共查询到20条相似文献,搜索用时 31 毫秒
1.
Lupidi G Venardi G Bollettini M Marmocchi F Rotilio G 《Preparative biochemistry & biotechnology》2001,31(3):305-316
Glyoxalase I was purified to homogeneity from bovine brain using affinity chromatography on S-hexylglutathione-Sepharose 6B with a yield of 22%. The enzyme was a dimer (44,000 Daltons) composed of, apparently, identical subunits (22,000 Daltons), as shown by SDS electrophoresis, and contained one mole of Zn2+/monomer. The active site metal ion, Zn2+, was removed by dialysis against EDTA, but the activity of the apoenzyme obtained was not completely restored after addition of Co2+ and Zn2+ (<25%), while a recovery of 50% was obtained after addition of Mg2+. The enzyme was inhibited by S-bromobenzylglutathione and S-p-nitrobenzylglutathione with a Ki value of 21 microM and 32 microM, respectively. The highest dissociation constant observed for the brain enzyme with respect to that reported for human erythrocytes, or other mammalian forms of enzyme could be related to a tissue-specific dependence of the glyoxalase I activity. 相似文献
2.
Investigation of the effect of metal ions on the reactivity of thiol groups in human 5-aminolaevulinate dehydratase. 总被引:5,自引:1,他引:4
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The reaction of human 5-aminolaevulinate dehydratase with 5,5'-dithiobis-(2-nitrobenzoic acid) (Nbs2) results in the release of 4 molar equivalents of 5-mercapto-2-nitrobenzoic acid (Nbs) per subunit. Two of the thiol groups reacted very rapidly (groups I and II), and their rate constants were determined by stopped-flow spectrophotometry; the other two thiol groups (groups III and IV) were observed by conventional spectroscopy. Titration of the enzyme with a 1 molar equivalent concentration of Nbs2 resulted in the release of 2 molar equivalents of Nbs and the concomitant formation of an intramolecular disulphide bond between groups I and II. Removal of zinc from the holoenzyme increased the reactivity of groups I and II without significantly affecting the rate of reaction of the other groups. The reactions of the thiol groups in both the holoenzyme and apoenzyme were little affected by the presence of Pb2+ ions at concentrations that strongly inhibit the enzyme, suggesting that Zn2+ and Pb2+ ions may have independent binding sites. Protein fluorescence studies with Pb2+ and Zn2+ have shown that the binding of both metal ions results in perturbation of the protein fluorescence. 相似文献
3.
Ferrochelatase (EC 4.99.1.1) was purified 2000-fold to apparent homogeneity from isolated chicken erythrocyte mitochondria. The purified enzyme yields a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an apparent Mr of 42 000. The enzyme utilizes proto-, meso- and deutero-porphyrin with Km values of 37, 51 and 80 microM respectively. The disubstituted porphyrins 2,4-bisglycol deutero-porphyrin and 2,4-disulphonic deuteroporphyrin were not substrates. Mn2+, Hg2+, Pb2+ and Co2+ were strong inhibitors of the purified enzyme. Palmitic acid and oleic acid stimulated activity, whereas linoleic acid inhibited and phospholipids had variable effects. Chicken ferrochelatase was inhibited by N-ethylmaleimide and iodoacetamide. Inhibition by iodoacetamide was pseudo-first-order, but inhibition by N-ethylmaleimide appeared to be biphasic in nature with an initial high rate followed by a much lower rate of inactivation. The characteristics of the chicken erythrocyte enzyme are compared with those previously reported for mammalian liver ferrochelatase. 相似文献
4.
