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1.
Purification and characterization of protein phosphatase 1I activating kinase from bovine brain cytosolic and particulate fractions 总被引:4,自引:0,他引:4
The activating kinase of protein phosphatase 1I is distributed in approximately equal amounts between the cytosolic and particulate fractions of bovine brain homogenates. Both species of this protein kinase have been purified to near homogeneity. The cytosolic form, purified about 7,000-fold, has an apparent Mr = approximately 75,000, as estimated by gel filtration chromatography on Sephacryl S-300. The enzyme contains two subunits, with apparent Mr = 52,000 and 46,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both subunits undergo phosphorylation when the enzyme is incubated with Mg2+ and [gamma-32P]ATP. Peptide maps of the two subunits are different, and rabbit antibodies to the 52-kDa subunit show only very minor cross-reactivity to the 46-kDa subunit. These observations indicate that the two subunits are different. The species of protein phosphatase 1I activating kinase that is associated with the membrane fraction has an apparent Mr = approximately 105,000 as estimated by gel filtration. This species also contains two subunits, with apparent Mr = 52,000 and 46,000, the properties of which are very similar, if not identical, to those of the two subunits comprising the cytosolic form of the protein kinase. 相似文献
2.
M A Porter S Milanez C D Stringer F C Hartman 《Archives of biochemistry and biophysics》1986,245(1):14-23
An efficient purification procedure utilizing affinity chromatography is described for spinach ribulose-5-phosphate kinase, a light-regulated chloroplastic enzyme. Gel filtration and polyacrylamide gel electrophoresis of the purified enzyme reveal a dimeric structure of 44,000 Mr subunits. Chemical crosslinking with dimethyl suberimidate confirms the presence of two subunits per molecule of native kinase, which are shown to be identical by partial NH2-terminal sequencing. Based on sulfhydryl titrations and on amino acid analyses, each subunit contains four to five cysteinyl residues. The observed slow loss of activity during spontaneous oxidation in air-saturated buffer correlates with the intramolecular oxidation of two sulfhydryl groups, presumably those involved in thioredoxin-mediated regulation. 相似文献
3.
Leucine dehydrogenase was inhibited by p-chioromercuribenzoate and HgCl2, but not by 5,5′-dithiobis(2-nitrobenzoic acid), 4,4′-dithiopyridine and N-ethylmaleimide. Modification of sulfhydryl groups of the enzyme with p-chloromercuribenzoate and HgCl2 was accompanied with a loss of the enzyme activity. The 6 reactive sulfhydryl groups per enzyme molecule play an essential role for catalysis. Approximately 12 sulfhydryl groups were titrated per molecule in the presence of 8 m urea: the enzyme contains 2 sulfhydryl groups per subunit, and one of them participates in the catalytic action. Fluorometric and gel filtration studies on binding of NADH to the enzyme revealed that the enzyme contains 6 coenzyme binding sites per molecule.These results are compatible with the hexameric structure of leucine dehydrogenase composed of identical subunits, showing that each subunit has one catalytic site and one indispensable sulfhydryl group. 相似文献
4.
M Kandel A G Gornall D L Cybulsky S I Kandel 《The Journal of biological chemistry》1978,253(3):679-685
Spinach carbonic anhydrase has been purified by modification and extension of a published method (Pocker, Y., and Ng. J. S. U. (1973) Biochemistry 12, 5127-5134), using (NH4)2SO4 precipitation and chromatography on DEAE-cellulose, agarose, and DEAE-Sephadex. The enzyme so obtained was homogeneous by criteria of both standard and sodium dodecyl sulfate polyacrylamide gel electrophoresis and of constant specific activity throughout the elution profile on DEAE-Sephadex chromatography. The enzyme has an apparent Mr of 212,000 by gel filtration on Sephadex G-200, a Mr of 26,000 by sodium dodecyl sulfate electrophoresis, and each of the subunits contains approximately 1 g atom of zinc. These data and the excellent correlation between the number of lysine and arginine residues per subunit, and the number of tryptic peptides obtained by peptide mapping, suggest that spinach carbonic anhydrase is an octamer consisting of identical or very similar subunits. Its amino acid composition is similar to parsley carbonic anhydrase; both contain large numbers of half-cystine residues relative to erythrocyte carbonic anhydrases. The spinach enzyme is devoid of disulfide bonds. The enzyme is stable around neutrality at -14 degrees, as a suspension in saturated (NH4)2SO4 solution. 相似文献
5.
