Mathematical modelling of negative feedback regulation by carboxyltransferase.

Acetyl-CoA carboxylase catalyses the first committed step in fatty acid synthesis in all organisms. The chemistry is accomplished in two half-reactions: activation of biotin via carboxylation by biotin carboxylase, followed by the carboxyltransferase-catalysed transfer of the carboxyl moiety from carboxybiotin to acetyl-CoA to generate malonyl-CoA. The Escherichia coli form of the carboxyltransferase subunit was recently found to regulate its own activity and expression by binding its own mRNA. By binding acetyl-CoA or the mRNA encoding its own subunits, carboxyltransferase is able to sense the metabolic state of the cell and attenuate its own translation and enzymatic activity using a negative feedback mechanism. Here, the network of these interactions is modelled mathematically with a set of non-linear differential equations. Numerical simulations of the model show that it qualitatively and quantitatively agrees with the experimental results for both inhibition of carboxyltransferase by mRNA and attenuation of translation. The modelling of the autoregulatory function of carboxyltransferase confirms that it is more than isolated interactions, but functions as a single dynamic system.

Function of Escherichia coli biotin carboxylase requires catalytic activity of both subunits of the homodimer.

Biotin carboxylase catalyzes the ATP-dependent carboxylation of biotin and is one component of the multienzyme complex acetyl-CoA carboxylase that catalyzes the first committed step in fatty acid synthesis. The Escherichia coli biotin carboxylase is readily isolated from the other components of the acetyl-CoA carboxylase complex such that enzymatic activity is retained. The three-dimensional structure of biotin carboxylase, determined by x-ray crystallography, demonstrated that the enzyme is a homodimer consisting of two active sites in which each subunit contains a complete active site. To understand how each subunit contributes to the overall function of biotin carboxylase, we made hybrid molecules in which one subunit had a wild-type active site, and the other subunit contained an active site mutation known to significantly affect the activity of the enzyme. One of the two genes encoded a poly-histidine tag at its N terminus, whereas the other gene had an N-terminal FLAG epitope tag. The two genes were assembled into a mini-operon that was induced to give high level expression of both enzymes. "Hybrid" dimers composed of one subunit with a wild-type active site and a second subunit having a mutant active site were obtained by sequential chromatographic steps on columns of immobilized nickel chelate and anti-FLAG affinity matrices. In vitro kinetic studies of biotin carboxylase dimers in which both subunits were wild type revealed that the presence of the N-terminal tags did not alter the activity of the enzyme. However, kinetic assays of hybrid dimer biotin carboxylase molecules in which one subunit had an active site mutation (R292A, N290A, K238Q, or E288K) and the other subunit had a wild-type active site resulted in 39-, 28-, 94-, and 285-fold decreases in the activity of these enzymes, respectively. The dominant negative effects of these mutant subunits were also detected in vivo by monitoring the rate of fatty acid biosynthesis by [(14)C]acetate labeling of cellular lipids. Expression of the mutant biotin carboxylase genes from an inducible arabinose promoter resulted in a significantly reduced rate of fatty acid synthesis relative to the same strain that expressed the wild type gene. Thus, both the in vitro and in vivo data indicate that both subunits of biotin carboxylase are required for activity and that the two subunits must be in communication during enzyme function.

Site-directed mutagenesis of ATP binding residues of biotin carboxylase. Insight into the mechanism of catalysis.

