Purification and Properties of a Self-associating, 50-kDa Copper-binding Protein from Brindled Mouse Livers*

The brindled mouse is an animal model of Menkes disease, a fatal, X-linked disease of copper metabolism. A self-associating, 50-kDa copper-binding protein (CuBP) was purified from brindled mouse hepatic cy- tosols, and some of its properties were determined. When 64Cu-labeled whole hepatic cytosols were fractionated on Superose, statistically significantly less than normal “Cu binding was detected in both the fraction which contained the tetramer plus dimer ( ~ 2 6 % less) and the fraction containing the monomer of CuBP (~37% less). CuBP was purified from brindled mouse hepatic cytosols by successive Mono Q, chelating Superose, and phenyl-Superose columns using the same methods used to purify the protein from normal mice. However, CuBP from the brindled mice was somewhat unstable during the purification. Also, CuBP from the brindled mouse eluted abnormally from the phenyl-Superose column. Thus, while the protein from normal mice eluted at =20 min after starting the final water elution step, the brindled mouse protein eluted by -5 min. This seemed to be due to abnormal self- association in the column buffers. Consistent with the results using whole cytosols, the purified CuBP from the brindled mouse showed decreased copper binding in both the tetramer and monomer fractions from Su- perose. Moreover, under the same conditions, CuBP from the brindled mice seemed to have relatively less tetramer and more dimer than normal. The results are consisient with a significant role for CuBP


Purification and Properties of a Self-associating, 50-kDa Copper-binding Protein from Brindled Mouse Livers*
(Received for publication, July 24, 1992) Hee Chan Seo and Murray J. EttingerS The brindled mouse is an animal model of Menkes disease, a fatal, X-linked disease of copper metabolism. A self-associating, 50-kDa copper-binding protein (CuBP) was purified from brindled mouse hepatic cytosols, and some of its properties were determined. When 64Cu-labeled whole hepatic cytosols were fractionated on Superose, statistically significantly less than normal "Cu binding was detected in both the fraction which contained the tetramer plus dimer (~2 6 % less) and the fraction containing the monomer of CuBP (~3 7 % less). CuBP was purified from brindled mouse hepatic cytosols by successive Mono Q , chelating Superose, and phenyl-Superose columns using the same methods used to purify the protein from normal mice. However, CuBP from the brindled mice was somewhat unstable during the purification. Also, CuBP from the brindled mouse eluted abnormally from the phenyl-Superose column. Thus, while the protein from normal mice eluted at =20 min after starting the final water elution step, the brindled mouse protein eluted by -5 min. This seemed to be due to abnormal selfassociation in the column buffers. Consistent with the results using whole cytosols, the purified CuBP from the brindled mouse showed decreased copper binding in both the tetramer and monomer fractions from Superose. Moreover, under the same conditions, CuBP from the brindled mice seemed to have relatively less tetramer and more dimer than normal. The results are consisient with a significant role for CuBP in intracellular copper metabolism, and an abnormal structure of CuBP may be the basic defect in the brindled mice and, by inference, Menkes disease. The brindled mouse model (1) of Menkes disease (2, 3) is a potentially useful tool for identifying copper-binding proteins with specific functions in cellular copper metabolism (4). Like Menkes disease, the brindled mouse defect is an X-linked, inherited disease of copper metabolism (1)(2)(3)(4). Although Menkes disease is fatal and untreatable, brindled mice survive and grow to adulthood if given a single dose of copper at days 7-9 of age ( 5 ) . Thus, the brindled mouse is amenable to studies of copper metabolism, and copper proteins whose structures, amounts, or copper binding may be directly or indirectly affected by the defect can be examined. The basic defect in the brindled mouse does not seem to be in membrane copper transport; the kinetic parameters are normal for both uptake * This work was supported by National Institutes of Health Grant DK19708. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$To whom correspondence and reprint requests should be addressed. Tel.: 716-829-2727; Fax: 716-829-2725. and efflux with brindled mouse hepatocytes (6), fibroblasts (7), or Menkes lymphoblasts (8). Therefore, the defect most likely involves an intracellular copper-transport or coppertrafficking protein (7, 9, 10). One possible function of this protein is the delivery of copper to the cellular sites where it is incorporated into copper enzymes because the activities of several copper enzymes are decreased in brindled mice and Menkes patients (1,3,11,12). Another possible function of the protein involved in the defect may be to mediate copper efflux because net copper efflux is impaired in Menkes or brindled mouse fibroblasts (7, 13) and Menkes lymphoblasts (14), and decreased copper efflux seems to be associated with the basic defect (7).