Transforming growth factor beta 1 treatment of AKR-2B cells is coupled through a pertussis-toxin-sensitive G-protein(s). 总被引:1,自引:0,他引:1
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Spermidine synthase was purified to apparent homogeneity from human spleens (8700-fold) by affinity chromatography. The native enzyme was composed of two subunits of identical Mr (35,000) and showed an apparent Mr of 62,000 in pore-gradient gel electrophoresis. Its pI was 5.1, Spermine synthase was purified to apparent homogeneity from placenta (5300-fold) and from kidney (4600-fold). The native enzyme was composed of two subunits of identical Mr (45,000) and showed an apparent Mr of 78,000 in pore-gradient gel electrophoresis. In isoelectric focusing it revealed two bands, with pI values of 4.9 and 5.0. Both synthases were present in all human tissues studied, but revealed a clear tissue-specific pattern. Mouse antisera against spermidine synthase revealed only one band, of Mr 35,000, in all purified enzyme preparations and in crude human tissue extracts in immunoblotting. Antisera against spermine synthase showed an immunoreactive band corresponding to the Mr of the subunit of spermine synthase. These antisera did not indicate any cross-reactivity in immunoblotting. Thus spermine synthase and spermidine synthase do not share homologous antigenic sites and are totally different proteins. 相似文献
6.
Purification and properties of pyruvate dehydrogenase phosphatase from bovine heart and kidney 总被引:10,自引:0,他引:10
Pyruvate dehydrogenase phosphatase was purified to apparent homogeneity from bovine heart and kidney mitochondria. The phosphatase has a sedimentation coefficient (S20,w) of about 7.4 S and a molecular weight (Mr) of about 150 000 as determined by sedimentation equilibrium and by gel-permeation chromatography. The phosphatase consists of two subunits with molecular weights of about 97 000 and 50 000 as estimated by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Phosphatase activity resides in the Mr 50 000 subunit, which is sensitive to proteolysis. The phosphatase contains approximately 1 mol of flavin adenine dinucleotide (FAD) per mol of protein of Mr 150 000. FAD is apparently associated with the Mr 97 000 subunit. The function of this subunit remains to be established. The phosphatase binds 1 mol of Ca2+ per mol of enzyme of Mr 150 000 at pH 7.0, with a dissociation constant (Kd) of about 35 microM as determined by flow dialysis. Use of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetate (EGTA) at pH 7.6 in conjunction with flow dialysis gave a Kd value for Ca2+ of about 8 microM. In the presence of both the phosphatase and the dihydrolipoyl transacetylase (E2) core of the pyruvate dehydrogenase complex, two equivalent and apparently non-interacting CA2+-binding sites were detected per unit of Mr 150 000, with a Kd value of about 24 microM in the absence and about 5 microM in the presence of EGTA. In the presence of 0.2 M KCl, which inhibits phosphatase activity about 95%, the phosphatase exhibited only one Ca2+-binding site, even in the presence of E2. The phosphatase apparently possesses an "intrinsic" Ca2+-binding site, and a second Ca2+-binding site is produced in the presence of E2. The second site is apparently altered by increasing the ionic strength. It is proposed that the second site may be at the interface between the phosphatase and E2, with Ca2+ acting as a bridging ligand for specific attachment of the phosphatase to E2. 相似文献
7.