Acetyl-CoA carboxylase catalyzes the first committed step in fatty acid synthesis in all plants, animals, and bacteria. The Escherichia coli form is a multimeric protein complex consisting of three distinct and separate components: biotin carboxylase, carboxyltransferase, and the biotin carboxyl carrier protein. The biotin carboxylase component catalyzes the ATP-dependent carboxylation of biotin using bicarbonate as the carboxylate source and has a distinct architecture that is characteristic of the ATP-grasp superfamily of enzymes. Included in this superfamily are d-Ala d-Ala ligase, glutathione synthetase, carbamyl phosphate synthetase, N(5)-carboxyaminoimidazole ribonucleotide synthetase, and glycinamide ribonucleotide transformylase, all of which have known three-dimensional structures and contain a number of highly conserved residues between them. Four of these residues of biotin carboxylase, Lys-116, Lys-159, His-209, and Glu-276, were selected for site-directed mutagenesis studies based on their structural homology with conserved residues of other ATP-grasp enzymes. These mutants were subjected to kinetic analysis to characterize their roles in substrate binding and catalysis. In all four mutants, the K(m) value for ATP was significantly increased, implicating these residues in the binding of ATP. This result is consistent with the crystal structures of several other ATP-grasp enzymes, which have shown specific interactions between the corresponding homologous residues and cocrystallized ADP or nucleotide analogs. In addition, the maximal velocity of the reaction was significantly reduced (between 30- and 260-fold) in the 4 mutants relative to wild type. The data suggest that the mutations have misaligned the reactants for optimal catalysis.

Movement of the biotin carboxylase B-domain as a result of ATP binding.

Acetyl-CoA carboxylase catalyzes the first committed step in fatty acid synthesis. In Escherichia coli, the enzyme is composed of three distinct protein components: biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase. The biotin carboxylase component has served for many years as a paradigm for mechanistic studies devoted toward understanding more complicated biotin-dependent carboxylases. The three-dimensional x-ray structure of an unliganded form of E. coli biotin carboxylase was originally solved in 1994 to 2.4-A resolution. This study revealed the architecture of the enzyme and demonstrated that the protein belongs to the ATP-grasp superfamily. Here we describe the three-dimensional structure of the E. coli biotin carboxylase complexed with ATP and determined to 2.5-A resolution. The major conformational change that occurs upon nucleotide binding is a rotation of approximately 45(o) of one domain relative to the other domains thereby closing off the active site pocket. Key residues involved in binding the nucleotide to the protein include Lys-116, His-236, and Glu-201. The backbone amide groups of Gly-165 and Gly-166 participate in hydrogen bonding interactions with the phosphoryl oxygens of the nucleotide. A comparison of this closed form of biotin carboxylase with carbamoyl-phosphate synthetase is presented.

Do cysteine 230 and lysine 238 of biotin carboxylase play a role in the activation of biotin?

Biotin carboxylase from Escherichia coli catalyzes the ATP-dependent carboxylation of biotin and is one component of the multienzyme complex acetyl-CoA carboxylase, which catalyzes the committed step in long-chain fatty acid synthesis. For the carboxylation of biotin to occur, biotin must be deprotonated at its N1' position. Kinetic investigations, including solvent isotope effects and enzyme inactivation by N-ethylmaleimide, suggested a catalytic role for a cysteine residue and led to the proposal of a mechanism for the deprotonation of biotin. The proposed pathway suggests a catalytic base removes a proton from a nearby cysteine residue, forming a thiolate anion, which then abstracts the proton from biotin. Inactivation studies of pyruvate carboxylase, which has an analogous mode of action to biotin carboxylase, suggests the catalytic base in this reaction is a lysine residue. Using the crystal structure of biotin carboxylase, cysteine 230 and lysine 238 were identified as the likely active-site residues that act as this acid-base pair. To test the hypothesis that cysteine 230 and lysine 238 act as an acid-base pair to deprotonate biotin, site-directed mutagenesis was used to mutate cysteine 230 to alanine (C230A) and lysine 238 to glutamine (K238Q). Mutations at either residue resulted in a 50-fold increase in the K(m) for ATP. The C230A mutation had no effect on the formation of carboxybiotin, indicating that cysteine 230 does not play a role in the deprotonation of biotin. However, the K238Q mutation resulted in no formation of carboxybiotin, which showed that lysine 238 has a role in the carboxylation reaction. N-Ethylmaleimide was found to inactivate the C230A mutant but not the K238Q mutant, suggesting that N-ethylmaleimide is reacting with lysine 238 and not cysteine 230. The pH dependence of N-ethylmaleimide inactivation revealed that the pK value for lysine 238 was 9.4 or higher, suggesting lysine 238 is not a catalytic base. Thus, the results suggest that cysteine 230 and lysine 238 do not act as an acid-base pair in the deprotonation of biotin.