We reported in an accompanying paper (15) the identification and purification of a self-associating, 50-kDa copperbinding protein (CuBP)' from mouse liver. Here we report that purified CuBP from brindled mouse liver has several abnormal properties including instability, reduced copper affinity, and abnormal chromatographic properties. The inherent instability of this protein most likely accounts for detecting decreased levels of the partially purified protein in earlier studies (16). The data reported here are consistent with significant roles of this protein in normal copper metabolism and the brindled mouse defect.

EXPERIMENTAL PROCEDURES
Materials-Columns (Superose-12 HR 10/30, Mono Q HR 5/5, chelating Superose HR 10/2, and phenyl-Superose HR 5/5), the HPLC pump (Model 2150), the HPLC controller (Model 2152), and all other HPLC accessories including a titanium prefilter were from Pharmacia LKB Biotechnology Inc. 64C~(N03)2 was from the Buffalo Materials Research Center of the State University of New York at Buffalo. The specific activity at the time of shipment was ~1 4 mCi/ mg copper. EDTA (disodium salt), HEPES, glycine, Tris, pepsin, and molecular weight standards were from Sigma. Silver nitrate (99.9+%) was from Alfa (Danvers, MA). All other chemicals were from Fisher.
Animals-Brindled male mice and their normal male littermates were bred as described previously (6,7). The brindled males were treated with a single injection of CuCI2 in propylene glycol (10 pg of CuC12/g) at 7-8 days after birth (5). The survival rate was 91%. Littermates were used as the controls for all experiments involving the purification of CuBP and the properties of the purified protein.
Age-and weight-matched C57 BL/6 mice from West Seneca Laboratory (West Seneca, NY) were used as the controls in the other experiments described.
Preparation of Cytosols-Hepatic cytosols from normal and brindled mice were prepared as previously described (15,16). Each frozen sample (=I ml) was thawed and used only once because the protein from the brindled mice was found to be unstable to thawing and refreezing.
Superose and Mono Q HPLC-The copper binding properties of whole hepatic cytosols and CuBP after partial or complete purification were examined by Superose columns. The Superose column was equilibrated with at least two column volumes (48 ml) of HEPES buffer (0.05 M HEPES, 0.1 M NaC1, pH 7.4). Samples were thawed and filtered, and 64Cu was added to a final volume of 250 pl. After 5 min at room temperature, 200-pl samples were injected through a titanium loop. Fraction collecting began 16 min after the sample was applied. The flow rate was 0.4 ml/min, and 220 p1 fractions were collected into test tubes containing 10 p1 of a phenylmethylsulfonyl fluoride (1.0 mg/ml) and leupeptin (10 rg/ml) solution to inhibit proteolysis. Protein absorbance was monitored at 280 nm by a single path monitor (Pharmacia, UV-1) using a 10-mm, 7.9-pl flow cell. Column fractions were counted for @Cu, and stored at -20 "C for electrophoresis. The following molecular weight markers were used to calibrate the column: ferritin (445,000), aldolase (161,000), ovalbumin (45,000), and myoglobin (17,000).
The concentrations of copper that were added to the whole hepatic cytosols were based on prior experiments with intact mouse hepatocytes (19). After hepatocytes were incubated for 5 min with 2 or 10 pM %u, the cytosols contained 0.65 p M or 3.25 pM @cu, respectively. Indistinguishable 64Cu-binding column profiles were obtained with 64Cu-labeled cytosols from cells which were incubated with %u or with cytosols isolated from cells to which equivalent amounts of copper were added (19). The properties of CuBP after partial purification by successive Mono Q, rechromatography on Mono Q, and Superose steps were also determined. The experimental conditions for the Mono Q steps were as described previously (15, 16).