Affinity purification and characterization of 2,4-dichlorophenol hydroxylase from Pseudomonas cepacia 总被引:3,自引:0,他引:3
V Radjendirane M A Bhat C S Vaidyanathan 《Archives of biochemistry and biophysics》1991,288(1):169-176
2,4-Dichlorophenol hydroxylase, a flavoprotein monooxygenase from Pseudomonas cepacia grown on 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, was purified to homogeneity by a single-step affinity chromatography on 2,4-DCP-Sepharose CL-4B. The enzyme was eluted from the affinity matrix with the substrate 2,4-dichlorophenol. The enzyme has a molecular weight of 275,000 consisting of four identical subunits of molecular weight 69,000 and requires exogenous addition of FAD for its complete catalytic activity. The enzyme required an external electron donor NADPH for hydroxylation of 2,4-dichlorophenol to 3,5-dichlorocatechol. NADPH was preferred over NADH. The enzyme had Km value of 14 microM for 2,4-dichlorophenol, and 100 microM for NADPH. The enzyme activity was significantly inhibited by heavy metal ions like Hg2+ and Zn2+ and showed marked inhibition with thiol reagents. Trichlorophenols inhibited the enzyme competitively. The hydroxylase activity decreased as a function of increasing concentrations of Cibacron blue and Procion red dyes. The apoenzyme prepared showed complete loss of FAD when monitored spectrophotometrically and had no enzymatic activity. The inactive apoenzyme was reconstituted with exogenous FAD which completely restored the enzyme activity. 相似文献
5.
A calcium-activated neutral proteinase has been purified to homogeneity from human placenta. The purified enzyme is a dimer composed of Mr 73 000 and 30 000 subunits. Half-maximal activity is observed at 250 microM Ca2+. It requires reduced sulfhydryl groups and neutral pH for optimal activity. Leupeptin, antipain, E-64, sulfhydryl-blocking agents and endogenous proteinase inhibitor inhibit the purified enzyme. This paper is the first to describe the purification and characterization of a calcium-activated neutral proteinase from a human non-muscular parenchymatous organ. 相似文献
6.
Enzymic synthesis of lignin precursors. Further studies on cinnamyl-alcohol dehydrogenase from soybean-cell-suspension cultures 总被引:4,自引:0,他引:4
Isoenzyme 2 of cinnamyl-alcohol dehydrogenase from soybean suspension cultures was purified about 3800-fold to apparent homogeneity by an improved purification procedure involving biospecific elution of the enzyme from a NADP+-agarose column. On sodium dodecylsulfate gels the dehydrogenase showed only one protein band with Mr 40 000 +/- 500. The enzyme is strongly inhibited by thiol reagents. Various metal chelators as well as the nonchelating 7,8-benzoquinoline also inhibited enzyme activity. Inhibition by 10 mM 1,10-phenanthroline could be partially reversed by addition of Zn2+. 1,10-Phenanthroline and 7,8-benzoquinoline are non-competitive inhibitors with respect to NADP+. The presence of zinc in the dehydrogenase was proved by atomic absorption spectroscopy and by specific incorporation of 65Zn into the enzyme. In steady-state kinetics inhibition patterns were obtained which are consistent with an ordered bi-bi mechanism in which NADP(H) is the first substrate to bind and the last product released. The cinnamyl-alcohol dehydrogenase belongs to the A-specific dehydrogenases and removes the pro-R hydrogen from coniferyl alcohol. The enzyme shows many similarities with alcohol dehydrogenases from horse and rat liver and from yeast. 相似文献
7.
H F Haugen 《Biochimica et biophysica acta》1981,659(2):411-421
A previously unrecognized erythrocyte phosphodiesterase I with activity against thymidine-5'-monophospho-p-nitrophenyl ester is described. The enzyme is present in the soluble fraction of the erythrocyte, and was purified about 500-fold by chromatography using DEAE-cellulose, followed by gel chromatography with Sephadex G-200. Erythrocyte phosphodiesterase I has a molecular weight of about 70 000, when fully active as a monomer. Its pI is 5.4 and the pH optimum is 8.5. The Km value for thymidine-5'-monophospho-p-nitrophenyl ester is rather high, about 4 mmol/l. The enzyme has a barely detectable nucleotide pyrophosphatase activity. It is extremely sensitive to SH-inhibitors such as N-ethyl-maleimide, p-chloromercuribenzoate and disulphides (a reversible 50% inhibition was obtained by cystamine, 0.01 mmol/l). It is a metalloenzyme with loosely bound metal, and is stimulated by Mg2+. This activation by Mg2+ is counteracted by Zn2+. Gel chromatography revealed that the enzyme is a monomer in the presence of Mg2+. When inhibited by Zn2+, it forms polymers that can be reconverted to the monomer by thiols. All of the above properties of the erythrocyte enzyme support the conclusion that it is different from plasma membrane phosphodiesterase I (oligonucleate 5'-nucleotidohydrolase, EC 3.1.4.1). 相似文献
8.