The saxitoxin receptor of the sodium channel from rat brain. Evidence for two nonidentical beta subunits 总被引:9,自引:0,他引:9
R P Hartshorne D J Messner J C Coppersmith W A Catterall 《The Journal of biological chemistry》1982,257(23):13888-13891
The saxitoxin receptor of the sodium channel purified from rat bran contains three types of subunits: alpha with Mr approximately 270,000, beta 1 with Mr approximately 39,000, and beta 2 with Mr approximately 37,000. These are the only polypeptides which quantitatively co-migrate with the purified saxitoxin receptor during velocity sedimentation through sucrose gradients. beta 1 and beta 2 are often poorly resolved by gel electrophoresis in sodium dodecyl sulfate (SDS), but analysis of the effect of beta-mercaptoethanol on the migration is covalently attached to the alpha subunit by disulfide bonds while the beta 1 subunit is not. The alpha and beta subunits of the sodium channel were covalently labeled in situ in synaptosomes using a photoreactive derivative of scorpion toxin. Treatment of SDS-solubilized synaptosomes with beta-mercaptoethanol decreases the apparent molecular weight of the alpha subunit band without change in the amount of 125I-labeled scorpion toxin associated with either the alpha or beta subunit bands. These results indicate that the alpha and beta 1 subunits are labeled by scorpion toxin whereas beta 1 is not and that the beta 2 subunit is covalently attached to alpha by disulfide bonds in situ as well as in purified preparations. 相似文献
8.
Spermidine biosynthesis in Saccharomyces cerevisiae. Biosynthesis and processing of a proenzyme form of S-adenosylmethionine decarboxylase 总被引:4,自引:0,他引:4
K Kashiwagi S K Taneja T Y Liu C W Tabor H Tabor 《The Journal of biological chemistry》1990,265(36):22321-22328
We have cloned and sequenced the Saccharomyces cerevisiae gene for S-adenosylmethionine decarboxylase. This enzyme contains covalently bound pyruvate which is essential for enzymatic activity. We have shown that this enzyme is synthesized as a Mr 46,000 proenzyme which is then cleaved post-translationally to form two polypeptide chains: a beta subunit (Mr 10,000) from the amino-terminal portion and an alpha subunit (Mr 36,000) from the carboxyl-terminal portion. The protein was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme contains both the alpha and beta subunits. About half of the alpha subunits have pyruvate blocking the amino-terminal end; the remaining alpha subunits have alanine in this position. From a comparison of the amino acid sequence deduced from the nucleotide sequence with the amino acid sequence of the amino-terminal portion of each subunit (determined by Edman degradation), we have identified the cleavage site of the proenzyme as the peptide bond between glutamic acid 87 and serine 88. The pyruvate moiety, which is essential for activity, is generated from serine 88 during the cleavage. The amino acid sequence of the yeast enzyme has essentially no homology with S-adenosylmethionine decarboxylase of E. coli (Tabor, C. W., and Tabor, H. (1987) J. Biol. Chem. 262, 16037-16040) and only a moderate degree of homology with the human and rat enzymes (Pajunen, A., Crozat, A., J?nne, O. A., Ihalainen, R., Laitinen, P. H., Stanley, B., Madhubala, R., and Pegg, A. E. (1988) J. Biol. Chem. 263, 17040-17049); all of these enzymes are pyruvoyl-containing proteins. Despite this limited overall homology the cleavage site of the yeast proenzyme is identical to the cleavage sites in the human and rat proenzymes, and seven of the eight amino acids adjacent to the cleavage site are identical in the three eukaryote enzymes. 相似文献
9.
Electrophoretically pure hog kidney diamine oxidase has been isolated by an improved procedure and subjected to molecular weight and subunit analyses. Sedimentation/diffusion and sedimentation equilibrium ultracentrifugation clearly show that the native enzyme has a molecular weight of 172,000. Acrylamide gel electrophoresis indicates that the enzyme consists of two apparently identical subunits of 91,000 daltons each. The native enzyme contains two firmly bound Cu(II) ions. The isolation procedure described provides diamine oxidase in 50–60% yield of activity and of the highest specific activity yet reported (1.2 units/mg). 相似文献
10.
gamma-Glutamylcysteine synthetase (rat kidney; Mr approximately 104,000) is composed of 2 nonidentical subunits. In the present work, a procedure was developed for the reversible dissociation of the enzyme into its subunits (Mr = 73,000 and 27,700) under nondenaturing conditions. Students in which gel electrophoresis was used, in conjunction with an enzyme activity stain and elution and re-electrophoresis of protein bands, showed that the heavy subunit contains all of the structural requirements for enzymatic activity and also for feedback inhibition of the enzyme activity by glutathione. The light subunit, which may be formed from a precursor protein, has a significantly lower content of Trp, Phe, Tyr, Val, and Ala residues than the heavy subunit, while its content of Lys, His, Met, and Asx residues is higher. 相似文献
11.