Protein and @Cu Assays-Protein concentrations were determined by the BCA method with bovine serum albumin as the standard as described by the supplier (Pierce). MCu was determined with an LKB y counter (model 1282), and corrections for decay were by a program supplied with the counter.
Statistics-A two-tailed t-test was performed with a statistical program.

Copper Binding in Whole Hepatic Cytosols from Brindled
Mice-Radiolabeled copper (0.65 p~ 64Cu(II)) was added to the cytosols, and the 64Cu-labeled cytosols were fractionated on Superose (Fig. 1). No significant, reproducible differences between control and brindled mouse samples were detected in the protein elution profiles (absorbance at 280 nm). Three major %u-binding fractions were detected with both the normal and brindled mouse cytosols. These are labeled the IS, IIs, and MT (metallothionein) fractions in Fig. 1. The tetramer and dimer forms of CuBP elute in the Is fraction, and the monomer elutes in the 11s fraction (15). Less 'Wu was detected in both the IS and 11s fractions from the brindled mice ( Fig. 1 and Table I). The average decrease in the Is fraction was ~2 6 % and in the 11s fraction was ~3 7 % with samples from five different animal sets (Table I). The MT fraction showed a corresponding increase in copper binding (Table I); i.e. the increase in 'j4Cu in the MT fraction (1753 pg) about equaled the decrease in 64Cu bound to proteins in the IS plus 11s fractions (1635 pg, Table I). This result is consistent with normal hepatic MT levels in the brindled mice (20,21), i.e. the increase in MT-bound 64Cu is consistent with an increase in available 64Cu for MT binding due to decreased binding in the IS and 11s fractions rather than any abnormal property or amount of MT. Also, consistent with this interpretation, the sum of 64Cu bound to MT plus the Is and 11s fractions from the brindled mice (6078 pg) was similar to the sum from normal mice (5958 pg). It should be noted for the normal mouse sample is also shown. The 64Cu-binding peaks of the Is, IIS, and MT fractions are labeled, and the elution positions of the molecular weight standards are indicated at the top.

TABLE I
Distribution of radiolabeled cytosolic copper in Superose fractions of hepatic cytosols from normal and brindled mice Radiolabeled cytosols (0.65 JLM 64Cu) were applied to Superose columns, and 0.22-ml fractions were collected and counted. The 1s fraction was tubes 24-33; the 11s fraction, tubes 34-41; and the MTfraction, tubes 42-48 (Fig. 1). Values shown are means t S.D., n = 5 different sets of C57 (control) and Br (brindled) mouse cvtosols. that while some variability was detected in the relative amounts of 64Cu bound in the IS and 11s fractions from normal mice, the variability was greater with samples from the brindled mice as indicated by the higher percent standard deviations with the brindled mouse cytosol data ( Table I). As discussed further below, the variability that was detected may be due to variable self-association of the CuBP in the brindled mouse cytosols. The variability was not correlated with the amount of 64Cu in the MT fraction with either normal or brindled mice samples. Copper Concentration Dependence of Superose Profiles-When the 64Cu concentration in the hepatic cytosol was increased to 3.25 p~, smaller differences were detected in the IS and IIs fractions from normal and brindled mice (Fig. 2 A ) . The IS fraction was ~1 2 % less of normal and the 11s fraction was ~1 9 % less of normal when the data from five different animal sets were averaged. At 20 p~ %u, no significant differences were detected between the IS and 11s fractions from the normal and brindled mice (Fig. 2B) or by the averages from five animal sets. This was most likely due to saturation of the 64Cu-binding protein(s) involved and increased binding to additional proteins with lower copper affinities. At lower copper concentrations (0.2 pM)  differences were detected in the Superose fractions from normal and brindled mice as were detected a t 0.65 p~ 64Cu (data not shown). Also, a t 0.2 p~ 'j4Cu, a prominent shoulder on the trailing edge of the 11s fraction was detected with brindled mouse cytosols which was also often detected as a shoulder on the 11s fraction from normal mice a t 0.65 pM (tubes 38-

40).
