In situ determination of the molecular weight of hepatic gamma-glutamyl transferase and gamma-glutamyl hydrolase activities

The molecular weight of gamma-glutamyl transferase from normal rat liver and hepatoma tissue was determined by radiation-inactivation and found to be approx 100000 in each case. The molecular weight previously reported for the subunit containing the gamma-glutamyl binding site (22000) is considerably less than that of the holoenzyme, suggesting that in situ the large subunit is implicated in both transferase and hydrolase activities.     

Limited proteolysis of calf thymus terminal deoxynucleotidyl transferase

Controlled, limited proteolysis of homogeneous calf thymus terminal deoxynucleotidyl transferase (EC 2.7.7.31) using immobilized Staphylococcus aureus V-8 protease results in a low molecular weight form of the enzyme which possesses unaltered catalytic activity. Analysis of the products of limited proteolysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that only the large subunit, β, is modified from a molecular weight of 30,500 to 25,500. The small subunit, α, which has a molecular weight of 9500, is unchanged. A shift in the apparent isoelectric pH of the calf enzyme following proteolysis is observed from pI = 8.2 to 7.8. Both forms of the enzyme are homogeneous in the isoelectric focusing gel system, as determined by coincidence of single protein bands with terminal transferase activity on the gel. The specific activities of cleaved and uncleaved terminal transferase proteins, as well as their thermal stabilities, are comparable. These results suggest that the polypeptide domain involved in terminal transferase enzymatic activity can be probed further by novel methods involving limited proteolysis without concomitant loss in enzymatic function.     

Biochemical and immunological analysis of an abundant form of glutathione S-transferase,in mouse testis

One of the major forms of glutathione S-transferase (designated as Ft transferase) has been identified and purified to near homogeneity from mouse testis. The purification was achieved by ammonium sulfate fractionation, DEAE cellulose chromatography, hydroxylapatite chromatography and the preparative isoelectric focusing. Purified Ft transferase has an isoelectric point of 4.9 ± 0.3 and was shown to be a homodimer with a native molecular weight of about 50 000.Immunologically, antisera to Ft transferase do not crossreact with F2 or F3 transferase. However, a weak cross reactivity was observed between the antisera to F3 transferase and Ft transferase. Biochemical properties of purified Ft transferase are similar to those transferases isolated from mouse liver. Tissue distributions of the multiple forms of glutathione S-transferase were examined by column isoelectric focusing of various mouse tissue homogenates. It was found that mouse Ft transferase is present only in testis as a major form and in brain as a minor form, but not in other tissues that were examined.     

Glycosylation of nuclear and cytoplasmic proteins. Purification and characterization of a uridine diphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase.

Using a combination of conventional and affinity chromatographic techniques, we have purified a uridine diphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase (O-GlcNAc transferase) over 30,000-fold from rat liver cytosol. The transferase is soluble and very large, migrating with an apparent molecular weight of 340,000 on molecular sieve chromatography. Analysis of the purified enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals two protein species migrating at 110 (alpha subunit) and 78 (beta subunit) kDa in approximately a two-to-one ratio. Thus, the enzyme likely exists as a heterotrimer complex with two subunits of 110 kDa and one of 78 kDa (alpha 2 beta). The alpha subunit appears to contain the enzyme's active site since it is selectively radiolabeled by a specific photoaffinity probe (4-[beta-32P]thiouridine diphosphate). Photoinactivation and photolabeling of the enzyme are dependent on time and long wavelength ultraviolet light. Photolabeling of the alpha subunit is specifically blocked by UDP. The enzyme has an extremely high affinity for UDP-GlcNAc (Km = 545 nM). This unusually high affinity for the sugar nucleotide donor probably provides the enzyme an advantage over the nucleotide transporters in the endoplasmic reticulum and Golgi apparatus which compete for available cytoplasmic UDP-GlcNAc. The multimeric state and large size of the O-GlcNAc transferase imply that its activity may be highly regulated within the cell.     