Inhibition of biotin carboxylase by a reaction intermediate analog: implications for the kinetic mechanism.

The first committed step in long-chain fatty acid synthesis is catalyzed by the multienzyme complex acetyl CoA carboxylase. One component of the acetyl CoA carboxylase complex is biotin carboxylase which catalyzes the ATP-dependent carboxylation of biotin. The Escherichia coli form of biotin carboxylase can be isolated from the other components of the acetyl CoA carboxylase complex such that enzymatic activity is retained. The synthesis of a reaction intermediate analog inhibitor of biotin carboxylase has been described recently (Organic Lett. 1, 99-102, 1999). The inhibitor is formed by coupling phosphonoacetic acid to the 1'-N of biotin. In this paper the characterization of the inhibition of biotin carboxylase by this reaction-intermediate analog is described. The analog showed competitive inhibition versus ATP with a slope inhibition constant of 8 mM. Noncompetitive inhibition was found for the analog versus biotin. Phosphonoacetate exhibited competitive inhibition with respect to ATP and noncompetitive inhibition versus bicarbonate. Biotin was found to be a noncompetitive substrate inhibitor of biotin carboxylase. These data suggested that biotin carboxylase had an ordered addition of substrates with ATP binding first followed by bicarbonate and then biotin.

The biotin domain peptide from the biotin carboxyl carrier protein of Escherichia coli acetyl-CoA carboxylase causes a marked increase in the catalytic efficiency of biotin carboxylase and carboxyltransferase relative to free biotin.

Acetyl-CoA carboxylase catalyzes the first committed step in the biosynthesis of long-chain fatty acids. The Escherichia coli form of the enzyme consists of a biotin carboxylase activity, a biotin carboxyl carrier protein, and a carboxyltransferase activity. The C-terminal 87 amino acids of the biotin carboxyl carrier protein (BCCP87) form a domain that can be independently expressed, biotinylated, and purified (Chapman-Smith, A., Turner, D. L., Cronan, J. E., Morris, T. W., and Wallace, J. C. (1994) Biochem. J. 302, 881-887). The ability of the biotinylated form of this 87-residue protein (holoBCCP87) to act as a substrate for biotin carboxylase and carboxyltransferase was assessed and compared with the results with free biotin. In the case of biotin carboxylase holoBCCP87 was an excellent substrate with a K(m) of 0.16 +/- 0.05 mM and V(max) of 1000.8 +/- 182.0 min(-1). The V/K or catalytic efficiency of biotin carboxylase with holoBCCP87 as substrate was 8000-fold greater than with biotin as substrate. Stimulation of the ATP synthesis reaction of biotin carboxylase where carbamyl phosphate reacted with ADP by holoBCCP87 was 5-fold greater than with an equivalent amount of biotin. The interaction of holoBCCP87 with carboxyltransferase was characterized in the reverse direction where malonyl-CoA reacted with holoBCCP87 to form acetyl-CoA and carboxyholoBCCP87. The K(m) for holoBCCP87 was 0.45 +/- 0.07 mM while the V(max) was 2031.8 +/- 231.0 min(-1). The V/K or catalytic efficiency of carboxyltransferase with holoBCCP87 as substrate is 2000-fold greater than with biotin as substrate.

Mutations at four active site residues of biotin carboxylase abolish substrate-induced synergism by biotin.