Purification and properties of uroporphyrinogen III synthase from human erythrocytes 总被引:7,自引:0,他引:7
Uroporphyrinogen III synthase (hydroxymethylbilane hydro-lyase (cyclizing); EC 4.2.1.75), the fourth enzyme in the heme biosynthetic pathway, was purified to homogeneity from human erythrocytes. For enzyme purification and characterization, a sensitive coupled enzyme assay was used which generated the substrate, hydroxymethylbilane; the oxidized product, uroporphyrin III, was quantitated by high pressure liquid chromatography. Uroporphyrinogen III synthase was initially separated from delta-aminolevulinate dehydratase and hydroxymethylbilane synthase by a preparative anion exchange chromatographic step. Subsequent chromatography on hydroxyapatite, phenyl-Sepharose, and Sephadex G-100 purified the enzyme about 70,000-fold with an 8% yield. Homogeneous enzyme was obtained following a final C4-reversed phase high pressure liquid chromatographic step which removed a single major and several minor protein contaminants from the enzyme. The purified enzyme had a specific activity of over 300,000 units/mg, an isoelectric point of 5.5, and was thermolabile (t1/2 at 60 degrees C approximately 1 min). Molecular weight studies by gel filtration (Mr approximately equal to 30,000) and analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr approximately equal to 29,500) were consistent with the enzyme being a monomer. Using hydroxymethylbilane as substrate, the purified enzyme formed uroporphyrinogen III in the absence of hydroxymethylbilane synthase or other cofactors. The pH optimum was 7.4 and the Km for hydroxymethylbilane was 5-20 microM. The enzyme was activated by Na+, K+, Mg+, and Ca2+ and was inhibited by Cd2+, Cu2+, Hg2+, and Zn2+. Amino acid composition analysis was performed, and the N-terminal sequence, Met-Lys-Val-Leu-Leu-Leu, was determined by microsequencing. The availability of the purified enzyme should permit investigation of its reaction mechanism as well as facilitate biochemical and molecular studies of the genetic defect in congenital erythropoietic porphyria. 相似文献
9.
31P nuclear magnetic resonance spectra and enzymatic activities are compared for alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) species with different zinc contents. The enzyme containing two Zn2+ per protein dimer exists in two forms; one, prepared by dialysis of native enzyme, has full enzymatic activity and a 31P magnetic resonance spectrum similar to but distinguishable from that of the native enzyme containing four or more Zn2+. The other form, prepared by restoring two Zn2+ to apoenzyme, has low enzymatic activity and a 31P magnetic resonance spectrum that indicates stoichiometric binding of phosphate, but otherwise altered properties. Reconstituted enzyme with four Zn2+ is similar to but distinguishable from native enzyme with four Zn2+. Chromatography on DEAE-cellulose can separate apoenzyme and enzyme containing two Zn2+ and suggests that the binding of a pair of Zn2+ is cooperative. 相似文献
10.
Wang XT Chan TF Lam VM Engel PC 《Protein science : a publication of the Protein Society》2008,17(8):1403-1411
Human glucose 6-phosphate dehydrogenase, purified after overexpression in E. coli, was shown to contain one molecule/subunit of acid-extractable "structural" NADP+ and no NADPH. This tightly bound NADP+ was reduced by G6P, presumably following migration to the catalytic site. Gel-filtration yielded apoenzyme, devoid of bound NADP+ but, surprisingly, still fully active. Mr of the main component of "stripped" enzyme by gel filtration was approximately 100,000, suggesting a dimeric apoenzyme (subunit Mr = 59,000). Holoenzyme also contained tetramer molecules and, at high protein concentration, a dynamic equilibrium gave an apparent intermediate Mr of 150 kDa. Fluorescence titration of the stripped enzyme gave the K d for structural NADP+ as 37 nM, 200-fold lower than for "catalytic" NADP+. Structural NADP+ quenches 91% of protein fluorescence. At 37 degrees C, stripped enzyme, much less stable than holoenzyme, inactivated irreversibly within 2 d. Inactivation at 4 degrees C was partially reversed at room temperature, especially with added NADP+. Apoenzyme was immediately active, without any visible lag, in rapid-reaction studies. Human G6PD thus forms active dimer without structural NADP+. Apparently, the true role of the second, tightly bound NADP+ is to secure long-term stability. This fits the clinical pattern, G6PD deficiency affecting the long-lived non-nucleate erythrocyte. The Kd values for two class I mutants, G488S and G488V, were 273 nM and 480 nM, respectively (seven- and 13-fold elevated), matching the structural prediction of weakened structural NADP+ binding, which would explain decreased stability and consequent disease. Preparation of native apoenzyme and measurement of Kd constant for structural NADP+ will now allow quantitative assessment of this defect in clinical G6PD mutations. 相似文献
11.