Partial Purification of CuBP from the Brindled Mouse-In principle, decreased 64Cu binding in both the 1s and 11s fractions from the brindled mice could be due to decreased copper binding by multiple proteins in these fractions or to decreased copper binding to the different oligomeric forms of CuBP. To address these possibilities, hepatic cytosols from the brindled mice were partially purified by two successive Mono Q steps followed by Superose. Both the 64Cu-binding profiles and SDS-PAGE patterns with the brindled mouse samples were similar to what was obtained with the hepatic cytosols from normal mice (Fig. 3, A and B). However, less 64Cu binding was reproducibly detected with the brindled mouse samples (Fig. 3A). The difference seemed to be greater in @Cu-binding fractions B and C than A. Comparisons of &Cu binding and SDS-PAGE data with partially or fully purified CuBP from normal mice indicated that 64Cu-binding fractions A, B, and C were due to binding to the tetramer, dimer, and monomer forms, respectively, of CuBP. Thus, the results with the brindled mouse cytosols are consistent with decreased binding to at least the dimer and monomer forms of CuBP.
The similarities in the %Cu-binding profiles from Superose columns of the partially purified CuBP and the profiles with the whole cytosols (15) are consistent with the decreases in the IS and 11s fractions in Fig. 1 being due to decreased binding to the CuBP. Interestingly, at the lowest protein concentration tested, i.e. at the highest copper to protein ratio, no %u binding peak corresponding to the putative dimer of CuBP was detected in the brindled mouse sample, and a new 64Cubinding fraction was detected in the fractions which contained high concentrations of a =38-kDa protein or subunit (Fig. 4). This result may be due to low binding by the dimer of CuBP a t these protein concentrations and corresponding increased binding by the 38-kDa protein which was still present at a high concentration.
Purification of CuBP from the Brindled Mouse-The CuBP from brindled mouse hepatic cytosols was purified to homogeneity by the same protocol that was used to purify the protein from the normal mouse (15): successive Mono Q, chelating Superose, and phenyl-Superose HPLC. The protein from the brindled mouse behaved somewhat differently in some of the purification steps. One significant difference was in the overall yield of CuBP from the chelating Superose

FIG. 4. Copper binding by partially purified CuBP at low concentrations from normal and brindled mice.
The same samples obtained from Mono Q and Mono Q rechromatography that were used for Fig. 3 were diluted 4-fold with the 0.05 M HEPES buffer, pH 7.4, 0.1 M NaC1, and 64Cu(II) was added. The "Cu-labeled samples (2 p M "Cu) from normal (0) and brindled mice (0) were fractionated on Superose as described in Fig. 1. The elution position of the dimer is indicated by the arrow.
column which was ~2 0 % less from the brindled mouse than the control. This seemed to be due to instability during handling. The most significant chromatographic difference was that the CuBP from the brindled mouse eluted anomalously from the phenyl-Superose column (Fig. 5). While CuBP from the normal mouse began to elute at 15 min and peaked at 20 min after initiating the Hz0 step, the protein from the brindled mouse began to elute immediately after initiating the H20 step and peaked at 2.5 and 5 min (Fig. 5). Moreover, the samples from normal mice showed a major central peak with smaller shoulders on the trailing and leading edges while the brindled mouse sample showed relatively more protein in the first fraction (Fig. 5). Since these peaks most likely represent the three oligomeric forms of CuBP, the protein from the brindled mouse may have had different percentages of the oligomeric forms at this stage of the purification. It should be noted that the elution profiles of CuBP from the phenyl-Superose with samples from normal mice were highly reproducible, i.e. it is unlikely that the abnormal elution of the brindled mouse protein was due to the specific sample that was used for Fig. 5. Interestingly, the CuBP elutes in two fractions from chelating Superose (15) and while Fraction 11 eluted anomalously from phenyl-Superose, Fraction I eluted normally (data not shown). This could be due to differences in the relative amounts of tetramer, dimer, and monomer in these fractions. However, once the phenyl-Superose-purified CuBP was concentrated in water and applied to Superose, the properties of the protein obtained from the two chelating Superose fractions were indistinguishable. SDS-PAGE of the pooled and concentrated phenyl-Superose fractions from the brindled mouse showed a single, 50-kDa band.