Purification and cell-free translation of a unique high molecular weight form of the brain isozyme of creatine phosphokinase from mouse

Mouse brain creatine kinase was purified to homogeneity and shown to consist of two polypeptide chains of 50,000 daltons. This protein thus differs in size from all other creatine kinase molecules purified to data including the mouse muscle enzyme which shows a molecular weight between 39,000 and 42,000. The high molecular weight isozyme has been shown to represent the primary translation product of creatine phosphokinase mRNA from mouse brain. The unusual size of this creatine phosphokinase subunit provides unique tools for the study of the differential regulation of creatine kinase gene expression and for the study of subunit interactions in creatine kinase isozymes.     

Purification and characterization of dimethylallyl pyrophosphate: aspulvinone dimethylallyltransferase from Aspergillus terreus

Dimethylallyl pyrophosphate:aspulvinone dimethylallyltransferase, the prenylation enzyme for the biosynthesis of aspulvinone pigments, has been purified from mycelia of Aspergillus terreus. The transferase catalyzed the transfer of the dimethylallyl moiety from dimethylallyl pyrophosphate to either of the two aromatic rings of aspulvinone E to give the mono- and diprenylated derivatives which were identified with the metabolites aspulvinone I and aspulvinone H, respectively. Aspulvinone G, another fundamental metabolite of this series, also acted as substrate to afford the corresponding diprenylated derivative, which is assumed to be a precursor for aspulvinone C, D, and F. The molecular weight of the enzyme was estimated to be 240 000--270 000 by gel filtration. Since the subunit molecular weight determined by NaDodSO4-polyacrylamide disc gel electrophoresis was 45 000, the native enzyme appears to be a hexomeric protein composed of identical molecular weight subunits. The apparent Km values for aspulvinone E, aspulvinone G, and dimethylallyl pyrophosphate were 13.7, 7.7, and 40.0 micron, respectively. The enzyme shows the maximum activity at pH 7.0, and no metal ion is necessary for the activation. Sulfhydryl blocking agents or mercaptoethanol has no effect. Bromophenol blue binds specifically to the transferase and strongly inhibits the enzyme activity.     

Subunit structure of high molecular weight mouse nerve growth factor

Studies from several laboratories have shown that mouse submandibular glands and mouse saliva contain nerve growth factor (NGF) as part of a high molecular weight oligomeric macromolecule composed of three different subunits, termed alpha, beta, and gamma. The beta-subunit is the nerve growth-promoting protein. The gamma-subunit is a serine protease class enzyme of highly restricted substrate specificity. The alpha-subunit has no known function. This high molecular weight form of nerve growth factor is also a Zn(II)-containing metalloprotein. In the present study, measurements of multiple physicochemical parameters have been used to deduce the subunit structure of high molecular weight NGF. Results demonstrate that it contains two alpha-, one beta- and one gamma-subunit together with one tightly bound Zn(II) ion per molecule.     

臭味假单胞菌乙酰乙酰辅酶A硫解酶的提纯及性质研究

从臭味假单胞菌中提纯97倍的AcAcCoA硫解酶在聚丙烯酰胺凝胶电泳上是均一的一带。该酶分子量为170,000,每分子含有4个亚基,亚基分子量为42,000。该酶的等电点为pI6.7。它的N-末端为丙氨酸,N-末端是单一的。该酶催化反应的Km值为10.2μmol/L,最大反应速度为16.7μmol/min·mg。 臭味假单胞菌细胞粗提液透析后,经DEAE-纤维素(DE-52)柱色谱,从洗脱液中可同时得到四个酶的活力峰:乙酰乙酸琥珀酰辅酶A转移酶,AcAcCoA硫解酶,β-酮已二酸琥珀酰辅酶A转移酶和β-酮己二酸单酰辅酶A硫解酶。一般认为在细菌的芳径代谢中存在β-酮己二酸代谢途径,上述四个酶的活力峰同时存在说明除β-酮已二酸代谢途径外,还同时存在乙酰乙酸代谢途径。     

Identification of pyruvate in S-adenosylmethionine decarboxylase from rat liver

S-Adenosylmethionine decarboxylase has been purified to homogeneity (26,000-fold) from rat liver. The enzyme has a molecular weight of 155,000 and a subunit molecular weight of 42,000. One mole of covalently bound pyruvate was found to be present per mole of enzyme subunit. This is the first mammalian enzyme found to contain covalently linked pyruvate.     