Acetyl-CoA carboxylase catalyzes the first committed step in the biosynthesis of long-chain fatty acids. The Escherichia coli form of the enzyme consists of a biotin carboxylase protein, a biotin carboxyl carrier protein, and a carboxyltransferase protein. In this report a system for site-directed mutagenesis of the biotin carboxylase component is described. The wild-type copy of the enzyme, derived from the chromosomal gene, is separated from the mutant form of the enzyme which is coded on a plasmid. Separation of the two forms is accomplished using a histidine-tag attached to the amino terminus of the mutant form of the enzyme and nickel affinity chromatography. This system was used to mutate four active site residues, E211, E288, N290, and R292, to alanine followed by their characterization with respect to several different reactions catalyzed by biotin carboxylase. In comparison to wild-type biotin carboxylase, all four mutant enzymes gave very similar results in all the different assays, suggesting that the mutated residues have a common function. The mutations did not affect the bicarbonate-dependent ATPase reaction. In contrast, the mutations decreased the maximal velocity of the biotin-dependent ATPase reaction 1000-fold but did not affect the Km for biotin. The activity of the ATP synthesis reaction catalyzed by biotin carboxylase where carbamoyl phosphate reacts with ADP was decreased 100-fold by the mutations. The ATP synthesis reaction required biotin to stimulate the activity in the wild-type; however, biotin did not stimulate the activity of the mutant enzymes. The results showed that the mutations have abolished the ability of biotin to increase the activity of the enzyme. Thus, E211, E288, N290, and R292 were responsible, at least in part, for the substrate-induced synergism by biotin in biotin carboxylase.

Synthesis of a reaction intermediate analogue of biotin-dependent carboxylases via a selective derivatization of biotin.

[formula: see text] An efficient and practical synthesis of 1, a unique reaction intermediate analogue of biotin-dependent carboxylases, is described. The synthesis features a selective acylation of the 1'-N of biotin. Target 1 inhibits the activity of the biotin carboxylase component of acetyl CoA carboxylase. It is the first known biotin-derived inhibitor of biotin carboxylase and should promote new kinetic and structural studies of the biotin-dependent carboxylases.

Overexpression and kinetic characterization of the carboxyltransferase component of acetyl-CoA carboxylase.

Acetyl-CoA carboxylase catalyzes the first committed step in the biosynthesis of fatty acids. The Escherichia coli form of the enzyme consists of a biotin carboxylase protein, a biotin carboxyl carrier protein, and a carboxyltransferase protein. In this report the overexpression of the genes for the carboxyltransferase component is described. The steady-state kinetics of the recombinant carboxyltransferase are characterized in the reverse direction, in which malonyl-CoA reacts with biocytin to form acetyl-CoA and carboxybiocytin. The initial velocity patterns indicated that the kinetic mechanism is equilibrium-ordered with malonyl-CoA binding before biocytin and the binding of malonyl-CoA to carboxyltransferase at equilibrium. The biotin analogs, desthiobiotin and 2-imidazolidone, inhibited carboxyltransferase. Both analogs exhibited parabolic noncompetitive inhibition, which means that two molecules of inhibitor bind to the enzyme. The pH dependence for both the maximum velocity (V) and the (V/K)biocytin parameters decreased at low pH. A single ionizing group on the enzyme with a pK of 6.2 or lower in the (V/K)biocytin profile and 7. 5 in the V profile must be unprotonated for catalysis. Carboxyltransferase was inactivated by N-ethylmaleimide, whereas malonyl-CoA protected against inactivation. This suggests that a thiol in or near the active site is needed for catalysis. The rate of inactivation of carboxyltransferase by N-ethylmaleimide decreased with decreasing pH and indicated that the pK of the sulfhydryl group had a pK value of 7.3. It is proposed that the thiolate ion of a cysteine acts as a catalytic base to remove the N1' proton of biocytin.

Three-dimensional structure of the biotin carboxylase subunit of acetyl-CoA carboxylase.