One form of calcium-activated neutral protease (CANP) highly sensitive to calcium ions was purified by column chromatographic procedures to homogeneity. The purified enzyme required microM order Ca2+ (mu CANP), and the half-maximum activity was attained at 50 microM Ca2+. The electrophoretic mobility in a non-denaturing buffer showed that this enzyme is less acidic than another CANP which required mM order Ca2+ (mCANP). On SDS-polyacrylamide gel electrophoresis, the enzyme separated into two components with molecular weights of 79,000 and 28,000, respectively. Of these, the former was slightly larger than the counterpart of mCANP (Mr 76,000). Thus, mu CANP cannot be derived from mCANP by limited autolysis. 相似文献
12.
B. F. Cooper V. Sideraki D. K. Wilson D. Y. Dominguez S. W. Clark F. A. Quiocho F. B. Rudolph 《Protein science : a publication of the Protein Society》1997,6(5):1031-1037
For murine adenosine deaminase, we have determined that a single zinc or cobalt cofactor bound in a high affinity site is required for catalytic function while metal ions bound at an additional site(s) inhibit the enzyme. A catalytically inactive apoenzyme of murine adenosine deaminase was produced by dialysis in the presence of specific zinc chelators in an acidic buffer. This represents the first production of the apoenzyme and demonstrates a rigorous method for removing the occult cofactor. Restoration to the holoenzyme is achieved with stoichiometric amounts of either Zn2+ or Co2+ yielding at least 95% of initial activity. Far UV CD and fluorescence spectra are the same for both the apo- and holoenzyme, providing evidence that removal of the cofactor does not alter secondary or tertiary structure. The substrate binding site remains functional as determined by similar quenching measured by tryptophan fluorescence of apo- or holoenzyme upon mixing with the transition state analog, deoxycoformycin. Excess levels of adenosine or N6- methyladenosine incubated with the apoenzyme prior to the addition of metal prevent restoration, suggesting that the cofactor adds through the substrate binding cleft. The cations Ca2+, Cd2+, Cr2+, Cu+, Cu2+, Mn2+, Fe2+, Fe3+, Pb2+, or Mg2+ did not restore adenosine deaminase activity to the apoenzyme. Mn2+, Cu2+, and Zn2+ were found to be competitive inhibitors of the holoenzyme with respect to substrate and Cd2+ and Co2+ were noncompetitive inhibitors. Weak inhibition (Ki > or = 1000 microM) was noted for Ca2+, Fe2+, and Fe3+. 相似文献
13.
Characterization and physiological function of glutathione reductase in Euglena gracilis z. 总被引:2,自引:0,他引:2
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The purified glutathione reductase was homogeneous on polyacrylamide-gel electrophoresis. It had an Mr of 79,000 and consisted of two subunits with a Mr of 40,000. The activity was maximum at pH 8.2 and 52 degrees C. It was specific for NADPH but not for NADH as the electron donor; the reverse reaction was not observed. The Km values for NADPH and GSSG were 14 and 55 microM respectively. The enzyme activity was markedly inhibited by thiol inhibitors and metal ions such as Hg2+, Cu2+ and Zn2+. Euglena cells contained total glutathione at millimolar concentration. GSH constituted more than 80% of total glutathione in Euglena under various growth conditions. Glutathione reductase was located solely in cytosol, as were L-ascorbate peroxidase and dehydroascorbate reductase, which constitute the oxidation-reduction cycle of L-ascorbate [Shigeoka et al. (1980) Biochem. J. 186, 377-380]. These results indicate that glutathione reductase functions to maintain glutathione in the reduced form and to accelerate the oxidation-reduction of L-ascorbate, which scavenges peroxides generated in Euglena cells. 相似文献
14.
