activity when added in vitro. However, this does not preclude an effect of copper on SAHH activity and its regulation in vivo or an effect of copper on cycling between NAD and NADH during catalytic turnover. Regardless of how copper binding affects CuBP/SAHH activity, decreased levels of copper in copper deficiency most likely decrease SAHH activity by reducing the steady state levels of CuBP/SAHH protein(2) .

The concentration of SAHH in the liver and its Kfor copper binding are consistent with a significant role of SAHH in regulating the distribution of intracellular copper through its copper binding activity. The affinity of CuBP/SAHH for copper is similar to that of albumin(47) , which is known to transport and deliver copper to the liver and other tissues(48, 49) . Thus, in analogy to copper delivery from albumin, the K for copper binding to CuBP/SAHH indicates that copper bound to CuBP/SAHH could equilibrate with various copper pools, copper enzymes, or other proteins involved in copper metabolism. Also, CuBP/SAHH constitutes approx0.5% of total hepatic cytosolic protein(1, 2) . This high concentration coupled to its high affinity for copper indicates that CuBP/SAHH is likely to compete well with other cellular copper binding proteins in the liver, and CuBP/SAHH seems to make a significant contribution to the total copper binding activity of hepatic cytosols(1) . Thus, the affinity of CuBP/SAHH for copper is high enough for it to compete well with other copper binding proteins in liver cells, yet low enough for CuBP/SAHH to supply its copper to copper pools, which are active in intracellular copper metabolism, and any changes in the levels of CuBP/SAHH or its binding affinity for copper should significantly alter the intracellular distribution of copper within the liver. SAHH may also bind other metals and play a role in their distribution.

The results obtained here suggest that CuBP/SAHH is a bifunctional protein with roles in copper metabolism and sulfur amino acid metabolism. Recently, several proteins have been identified as bifunctional proteins. Aconitase, an enzyme in the Krebs cycle, has been shown to be a posttranscriptional regulator of transferrin and ferritin mRNA expression(50) . Glyceraldehyde-3-phosphate dehydrogenase, an enzyme in glycolysis, also binds tRNA and may be involved in nuclear RNA export(51) . As discussed above, CuBP/SAHH may have a bifunctional role in copper metabolism through its enzymatic and copper binding activities. Moreover, the effects of copper on CuBP/SAHH levels may affect methionine and cysteine metabolism under some conditions(2) . In particular, methylation may be affected since SAHH activity is required to remove SAH, which is a potent competitive inhibitor of methyltransferases(52) .


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; SAH, S-adenosylhomocysteine; MOPS, 4-morpholinepropanesulfonic acid; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Dr. Michael Hershfield (Duke University, Durham, NC) for interest and generosity in providing the human placenta SAHH, recombinant SAHH, the three monoclonal antibodies to SAHH.