Acetyl-CoA carboxylase is found in all animals, plants, and bacteria and catalyzes the first committed step in fatty acid synthesis. It is a multicomponent enzyme containing a biotin carboxylase activity, a biotin carboxyl carrier protein, and a carboxyltransferase functionality. Here we report the X-ray structure of the biotin carboxylase component from Escherichia coli determined to 2.4-A resolution. The structure was solved by a combination of multiple isomorphous replacement and electron density modification procedures. The overall fold of the molecule may be described in terms of three structural domains. The N-terminal region, formed by Met 1-Ile 103, adopts a dinucleotide binding motif with five strands of parallel beta-sheet flanked on either side by alpha-helices. The "B-domain" extends from the main body of the subunit where it folds into two alpha-helical regions and three strands of beta-sheet. Following the excursion into the B-domain, the polypeptide chain folds back into the body of the protein where it forms an eight-stranded antiparallel beta-sheet. In addition to this major secondary structural element, the C-terminal domain also contains a smaller three-stranded antiparallel beta-sheet and seven alpha-helices. The active site of the enzyme has been identified tentatively by a difference Fourier map calculated between X-ray data from the native crystals and from crystals soaked in a Ag+/biotin complex. Those amino acid residues believed to form part of the active site pocket include His 209-Glu 211, His 236-Glu 241, Glu 276, Ile 287-Glu 296, and Arg 338.2+ represents the first X-ray model of a biotin-dependent carboxylase.

A 70-amino acid zinc-binding polypeptide fragment from the regulatory chain of aspartate transcarbamoylase causes marked changes in the kinetic mechanism of the catalytic trimer.

Interaction between a 70-amino acid and zinc-binding polypeptide from the regulatory chain and the catalytic (C) trimer of aspartate transcarbamoylase (ATCase) leads to dramatic changes in enzyme activity and affinity for active site ligands. The hypothesis that the complex between a C trimer and 3 polypeptide fragments (zinc domain) is an analog of R state ATCase has been examined by steady-state kinetics, heavy-atom isotope effects, and isotope trapping experiments. Inhibition by the bisubstrate ligand, N-(phosphonacetyl)-L-aspartate (PALA), or the substrate analog, succinate, at varying concentrations of substrates, aspartate, or carbamoyl phosphate indicated a compulsory ordered kinetic mechanism with carbamoyl phosphate binding prior to aspartate. In contrast, inhibition studies on C trimer were consistent with a preferred order mechanism. Similarly, 13C kinetic isotope effects in carbamoyl phosphate at infinite aspartate indicated a partially random kinetic mechanism for C trimer, whereas results for the complex of C trimer and zinc domain were consistent with a compulsory ordered mechanism of substrate binding. The dependence of isotope effect on aspartate concentration observed for the Zn domain-C trimer complex was similar to that obtained earlier for intact ATCase. Isotope trapping experiments showed that the compulsory ordered mechanism for the complex was attributable to increased "stickiness" of carbamoyl phosphate to the Zn domain-C trimer complex as compared to C trimer alone. The rate of dissociation of carbamoyl phosphate from the Zn domain-C trimer complex was about 10(-2) that from C trimer.(ABSTRACT TRUNCATED AT 250 WORDS)

Secondary 18O and primary 13C isotope effects as a probe of transition-state structure for enzymatic decarboxylation of oxalacetate.

A new method for directly measuring 18O isotope effects on decarboxylation reactions has been developed. By running the reaction under high vacuum (10(-5) torr), CO2 leaves the solution before exchange with the oxygens of water to an extent greater than 2%. Thus, the method permits determination of 18O isotope effects with the precision of the isotope ratio mass spectrometer, and without the necessity of resorting to the remote label method and its attendant required syntheses. The method is used to determine 18O isotope effects for decarboxylation of oxalacetate (OAA) by Mg2+, and enzymatically by OAA decarboxylase from Pseudomonas putida; 13C isotope effects are also reported for this enzyme, as well as for decarboxylation of OAA by pyruvate kinase. Initial velocity patterns and pH profiles are reported for the P. putida enzyme, and all available data are used to discuss the kinetic and chemical mechanism of decarboxylation.

Ionization of amino acid residues involved in the catalytic mechanism of aspartate transcarbamoylase.