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
15.
K Sorimachi 《The Journal of biological chemistry》1987,262(4):1535-1541
In Reuber rat hepatoma cells (R-Y121B), alkaline phosphatase activity increased without de novo enzyme synthesis (Sorimachi, K., and Yasumura, Y. (1986) Biochim. Biophys. Acta 885, 272-281). The enzyme was partially purified by butanol extraction from the particulate fractions. The incubation of the extracted alkaline phosphatase with the cytosol fraction induced a large increase in enzyme activity (5-10-fold of control). The dialyzed cytosol was more effective than the undialyzed cytosol during an early period of incubation at 37 degrees C. This difference between the dialyzed and the undialyzed cytosol fractions was due to endogenous Na+. For maximal activation of the enzyme, both Mg2+ above 1 mM and Zn2+ at low concentrations (below 0.01 mM) were needed, although Zn2+ at high concentrations (above 0.1 mM) showed an inhibitory effect. Zn2+ and Mg2+ alone slightly increased alkaline phosphatase activity. This activation of the enzyme was temperature dependent and was not observed at 0 or 4 degrees C. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the increase in alkaline phosphatase activity did not involve the fragmentation of the enzyme and that 65Zn2+ bound to it during enzyme activation with 65Zn2+ and Mg2+. The cytosol fraction not only supplied Zn2+ to the nascent enzyme but also increased the maximal enzyme activity more than did direct addition of metal ions. Ferritin and metallothionein contributed to the activation of alkaline phosphatase with the metal ions. Since the binding of Zn2+ and Mg2+ to the nascent alkaline phosphatase is disturbed in Reuber rat hepatoma cells (R-Y121B), the apoenzyme is accumulated inside the cells. The binding of Zn2+ and Mg2+ to the apoenzyme readily takes place in the cell homogenates accompanied by an increase in catalytic activity without new enzyme synthesis. 相似文献
16.
A new enzymatic method has been developed to determine trace amounts of Zn2+ in vegetables. The basis of the method is that apocarbonic anhydrase regains its activity in proportion to the concentration of Zn2+ present in solution. Bovine carbonic anhydrase was purified from erythrocyte haemolysate by affinity chromatography and the bound Zn2+ removed by dialysis of purified enzyme against a solution of pyridine-2, 6-dicarboxylic acid. Pure (100%) apoenzyme was obtained. The concentration of Zn2+ in vegetable samples was determined using the enzymatic method and by atomic absorption spectroscopy. Determinations made using the two methods were not significantly different one from another. 相似文献
17.
A G Larsen A C Ostvold J Holtlund T Kristensen S G Laland 《The Biochemical journal》1982,203(2):511-513
Zn2+ inhibits purified poly(ADP-ribose) polymerase (50% inhibition at 10 microM). Furthermore poly (ADP-ribose) polymerase present in nuclei and metaphase chromosome clusters is also inhibited by Zn2+. The inactivated enzyme could be re-activated by dithiothreitol. The concentration of Zn2+ needed to affect the enzyme activity in the organelles is sufficiently low for it to have a possible role in controlling the activity of this chromatin-bound enzyme. 相似文献
18.
Uroporphyrinogen decarboxylase (EC 4.1.1.37) has been purified to homogeneity from bovine liver by using isoelectric and salt precipitations, followed by chromatography on DEAE-cellulose, phenyl-Sepharose, hydroxylapatite, and Sephacryl S-200. The purified enzyme is a monomer with an Mr approximately 57 000 and an isoelectric point at pH 4.6. Enzyme activity is optimal in buffers having an ionic strength of approximately 0.1 M and a pH of 6.8. The purified enzyme has a specific activity (expressed as the disappearance of uroporphyrinogen I) of 936 nmol X h-1 X (mg of protein)-1. The purified enzyme catalyzes all four decarboxylation reactions in the conversion of uroporphyrinogen I or III to the corresponding coproporphyrinogen. The rate-limiting step in the physiologically significant conversion of uroporphyrinogen III to coproporphyrinogen III is the decarboxylation of heptacarboxylate III. Kinetic data suggest that the enzyme has at least two noninteracting active sites. At least one sulfhydryl group is required for catalytic activity. The enzyme is inhibited by sulfhydryl-specific reagents and by divalent metal ions including Fe2+, Co2+, Cu2+, Zn2+, and Pb2+. The pattern of accumulation of intermediate (hepta-, hexa-, and pentacarboxylate porphyrinogens) and final (coproporphyrinogen) decarboxylation products is affected by the ratio of substrate (uroporphyrinogen I or III) concentration to enzyme concentration. Under physiologic conditions where the uroporphyrinogen to enzyme ratio is low, the substrate is nearly quantitatively decarboxylated, and the major product is coproporphyrinogen. If the ratio of uroporphyrinogen to enzyme is high, intermediates accumulate, and heptacarboxylate porphyrinogen becomes the major decarboxylation product.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
19.
