(Received for publication, July 15, 1994; and in revised form, June 26, 1995)
From the
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
10
M, and the stoichiometry was
one g atom of copper per 48-kDa subunit. Western blots indicated that
the liver contains
12 times more SAHH than the kidney, which in
turn contains
5 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
42% the amount of SAHH detected in controls, and hepatic
levels of SAHH were also decreased by
45% 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.
A cDNA was recently cloned, which encodes a major copper binding
protein (CuBP) ()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.
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 200
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
NHCl buffer (50 mM NaOAc, 50 mM MOPS, 50
mM MES, 250 mM NaCl, 1 M NH
Cl,
pH 6.4), and a 2 M NH
Cl buffer (50 mM NaOAc, 50 mM MOPS, 50 mM MES, 250 mM NaCl, 2 M NH
Cl, pH 4.9). 7-9 ml
(4-5 column volumes) of 0.1 M ZnSO
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
Cl buffer, and then 4-6 column volumes of
starting buffer. The charged column was used
10 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 (
30
min), and the remaining proteins were eluted by a stepwise gradient of
0-6 min, 0.7 M NH
Cl (pH 6.4); 6-16
min, 1 M NH
Cl (pH 6.4); 16-32 min, 1.4 M NH
Cl (pH 4.9); 32-39 min, 1.85 M
NH
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)
SO
,
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
)
SO
; 10-17.5 min, 0.15 M(NH
)
SO
; 17.5-38
min, 0 M(NH
)
SO
, 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
. 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 0.5% 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.
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.
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.
where P is the concentration of
the ligand bound to protein, B
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.
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
µM
Cu.
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
10
M and
[Cu]
= 0.23 ± 0.016
µM.
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
1.0 equivalent of copper was added and then reached a
plateau at
1.1 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 µM
Cu. 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.
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.
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 7 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.
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).
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).
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;
-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).
Figure 8:
Superose Cu binding profiles
of liver cytosol from moderately copper-deficient and control mice.
Liver cytosols were incubated with 0.65 µM
Cu
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
is where the tetramer and dimer of SAHH elute, and the II
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
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,
40% decrease in II
Cu binding (Table 4). Cytosols from two mice with the lowest hemoglobin
concentrations showed increased I
Cu binding
(see ``Discussion''), while other severely copper-deficient
cytosols showed decreases in I
Cu binding.
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
10
M) per 48-kD
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
10
M (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
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
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
50% 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 ()indicate that SAHH mRNA levels are decreased by
30%
under conditions that decrease SAHH protein levels by
40%. 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. ()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.