REFERENCES

  1. Seo, H. C., and Ettinger, M. J. (1993) J. Biol. Chem. 268,1151-1159 [Abstract/Free Full Text]
  2. Bethin, K. E., and Ettinger, M. J. (1995) J. Biol. Chem. 270,20703-20711 [Abstract/Free Full Text]
  3. Seo, H. C., and Ettinger, M. J. (1993) J. Biol. Chem. 268,1160-1165 [Abstract/Free Full Text]
  4. Hunt, D. M. (1974) Nature 249,852-853 [Medline] [Order article via Infotrieve]
  5. Menkes, J. H., Alter, M., Steigleder, G. K., Weakley, D. R., and Sung, J. H. (1962) Pediatrics 29,764-779 [Abstract]
  6. 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, NY
  7. Ettinger, M. J. (1984) Life Chem. Rep. 5,169-186
  8. Vulpe, C., Levinson, B., Whitney, S., Packman, S., and Gitschier, J. (1993) Nat. Genet. 3,7-13 [Medline] [Order article via Infotrieve]
  9. Chelly, J., Tümer, Z., T, 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]
  10. 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]
  11. Yamaguchi, Y., Heiny, M. E., and Gitlin, J. D. (1993) Biochem. Biophys. Res. Commun. 197,271-277 [CrossRef][Medline] [Order article via Infotrieve]
  12. 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]
  13. 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]
  14. 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]
  15. Nucifora, G., Chu, L., Misra, T. K., and Silver, S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86,3544-3548 [Abstract]
  16. Odermatt, A., Suter, H., Krapf, R., and Solioz, M. (1993) J. Biol. Chem. 268,12775-12779 [Abstract/Free Full Text]
  17. 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
  18. 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]
  19. 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]
  20. Coulter-Karis, D. E., and Hershfield, M. S. (1989) Ann. Hum. Genet. 53,169-175 [Medline] [Order article via Infotrieve]
  21. Gomi, T., Ishiguro, Y., and Fujioka, M. (1985) J. Biol. Chem. 260,2789-2793 [Abstract]
  22. Gomi, T., Date, T., Ogawa, H., Fujioka, M., Askamit, R. R., Backlund, P. S., Jr., and Cantoni, G. L. (1989) J. Biol. Chem. 264,16138-16142 [Abstract/Free Full Text]
  23. de la Haba, G., and Cantoni, G. L. (1959) J. Biol. Chem. 234,603-608 [Free Full Text]
  24. Squibb, K. S., Cousins, R. J., and Feldman, S. L. (1977) Biochem. J. 164,223-228 [Medline] [Order article via Infotrieve]
  25. Darwish, H. M., Hoke, J. E., and Ettinger, M. J. (1983) J. Biol. Chem. 258,13621-13626 [Abstract/Free Full Text]
  26. Waldrop, G. L., and Ettinger, M. J. (1990) Biochem. J. 267,417-422 [Medline] [Order article via Infotrieve]
  27. Mann, J. R., Camakaris, J., Danks, D. M., and Walliczek, E. F. (1979) Biochem. J. 180,605-612 [Medline] [Order article via Infotrieve]
  28. 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]
  29. Laemmli, U. K. (1970) Nature 227,680-685 [Medline] [Order article via Infotrieve]
  30. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76,4350-4354 [Abstract]
  31. Hershfield, M. S. (1979) J. Biol. Chem. 254,22-25 [Abstract]
  32. Palida, F. A., and Ettinger, M. J. (1991) J. Biol. Chem. 266,4586-4592 [Abstract/Free Full Text]
  33. Doskeland, S. O., and Ueland, P. M. (1982) Biochim. Biophys. Acta 708,185-193 [Medline] [Order article via Infotrieve]
  34. Hershfield, M. S., Aiyar, V. N., Premakumar, R., and Small, W. C. (1985) Biochem. J. 230,43-52 [Medline] [Order article via Infotrieve]
  35. Gomi, T., Takata, Y., Date, T., Fujioka, M., Aksamit, R. R., Backlund, P. S., Jr., and Cantoni, G. L. (1990) J. Biol. Chem. 265,16102-16107 [Abstract/Free Full Text]
  36. de la Haba, G., Agostini, S., Bozzi, A., Merta, A., Unson, C., and Cantoni, G. (1986) Biochemistry 25,8337-8342 [Medline] [Order article via Infotrieve]
  37. Porter, D. J. T., and Boyd, F. L. (1992) J. Biol. Chem. 267,3205-3213 [Abstract/Free Full Text]
  38. Palmer, J. J., and Abeles, R. H. (1979) J. Biol. Chem. 254,1217-1226 [Abstract]
  39. Abeles, R. H., Fish, S., and Lapinskas, B. (1982) Biochemistry 21,5557-5562 [Medline] [Order article via Infotrieve]
  40. Cooper, A. J. L. (1983) Annu. Rev. Biochem. 52,187-222 [CrossRef][Medline] [Order article via Infotrieve]
  41. Pegg, A. E. (1988) Cancer Res. 48,759-774 [Abstract]
  42. Metzler, D. E. (1977) Biochemistry , pp. 846-849, Academic Press, NY
  43. Kagi, J. H. R., and Kojima, Y. (1979) in Metallothionein II (Kagi, H. R., and Kojima, X., eds) pp. 25-61, Birkhauser-Verlag, Basel _
  44. Ciriolo, M. R., Desideri, A., Paci, M., and Rotilio, G. (1990) J. Biol. Chem. 265,11030-11034 [Abstract/Free Full Text]
  45. Freedman, J. H., Ciriolo, M. R., and Peisach, J. (1989) J. Biol. Chem. 264,5598-5605 [Abstract/Free Full Text]
  46. 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]
  47. Lau, S. J., and Sarkar, B. (1971) J. Biol. Chem. 246,5938-5943 [Abstract/Free Full Text]
  48. Bearn, A. G., and Kunkel, H. G. (1954) Proc. Soc. Exp. Biol. Med. 85,44-48
  49. Marceau, N., Aspin, N., and Sass-Kortsak, A. (1970) Am. J. Physiol. 218,377-383 [Medline] [Order article via Infotrieve]
  50. Haile, D. J., Rouault, T. A., Tang, C. K., Chin, J., Harford, J. B., and Klausner, R. D. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,7536-7540 [Abstract]
  51. Singh, R., and Green, M. R. (1993) Science 259,365-368 [Medline] [Order article via Infotrieve]
  52. Liu, S. L., Wolfe, M. S., and Borchardt, R. T. (1992) Antiviral Res. 19,247-265 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.