The chemical and kinetic mechanisms of the reaction catalyzed by the catalytic trimer of aspartate transcarbamoylase have been examined. The variation of the kinetic parameters with pH indicated that at least four ionizing amino acid residues are involved in substrate binding and catalysis. The pH dependence of K(ia) for carbamoyl phosphate and the K(i) for N-(phosphonoacetyl)-L- aspartate revealed that a protonated residue with a pK value of 9.0 is required for the binding of carbamoyl phosphate. However, the variation with pH of K(i) for succinate, a competitive inhibitor of aspartate, and for cysteine sulfinate, a slow substrate, showed that a single residue with a pK value of 7.3 must be protonated for binding these analogues and, by inference, aspartate. The profile of log V against pH displayed a decrease in reaction rate at low and high pH, suggesting that two groups associated with the Michaelis complex, a deprotonated residue with a pK value of 7.2 and a protonated group with a pK value of 9.5, are involved in catalysis. By contrast, the catalytically productive form of the enzyme-carbamoyl phosphate complex, as illustrated in the bell-shaped pH dependence of log (V/K)(asp), is one in which a residue with a pK value of 7.0 must be protonated while a group with a pK value of 9.1 is deprotonated. This interpretation is supported by the results from the temperature dependence of the V and V/K profiles and from the pH dependence of pK(i) for the aspartate analogues.(ABSTRACT TRUNCATED AT 250 WORDS)

The contribution of threonine 55 to catalysis in aspartate transcarbamoylase.

Heavy-atom isotope effects and steady-state kinetic parameters were measured for the catalytic trimer of an active site mutant of aspartate transcarbamoylase, T55A, to assess the role of Thr 55 in catalysis. The binding of carbamoyl phosphate to the T55A mutant was decreased by 2 orders of magnitude relative to the wild-type enzyme whereas the affinities for aspartate and succinate were not markedly altered. This indicates that Thr 55 plays a significant role in the binding of CbmP. If, as had been suggested previously, Thr 55 assists in the polarization of the carbonyl group of CbmP, the carbon isotope effect for the T55A mutant should increase relative to that observed for the wild-type enzyme. However, the opposite is seen, indicating that Thr 55 is not involved in stabilizing the oxyanion in the transition state. Quantitative analysis of a series of 13C and 15N isotope effects suggested that the rate-determining step in the reaction catalyzed by T55A trimer may be a conformational change in the protein subsequent to formation of the Michaelis complex. Thus, Thr 55 may facilitate a conformational change in the enzyme that is a prerequisite for catalysis. An altered active site environment in the binary and Michaelis complexes with T55A trimer is reflected in the pH profiles for log V, log (V/K)asp, and pK(i) succinate, show a displacement in the pK values of ionizing residues involved in aspartate binding and catalysis relative to the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Steady-state kinetics and isotope effects on the mutant catalytic trimer of aspartate transcarbamoylase containing the replacement of histidine 134 by alanine.

A detailed kinetic analysis of the catalytic trimer of aspartate transcarbamoylase containing the active site substitution H134A was performed to investigate the role of His 134 in the catalytic mechanism. Replacement of histidine by alanine resulted in decreases in the affinities for the two substrates, carbamoyl phosphate and aspartate, and the inhibitor succinate, by factors of 50, 10, and 6, respectively, and yielded a maximum velocity that was 5% that of the wild-type enzyme. However, the pK values determined from the pH dependence of the kinetic parameters, log V and log (V/K) for aspartate, the pK(i) for succinate, and the pK(ia) for carbamoyl phosphate, were similar for both the mutant and the wild-type enzymes, indicating that the protonated form of His 134 does not participate in binding and catalysis between pH 6.2 and 9.2. 13C and 15N isotope effects were studied to determine which steps in the catalytic mechanism were altered by the amino acid substitutions. The 13(V/K) for carbamoyl phosphate exhibited by the catalytic trimer containing alanine at position 134 revealed an isotope effect of 4.1%, probably equal to the intrinsic value and, together with quantitative analysis of the 15N isotope effects, showed that formation of the tetrahedral intermediate is rate-determining for the mutant enzyme. Thus, His 134 plays a role in the chemistry of the reaction in addition to substrate binding. The initial velocity pattern for the reaction catalyzed by the H134A mutant intersected to the left of the vertical axis, negating an equilibrium ordered kinetic mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of albumin and histidine on kinetics of copper transport by fibroblasts.