The structural subunit of glucose-6-phosphate dehydrogenase (baker's yeast).

The subunit molecular weight of glucose-6-phosphate dehydrogenase (G6PD) from baker's yeast has been evaluated. The subunit molecular weight value is shown to be 25,500 daltons by analytical ultracentrifugation, SDS-polyacrylamide gel electrophoresis, and the number of peptides produced by CNBr cleavage. The number of NADP binding sites was determined to be one per 25,500 dalton unit.     

Pig heart CoA transferase exists as two oligomeric forms separated by a large kinetic barrier

Pig heart CoA transferase (EC 2.8.3.5) has been shown previously to adopt a homodimeric structure, in which each subunit has a molecular weight of 52 197 and consists of N- and C-domains linked by a hydrophilic linker or "hinge". Here we identify and characterize a second oligomeric form constituent in purified enzyme preparations, albeit at low concentrations. Both species catalyze the transfer of CoA with similar values for k(cat) and K(M). This second form sediments more rapidly than the homodimer under the conditions of conventional sedimentation velocity and active enzyme centrifugation. Apparent molecular weight values determined by sedimentation equilibrium and gel filtration chromatography are 4-fold greater than the subunit molecular weight, confirming that this form is a homotetramer. The subunits of both oligomeric forms are indistinguishable with respect to molecular mass, far-UV CD, intrinsic tryptophan fluorescence, and equilibrium unfolding. Dissociation of the homotetramer to the homodimer occurs very slowly in benign solutions containing high salt concentrations (0.25-2.0 M KCl). The homotetramer is fully converted to homodimer during refolding from denaturant at low protein concentrations. Disruption of the hydrophilic linker between the N- and C-domains by mutagenesis or mild proteolysis causes a decrease in the relative amount of the larger conformer. The homotetramer is stabilized by interactions involving the helical hinge region, and a substantial kinetic barrier hinders interconversion of the two oligomeric species under nondenaturing conditions.     

Distinction between mouse DNA polymerases alpha and beta by tryptic peptide mapping.

Results presented here and in a previous paper (Tanabe et al. (1979) Biochemistry 18, 3401--3406) indicate that mouse beta-polymerase is a single polypeptide with an apparent molecular weight of 40,000. This polypeptide has now been analyzed by tryptic peptide mapping. Comparison of the results with identical analysis of mouse alpha-polymerase reveals that the tryptic peptides derived from the two enzymes are different. These results indicate that beta-polymerase is neither a subunit of alpha-polymerase nor a proteolytic degradation product of alpha-polymerase.     

Uridine kinase: altered subunit size or enzyme expression as a function of cell type, growth stimulation, or mutagenesis

Using antibody prepared against pure uridine kinase from Ehrlich ascites cells, we have measured the expression of enzyme protein by the Western blot technique. Variations were observed in the Mr of the enzyme subunit for uridine kinase from different species: 32,000 (mouse Ehrlich ascites cells), 30,000 (normal human lymphocytes), 28,000 (mouse tissues), 27,500 (rat tissues). For different normal tissues from the same species, there was no significant variation in the subunit size. Transformed human and mouse cell lines, selected for a deficiency of uridine kinase activity in the presence of inhibitors activated by this enzyme, expressed two cross-reacting proteins, one with a normal (30,000) and one with a smaller (21,000) subunit molecular weight than was found in the parental cell line (human lymphoma), or only a smaller protein of Mr 25,000 (mouse lymphoma). Our results show that selection protocols using metabolite inhibitors do not always repress the expression of the enzyme but instead may lead to selection of those cells that have a mutation in the uridine kinase gene, resulting in the expression of an inactive enzyme. The expression of uridine kinase protein changes when cells are stimulated to divide. For both mouse fibroblasts and human lymphocytes, expression of uridine kinase protein as well as activity clearly increased after cells were stimulated to grow. In fibroblasts, increases are seen by 3 hr after stimulation, and plateau after 9 hr at a sevenfold increase. In lymphocytes, no change is seen until 12 hr after stimulation, and a plateau is not reached until 72 hr, with a total increase of approximately 50-fold. There has been considerable interest in the possibility of uridine kinase isozymes. Except for cells that have been mutagenized, the present results show that, as judged by subunit molecular weight, there appears to be only one enzyme form in normal and neoplastic cells or in cells in which uridine kinase activity is induced.     