Urease has been purified from the dehusked seeds of pigeonpea (Cajanus cajan L.) to apparent electrophoretic homogeneity with approximately 200 fold purification, with a specific activity of 6.24 x10(3) U mg(-1) protein. The enzyme was purified by the sequence of steps, namely, first acetone fractionation, acid step, a second acetone fractionation followed by gel filtration and anion-exchange chromatographies. Single band was observed in both native- and SDS-PAGE. The molecular mass estimated for the native enzyme was 540 kDa whereas subunit values of 90 kDa were determined. Hence, urease is a hexamer of identical subunits. Nickel was observed in the purified enzyme from atomic absorption spectroscopy with approximately 2 nickel ions per enzyme subunit. Both jack bean and soybean ureases are serologically related to pigeonpea urease. The amino acid composition of pigeonpea urease shows high acidic amino acid content. The N-terminal sequence of pigeonpea urease, determined up to the 20th residue, was homologous to that of jack bean and soybean seed ureases. The optimum pH was 7.3 in the pH range 5.0-8.5. Pigeonpea urease shows K(m) for urea of 3.0+/-0.2 mM in 0.05 M Tris-acetate buffer, pH 7.3, at 37 degrees C. The turnover number, k(cat), was observed to be 6.2 x 10(4) s(-1) and k(cat)/K(m) was 2.1 x 10(7) M(-1) s(-1). Pigeonpea urease shows high specificity for its primary substrate urea. 相似文献
12.
Molecular analysis of a novel glutamine synthetase of the anaerobe Bacteroides fragilis 总被引:12,自引:0,他引:12
R T Hill J R Parker H J Goodman D T Jones D R Woods 《Journal of general microbiology》1989,135(12):3271-3279
13.
Tyrosine hydroxylase purified to apparent homogeneity from the soluble fraction of bovine adrenal medulla had an apparent Mr of about 280,000 by Bio-Gel A-1.5m chromatography, and gave a single band with a Mr of 60,000 by sodium dodesyl sulfate polyacrylamide gel electrophoresis. The enzyme is considered to be composed of four identical subunits. Isoelectric point of purified enzyme was pH 6.0. The amino acid composition of the enzyme was characterized by fairly high contents of glutamic acid and alanine residues. The N-terminal amino acid was determined to be glutamic acid. 相似文献
14.
Purification and characterization of a type II casein kinase from Drosophila melanogaster 总被引:13,自引:0,他引:13
A cyclic nucleotide-independent protein kinase has been isolated from Drosophila melanogaster by chromatography on phosphocellulose and hydroxylapatite followed by gel filtration and glycerol gradient sedimentation. As determined by sodium dodecyl sulfate gel electrophoresis, the purified enzyme is greater than 95% homogeneous and is composed of two distinct subunits, alpha and beta, having Mr = 36,700 and 28,200, respectively. The native form of the enzyme is an alpha 2 beta 2 tetramer having a Stokes radius of 48 A, a sedimentation coefficient of 6.4 S, and Mr approximately 130,000. The purified kinase undergoes an autocatalytic reaction resulting in the specific phosphorylation of the beta subunit, exhibits a low apparent Km for both ATP and GTP as nucleoside triphosphate donor (17 and 66 microM, respectively), phosphorylates both casein and phosvitin but neither histones nor protamine, modifies both serine and threonine residues in casein, and is strongly inhibited by heparin (I50 = 21 ng/ml). These properties are remarkably similar to those of casein kinase II, an enzyme previously described in several mammalian and avian species. The strong similarities among the insect, avian, and mammalian enzymes suggest that casein kinase II has been highly conserved during evolution. 相似文献
15.