The kinetics of copper transport by fibroblasts were examined and compared with earlier data with hepatocytes to determine the basis of rapid, preferential copper uptake by the liver. The Km and maximal velocity (Vmax) parameters for copper transport by fibroblasts in serum-free media were comparable to the parameters with hepatocytes. As with hepatocytes, albumin markedly inhibited initial rates of copper transport by fibroblasts. Although the only effect of histidine on copper transport by hepatocytes in serum-free media is a small increase in Km, histidine as His2Cu decreases the Vmax of copper transport fivefold with fibroblasts. Moreover, although histidine increases copper accumulation by hepatocytes when transport is inhibited by albumin, histidine further inhibits copper accumulation by fibroblasts when albumin is in the medium. Thus the inhibitory effects of histidine and albumin on copper transport by fibroblasts are additive. The data are consistent with an intermediary role for the His2Cu complex in copper transport. Copper is transported from His2Cu as the free ion, and copper transport is strictly passive with both cell types. The data suggest that rapid uptake by the liver is in part due to the ability of hepatocytes to transport copper from His2Cu more rapidly than other cell types.

Effect of albumin on net copper accumulation by fibroblasts and hepatocytes.

The liver accumulates copper rapidly and preferentially from plasma. The effects of albumin on net copper accumulation by fibroblasts and hepatocytes were compared to determine whether preferential uptake involves hepatocyte-specific sequestering of copper. Although albumin inhibits the initial rates (30 s) of copper transport by fibroblasts and hepatocytes similarly, the effects of albumin on net copper accumulation (4 h) by these cell types were strikingly different. Fibroblasts accumulate only approximately 15% as much copper when equimolar albumin is present as from albumin-free media; hepatocytes accumulate about the same amount of copper with or without extracellular albumin present. Copper efflux data show that the special capacity of hepatocytes to accumulate copper in the presence of extracellular albumin is due to greater copper retention by hepatocytes than fibroblasts. The ability of hepatocytes to accumulate copper does not seem to be due to albumin-receptor-mediated uptake, since albumin was not co-transported with copper. The data are consistent with an equilibrium model of copper accumulation in which intracellular and extracellular copper are in equilibrium with intracellular and extracellular ligands. A high-affinity, copper-binding fraction that was previously identified in cytosols from hepatocytes was low or absent in fibroblasts. This may contain a liver-specific protein(s) that helps hepatocytes sequester and retain copper from albumin or serum-containing media. Irrespective of the exact species involved, the data are consistent with rapid, preferential copper uptake by the liver being due in part to a liver-specific, intracellular copper-binding protein(s) with a high binding affinity for copper.

The relationship of excess copper accumulation by fibroblasts from the brindled mouse model of Menkes disease to the primary defect.

Fibroblasts from the brindled mouse model of Menkes disease are known to accumulate excess copper. Most of the copper in the cytosol of these fibroblasts is bound to metallothionein (MT), which is elevated in Menkes or brindled mouse fibroblasts. Copper accumulation by normal fibroblasts containing excess MT was examined to determine if the excess copper accumulation phenotype was secondary to excess MT or associated with the primary defect in fibroblasts from the brindled mice. MT was induced in normal fibroblasts by copper, zinc or dexamethasone to levels comparable with those in brindled mice fibroblasts, as determined by radioimmunoassays. Normal fibroblasts containing excess MT accumulate copper normally, i.e. they do not exhibit the excess copper accumulation phenotype. Consistent with this result, copper efflux from normal fibroblasts containing excess MT was also normal. The data suggest that one function of the protein associated with the primary defect is to help determine how much copper is taken up and retained by fibroblasts and other cell types exhibiting the excess copper phenotype in Menkes disease. The capacity of this protein is apparently exceeded in normal fibroblasts if serum or albumin is not present extracellularly to limit total copper uptake. Consistent with a defect in an intracellular protein, the kinetics of copper transport by brindled mice fibroblasts were found to be normal.