Copper Binding to CuBP from Normal and Brindled Mouse
Hepatic Cyt~sols-~~Cu(II) was added to the phenyl-Superosepurified proteins from normal and brindled mice, and the radiolabeled proteins were applied to Superose. Since it was reasoned that the 64Cu added would bind to any protein present, EDTA was added to minimize nonspecific binding. Moreover, the apparent affinity of CuBP could be compared to the affinity of EDTA for copper. The control sample showed large 64Cu binding in the two column fractions containing the tetramer and monomer of CuBP; high 64Cu binding to the dimer was also detected as a prominent shoulder on the tetramer fraction (Fig. 6). The apparent binding constant of CuBP as estimated from the concentrations of protein, EDTA, and copper was similar to the complex formation constant of EDTA, i.e. =lo1' M-'. The apparent affinity for copper of CuBP from the brindled mouse was less than normal (Fig. 6). Less than normal 64Cu binding was detected in both the monomer (~1 5 % less) and tetramer plus dimer (~2 6 % less) fractions. The apparent greater difference on binding to the tetramer in the brindled mouse sample may be due to less tetramer in the brindled mouse sample. While the total areas under the Azso profiles with the control and brindled mouse samples were equal, the CuBP from control mice seemed to contain relatively more of the tetramer and less of the dimer than the protein from the brindled mice (Fig. 6). It should be noted that the CuBP used for Fig. 6 was purified from Fraction I from chelating Superose; the CuBP from Fraction I1 from the brindled mice gave the identical absorbance profile as with Fraction I, <.e. it also apparently contained relatively more dimer and less tetramer than CuBP from either chelating Superose fraction from normal mice.

DISCUSSION
CuBP from the brindled mouse appears to have several abnormal properties. The apparent copper affinity of CuBP from the brindled mouse is less than normal. and the brindled mouse protein seems to have a somewhat smaller tendency to form the tetramer from the dimer than normal. Also, CuBP from the brindled mouse seemed unstable since the overall yield during the chelating Superose step was less than normal. Perhaps the most striking abnormality of the brindled mouse protein was its elution from phenyl-Superose. While the normal CuBP eluted only after 20 min of the 100% water step, the CuBP from the brindled mouse began to elute immediately after the HzO step began, and the entire CuBP from the brindled mouse eluted before the CuBP from normal mice would begin to elute. These results suggest that the surface of the CuBP from the brindled mouse is less hydrophobic than normal. This could be due to a different mixture of tetramer, dimer, and monomer or different conformations of the oligomeric forms of the brindled mouse protein. An abnormal CuBP could also account for the variability that was observed with the Superose profiles with the whole cytosols from the brindled mice. Any effect on self-association and/or the stability of the CuBP would be expected to have significant effects on the relative amounts of ' %u in the Is and IIs fractions.
The simplest interpretation of the Superose results with the whole hepatic cytosols is that the decreases in 64Cu in the 1s and 11s fractions that were detected were due to the decreased affinity of the CuBP rather than to decreased binding t o multiple proteins in these fractions. This follows from the fact that the affinities of the tetramer, dimer, and monomer from the brindled mice are each decreased, and that the monomer elutes at or near the peak of the 11s fraction from whole hepatic cytosols while the tetramer and dimer elute in the IS fraction (15). Moreover, the fact that similar quantitative differences in copper binding between normal and brindled mice were detected in the Superose fractions of the whole cytosol, the Superose fractions after two Mono Q purification steps, HPLC fractions containing CuBP after various other partial purification protocols involving Mono Q and Superose columns (22), and the fully purified protein indicates that the lower affinity of CuBP purified from the brindled mice was not an artifact of the purification procedure. These results are also consistent with CuBP being a major contributor to the copper binding in both of the Superose fractions which elute before M T with the whole hepatic cytosol. If CuBP were a minor contributor, then decreased binding to the CuBP would not be detected in the Superose profiles. The apparently greater effect of the brindled mouse defect on the IIs fraction than the IS fraction with whole hepatic cytosols is consistent with the monomer having a lower affinity than the tetramer in both normal and brindled mice. This is also consistent with the concentration dependence of copper binding to the fractions from the Mono Q-Mono Q-Superose protocol which also indicated that the tetramer has a higher affinity than the monomer (data not shown). Also, the competitive effect of EDTA on 64Cu binding to the monomer was greater than its effect on binding to the dimer or tetramer.