Larval-specific protein (LSP) is the most abundant protein in the hemolymph of cockroaches shortly before molting, but is rapidly cleared from the hemolymph during the molt (Kunkel, J. G., and Lawler, D. M. (1974) Comp. Biochem. Physiol. 47B, 697-710). Blatta orientalis LSP was purified by sedimentation in preparative sucrose gradients followed by 2-hydroxypropylamino-cellulose anion-exchange chromatography and gel filtration on a column of Bio-Gel A-1.5m. The amino acid composition of LSP includes 16.3 mol % tyrosine and 4.9 mol % phenylalanine, but virtually no cysteine and little methionine. The following physical properties were determined for LSP: R8 = 68.3 A, 8(20),w = 17.8, and V = 0.723. From these values an Mr = 507,900 was calculated. In electron micrographs, LSP appears as rectangular particles of 121 by 134 A. In disc polyacrylamide gel electrophoresis, native LSP exhibits a single band, but in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, LSP is resolved into a doublet of closely spaced bands of Mr = 88,100 and 84,400 present in a ratio of 1.38:1. These data indicate that native B. orientalis LSP is a hexamer of subunits averaging approximately Mr = 86,000. Crossed immunoelectrophoresis of Blattella germanica larval serum indicates that LSP in that species is a hexamer composed of a random assortment of two subunits of different charge in the ratio 1.25:1. The amino acid composition and physical properties of LSP suggest that LSP may be the hemimetabolous analogue of the tyrosine- and phenylalanine-rich storage proteins of holometabolous insects. 相似文献
16.
X-ray structure of nucleoside diphosphate kinase. 总被引:8,自引:0,他引:8
C Dumas I Lascu S Morra P Glaser R Fourme V Wallet M L Lacombe M Vron J Janin 《The EMBO journal》1992,11(9):3203-3208
The X-ray structure of a point mutant of nucleoside diphosphate kinase (NDP kinase) from Dictyostelium discoideum has been determined to 2.2 A resolution. The enzyme is a hexamer made of identical subunits with a novel mononucleotide binding fold. Each subunit contains an alpha/beta domain with a four stranded, antiparallel beta-sheet. The topology is different from adenylate kinase, but identical to the allosteric domain of Escherichia coli ATCase regulatory subunits, which bind mononucleotides at an equivalent position. Dimer contacts between NDP kinase subunits within the hexamer are similar to those in ATCase. Trimer contacts involve a large loop of polypeptide chain that bears the site of the Pro----Ser substitution in Killer of prune (K-pn) mutants of the highly homologous Drosophila enzyme. Properties of Drosophila NDP kinase, the product of the awd developmental gene, and of the human enzyme, the product of the nm23 genes in tumorigenesis, are discussed in view of the three-dimensional structure and of possible interactions of NDP kinase with other nucleotide binding proteins. 相似文献
17.
Aminopropionaldehyde dehydrogenase was purified to apparent homogeneity from 1,3-diaminopropane-grown cells of Arthrobacter sp. TMP-1. The native molecular mass and the subunit molecular mass of the enzyme were approximately 20,5000 and 52,000, respectively, suggesting that the enzyme is a tetramer of identical subunits. The apparent Michaelis constant (K(m)) for 1,3-diaminopropane was approximately 3 microM. The enzyme equally used both NAD(+) and NADP(+) as coenzymes. The apparent K(m) values for NAD(+) and NADP(+) were 255 microM and 108 microM, respectively. The maximum reaction rates (V(max)) for NAD(+) and NADP(+) were 102 and 83.3 micromol min(-1) mg(-1), respectively. Some tested aliphatic aldehydes and aromatic aldehydes were inert as substrates. The optimum pH was 8.0-8.5. The enzyme was sensitive to sulfhydryl group-modifying reagents. 相似文献
18.