Purification and characterization of mouse kidney beta-glucuronidase.

Beta-Glucuronidase has been purified from mouse kidneys previously induced by gonadotrophin to a specific enzyme activity 15 times higher than the non-induced kidney. The purification procedure includes ultrasonication to solubilize the enzyme, acid and ammonium sulfate precipitations, gel filtration in Sephadex G-200, DEAE-ion exchange chromatography, and isoelectric focusing. The resulting product has a specific activity of 284,000 Fishman units/mg of protein, representing a 1,090-fold purification and is 17,000-fold higher than the level in the non-induced kidney. The purified beta-glucuronidase is apparently homogeneous by criteria of gel filtration, sodium dodecyl sulfate gel electrophoresis, and immunodiffusion. Characterization of the purified enzyme showed that it is identical with the lysosomal isoenzymic from electrophoretically, has subunit molecular weight of 74,000 (estimated by sodium dodecyl sulfate gel electrophoresis) and oligomer molecular weight of 300,000. The purified enzyme is stable at high temperature (up to 55 degrees) and at wide range of pH (from 4 to 11). It has a pH optimum for its activity at 4.7 and a Km of 1.18 times 10- minus 4 M. The purification and characterization of this enzyme from mouse kidney will have significance in the understanding of the molecular nature of the isoenzymes of beta-glucuronidase and will be useful in future studies on the mechanism of intracellular transport and distribution of this hydrolase.     

Glutathione transferase of human breast is closely related to transferase of human placenta and erythrocytes

An acidic form (pI 4.6) of glutathione transferase has been purified to homogeneity from normal and tumor specimens of human breast. The two proteins did not differ significantly in their molecular and catalytic properties. The enzyme has a molecular weight of 46,000 and is composed of two identical subunits. The data presented, including amino acid composition, substrate specificity and immunological studies, give strong evidence that the glutathione transferase of human breast, placenta and erythrocytes are similar if not identical proteins.     

Isolation of murine neuron-specific and non-neuronal enolase cDNA clones

cDNA clones corresponding to subunits of neuron-specific (gamma gamma and alpha gamma) and non-neuronal (alpha alpha) enolase isozymes were characterized from two mouse brain cDNA libraries. Our hybridization data revealed a partial homology of the coding sequences of mouse alpha, mouse gamma and rat gamma mRNAs. The noncoding sequences, however, appear to be specific for each mouse mRNA. Although coding for two polypeptides of the same molecular weight, the mRNA for the gamma subunit (2600 bases) is larger than that for the alpha subunit (1900 bases). The noncoding sequences for neuron-specific gamma mRNA (about 1300 bases) are therefore longer than those of the non-nervous tissue specific alpha mRNA (about 600 bases).     

Purification and characterization of five forms of glutathione transferase from human uterus

Five glutathione transferase (GST) forms were purified from human uterus by glutathione-affinity chromatography followed by chromatofocusing, and their structural, kinetic and immunological properties were investigated. Upon SDS/polyacrylamide slab gel electrophoresis all forms resulted composed of two subunits of identical molecular size. GST V (pI 4.5) is a dimer of 23-kDa subunits. GST I (pI 6.8) and GST IV (pI 4.9) are dimers of 24-kDa subunits whereas GST II (pI 6.1) and GST III (pI 5.5) are dimers of 26.5-kDa subunits. GST V accounts for about 85-90% of the activity whereas the other isoenzymes are present in trace quantities. On the basis of the molecular mass of the subunits, amino acid composition, substrate specificities, sensitivities to inhibitors, CD spectra and immunological studies, GST V appeared very similar to transferase pi. Structural and immunological studies provide evidence that GST IV is closely related to the less 'basic' transferase (GST pI 8.5) of human skin. Extensive similarities have been found between GST II and GST III. The comparison includes amino acid compositions, subunits molecular size and immunological properties. The two enzymes, however, are kinetically distinguishable. The data presented also indicate that GST II and GST III are related to transferase mu and to transferase psi of human liver. Even though GST I has a subunit molecular mass identical to GST IV, several lines of evidence, including catalytic and immunological properties, indicate that they are different from each other. GST I seems not to be related to any of known human transferases, suggesting that it may be specific for the uterus.     