An abnormal structure of CuBP may be the primary defect in the brindled mouse. Clearly, a decreased affinity for copper, instability, abnormal aggregation, or abnormal conformation of CuBP could significantly decrease the activity of this protein in intracellular copper metabolism and give rise to the biochemical abnormalities that are associated with the disease. However, it is also possible that the abnormalities that were detected with CuBP from the brindled mouse are secondary consequences of the primary defect. For example, CuBP may play an important role in intracellular copper metabolism, but, in uiuo, it may bind copper that is donated to it by the protein that is actually responsible for the basic defect in the brindled mouse. If CuBP from the brindled mouse had less than its normal complement of copper, when isolated, its stability, aggregation, and elution from phenyl-Superose could all be affected. Clearly, further analyses of the protein and the gene encoding its sequence are necessary to determine if the abnormal properties of CuBP from the brindled mouse are primary or secondary to the basic defect in the brindled mouse.
We previously reported that the amount of a partially purified 48-kDa protein was less in brindled mouse hepatic and renal cytosols than in the normal mouse (16). The migrations of the 48-kDa and 50-kDa CuBP on SDS-PAGE gels and their elution positions from Mono Q columns indicate that these proteins are identical. Previously, the cytosols that were used for the HPLC columns were thawed and refrozen for further use. Here, aliquots sufficient for single HPLC columns were stored at -70 "C and used once. Also, cytosols were isolated and frozen more rapidly than in the initial studies. These precautions markedly reduced the differences that were detected in the amounts of the protein from the brindled and normal mice and are consistent with the previously reported results being due to instability of the protein from the brindled mice during handling rather than decreased steady state levels of CuBP in uiuo.
Several functions can be postulated for CuBP. The estimated affinity constant of CuBP for copper, i.e. -lo1' "I, is consistent with an intracellular copper-transport function. The stability constant of albumin-Cu2+, which is a major plasma copper transport protein, is 6 X 10" I " ' (23). Thus, the CuBP could transport copper to specific cellular compartments and specific apocopper enzymes. Irrespective of whether the abnormal properties of CuBP from the brindled mouse are primary or secondary to the basic defect, the copper-metabolic abnormalities in the brindled mouse defect suggest several possible functions for CuBP. CuBP may be involved in delivering copper to the cellular sites where copper is incorporated into essential copper enzymes because the activities of several copper enzymes are decreased in the brindled mice (1,3,11,12). Also, a role for a liver protein(s) in helping the liver preferentially accumulate and trap copper was recently postulated (24). Since the liver is known to be Brindled Mouse Copper-binding Protein 1165 low in copper in the brindled mice, decreased copper binding by CuBP or decreased ability to deliver copper to the sites where it is retained may contribute to their decreased hepatic copper levels. In addition, this protein may mediate copper efflux from, e.g. fibroblasts, lymphoblasts, and kidney cells ( 7 ) ; preliminary results with antibodies raised to the mouse liver CuBP indicate that mouse kidney and human lymphoblasts also contain CuBP. The liver may either have more of this protein or another protein(s) which can mediate copper efflux which would account for normal efflux from the liver, but not from the kidney and other cell types in the brindled mouse ( 7 ) . Thus, CuBP may be a general copper trafficking protein with several related functions. Although it has been known since the early 1970s that copper binds to high molecular weight proteins and MT when it first enters most organs (25-28), CuBP is the first protein which has been identified and purified other than MT that potentially plays a pivotal role in cellular copper metabolism, and an abnormal CuBP may be the basic defect in the brindled mouse and Menkes disease.