Purification and characterization of a high-Mr carbonic anhydrase from sheep parotid gland. 总被引:1,自引:0,他引:1
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Approximately half the carbonic anhydrase activity of sheep parotid-gland homogenate is derived from a high-Mr protein [Fernley, Wright & Coghlan (1979) FEBS Lett. 105, 299-302]. This enzyme has now been purified to homogeneity, and its properties were compared with those of the well-characterized sheep carbonic anhydrase II. The protein has an apparent Mr of 540,000 as measured by gel filtration under non-denaturing conditions and an apparent subunit Mr of 45,000 as measured by SDS/polyacrylamide-gel electrophoresis. After deglycosylation with the enzyme N-glycanase the protein migrates with an apparent Mr of 36,000 on SDS/polyacrylamide-gel electrophoresis. The CO2-hydrating activity was 340 units/mg compared with 488 units/mg for sheep carbonic anhydrase II measured under identical conditions. This enzyme does not, however, hydrolyse p-nitrophenyl acetate. The enzyme contains 0.8 g-atom of zinc/mol of protein subunit. The peptide maps of the two carbonic anhydrases differ significantly from one another, indicating they are not related closely structurally. Unlike the carbonic anhydrase II isoenzyme, which has a blocked N-terminus, the high-Mr enzyme has a free glycine residue at its N-terminus. 相似文献
19.
Bovine heart phosphorylase kinase has been isolated by a procedure involving precipitation with polyethylene glycol, DEAE-Sephacel chromatography and calmodulin-Sepharose affinity chromatography. The isolated enzyme had a specific activity of 8.3 IU/mg of protein at pH 8.2 at 30 degrees C in the presence of 1% glycogen. The native enzyme had a sedimentation coefficient of 23 S and the Mr of the alpha', beta, gamma, and delta subunits, were 140,000, 130,000, 46,000, and 18,000, respectively. Activation of the phosphorylase kinase by the catalytic subunit of bovine heart cAMP-dependent protein kinase increases the pH 6.8/8.2 activity ratio from 0.01 to 0.32-0.38. Glycogen (1%) decreased the Km of the activated phosphorylase kinase at pH 6.8 for phosphorylase b from 5.5 to 1.25 mg/ml. Trypsin treatment increased the pH 6.8 activity but decreased the pH 8.2 activity. During this process the alpha' subunit was converted to a Mr 110,000 polypeptide and the enzyme activity was converted essentially to a 5.9 S species having an apparent Mr of 100,000 as determined by gel filtration. On extended trypsin treatment only one major polypeptide corresponding to the beta subunit remained. The same polypeptide was present in the active fractions following gel filtration of the trypsinized kinase. 相似文献
20.
The speEspeD operon of Escherichia coli. Formation and processing of a proenzyme form of S-adenosylmethionine decarboxylase 总被引:6,自引:0,他引:6
We have previously shown that the gene (speD) for S-adenosylmethionine decarboxylase is part of an operon that also contains the gene (speE) for spermidine synthase (Tabor, C. W., Tabor, H., and Xie, Q.-W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 6040-6044). We have now determined the nucleotide sequence of this operon and have found that speD codes for a polypeptide of Mr = 30,400, which is considerably greater than the subunit size of the purified enzyme. Our studies show that S-adenosylmethionine decarboxylase is first formed as a Mr = 30,400 polypeptide and that this proenzyme is then cleaved at the Lys111-Ser112 peptide bond to form a Mr = 12,400 subunit and a Mr = 18,000 subunit. The latter subunit contains the pyruvoyl moiety that we previously showed is required for enzymatic activity. Both subunits are present in the purified enzyme. These conclusions are based on (i) pulse-chase experiments with a strain containing a speD+ plasmid which showed a precursor-product relationship between the proenzyme and the enzyme subunits, (ii) the amino acid sequence of the proenzyme form of S-adenosylmethionine decarboxylase (derived from the nucleotide sequence of the speD gene), and (iii) comparison of this sequence of the proenzyme with the N-terminal amino acid sequences of the two subunits of the purified enzyme reported by Anton and Kutny (Anton, D. L., and Kutny, R. (1987) J. Biol. Chem. 262, 2817-2822). 相似文献