Carbonate dehydratase was detected dissolved in the hemolymph of the tarantula, Eurypelma californicum. The enzyme was purified 31-fold by gel filtration, anion-exchange chromatography, a second gel filtration, and finally, preparative polyacrylamide gel electrophoresis. Zinc content increased during purification to up to 2.4 mol Zn/100 000 g of protein (= 1.58 mg Zn/g protein). In the polyacrylamide electrophoresis of tarantula hemolymph under non-denaturing conditions three major protein bands were observed: hemocyanin, a 16 S lipoprotein and the active band which migrated closely behind the 16 S lipoprotein. After treatment with sodium dodecyl sulfate both the carbonate dehydratase-active protein and the lipoprotein revealed bands corresponding to Mr = 95 000 and 110 000, respectively, but the enzymatically active protein revealed an additional third band with Mr = 40 000. The latter band is though to represent the 'true' carbonate dehydratase protein. Upon isoelectric focusing of material containing carbonate dehydratase activity and lipoprotein, bands were obtained at pH 5.45, 5.6 and 5.7. The band at pH 5.6 contained the peak of enzyme activity, and upon dodecyl sulfate-polyacrylamide gel electrophoresis showed the highest proportion of the 40-kDa polypeptide. It is concluded that tarantula carbonate dehydratase, instead of forming a high molecular mass aggregate, is associated with the 16 S lipoprotein, the latter serving as a carrier for the enzyme. The lipoprotein is probably also involved in other transport processes. It is present in great excess and may therefore occur in two forms, charged with carbonate dehydratase or uncharged. Tarantula carbonate dehydratase is inhibited by acetazolamide and by dansylamide, but not by a number of other known inhibitors, most notably not by 4-(aminomethyl)benzenesulfonamide. Treatment with 1M urea does not affect specific enzyme activity, while 2M urea inhibits by 50%. 2-Mercaptoethanol inhibits activity by 50% at 0.1M. Like other carbonate dehydratases, the tarantula enzyme shows esterase activity. The Km for 4-nitrophenyl acetate is 5mM. 相似文献
20.
Purification and characterization of phosphatidylinositol kinase from porcine liver microsomes 总被引:5,自引:0,他引:5
Phosphatidylinositol kinase was solubilized and purified from porcine liver microsomes to apparent homogeneity. The purification procedure includes: solubilization of microsomes by 2% Triton X-100, ammonium sulfate precipitation (20-35% saturation), Reactive blue agarose chromatography, DEAE-Sephacel chromatography and two consecutive hydroxyapatite chromatographies. A total of 4900-fold purification with 8% recovery of enzyme activity was achieved. The molecular weight of the enzyme as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 55000. The enzyme is stimulated in a decreasing order by Mg2+, Fe2+, Mn2+, Fe3+ and Co2+. Ca2+ inhibited Mg2+-stimulated activity with an I50 of 0.4 mM. Apparent Km values for phosphatidylinositol and ATP are 120 and 60 microM, respectively. The enzyme is inhibited by adenosine (I50 = 70 microM), ADP (I50 = 120 microM) and quercetin (I50 = 100 microM). The enzyme is also sensitive to sulfhydryl inhibitors. Using the purified enzyme as an immunogen, we have successfully prepared antibodies for phosphatidylinositol kinase in rabbits. The antibodies appear to recognize an antigen of Mr 55000 on SDS-polyacrylamide gel electrophoresis from various porcine tissues in Western blot analysis. 相似文献