Purification and properties of ornithine carbamoyl transferase from liver of Squalus acanthias

Citrulline synthesis from ammonia by hepatic mitochondria in elasmobranchs involves intermediate formation of glutamine as the result of the presence of high levels of glutamine synthetase and a unique glutamine- and N-acetyl-glutamate-dependent carbamoyl phosphate synthetase, both of which have properties unique to the function of glutamine-dependent synthesis of urea, which is retained in the tissues of elasmobranchs at high concentrations for the purpose of osmoregulation [P.M. Anderson and C.A. Casey (1984) J. Biol. Chem. 259, 456-462; R.A. Shankar and P.M. Anderson (1985) Arch. Biochem. Biophys. 239, 248-259]. The objective of this study was to determine if ornithine carbamoyl transferase, which catalyzes the last step of mitochondrial citrulline synthesis and which has not been previously isolated from any species of fish, also has properties uniquely related to this function. Ornithine carbamoyl transferase was highly purified from isolated liver mitochondria of Squalus acanthias, a representative elasmobranch. The purified enzyme is a trimer with a subunit molecular weight of 38,000 and a native molecular weight of about 114,000. The effect of pH is significantly influenced by ornithine concentration; optimal activity is at pH 7.8 when ornithine is saturating. The apparent Km values for ornithine and carbamoyl phosphate at pH 7.8 are 0.71 and 0.05 mM, respectively. Ornithine displays considerable substrate inhibition above pH 7.8. The activity is not significantly affected by physiological concentrations of the osmolyte urea or trimethylamine-N-oxide or by a number of other metabolites. The results of kinetic studies are consistent with a steady-state ordered addition of substrates (carbamoyl phosphate binding first) and rapid equilibrium random release of products. Except for an unusually low specific activity, the properties of the purified elasmobranch enzyme are similar to the properties of ornithine carbamoyl transferase from mammalian ureotelic and other species and do not appear to be unique to its role in glutamine-dependent synthesis of urea for the purpose of osmoregulation.     

NADH-specific dihydropteridine reductase in mastocytoma P-815 cells

NADH-specific dihydropteridine reductase [EC 1.6.99.7] was purified from mouse mastocytoma P-815 cells. Km values for NADH and NADPH were determined to be 1.4 microM and 32 microM, respectively, using tetrahydro-6-methylpterin. Molecular weight was 50,000, and subunit molecular weight was 25,000. The enzymes from P-815 and liver of host mouse (DBA/2) showed similar electrophoretic mobility on polyacrylamide gel. The P-815 enzyme reacted with antiserum against bovine liver NADH-specific dihydropteridine reductase, forming a single precipitin line.     

Structure and function of the carboxyl-terminal oxygen-binding domain from the subunit of Octopus dofleini hemocyanin

The C-terminal domain, Od-1, of the 7-domain subunit of Octopus dofleini hemocyanin has been prepared by partial trypsinolysis followed by ion-exchange chromatography. It binds oxygen reversibly and is homogeneous in molecular weight. Its physical properties have been compared with those of the subunit. The domain molecular weight is found by sedimentation equilibrium to be 4.7 X 10(4), in excellent agreement with the result recently obtained in our laboratory from cDNA sequencing of this domain [Lang, W. H. (1988) Biochemistry (preceding paper in this issue)]. It has a sedimentation coefficient of 3.8 S. Both the molecular weight and sedimentation coefficient are consistent with the domain constituting approximately one-seventh of the Mr 3.5 X 10(5) subunit. Its amino acid composition and carbohydrate content differ significantly from that of the whole subunit, confirming the heterogeneity in domains previously established on an immunological basis. Circular dichroism predicts similar secondary structure for the domain and subunit. The domain does not self-associate in the presence of Mg2+ but does bind to the whole molecule in a ratio of approximately 1 domain/subunit. The oxygen affinity of this domain is quite low. It shows intrinsic magnesium and Bohr effects similar to those of the whole molecule but of greatly reduced magnitude.