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1.
A specific alkaline phosphatase from Saccharomyces cerevisiae with protein phosphatase activity 总被引:1,自引:0,他引:1
Boriyana Tuleva Evgenia Vasileva-Tonkova Danka Galabova 《FEMS microbiology letters》1998,161(1):139-144
In this paper, specific PHO13 alkaline phosphatase from Saccharomyces cerevisiae was demonstrated to possess phosphoprotein phosphatase activity on the phosphoseryl proteins histone II-A and casein. The enzyme is a monomeric protein with molecular mass of 60 kDa and hydrolyzes p-nitrophenyl phosphate with maximal activity at pH 8.2 with strong dependence on Mg2+ ions and an apparent Km of 3.6×10−5 M. No other substrates tested except phosphorylated histone II-A and casein were hydrolyzed at any significant rate. These data suggest that the physiological role of the p-nitrophenyl phosphate-specific phosphatase may involve participation in reversible protein phosphorylation. 相似文献
2.
《Journal of Fermentation Technology》1988,66(3):267-272
Glucose-dehydrogenase-poly(ethylene glycol)-NAD conjugate (GlcDH-PEG-NAD) was prepared and its kinetic properties as an NADH-regeneration unit were investigated. The conjugate has about two molecules of active and covalently linked NAD per tetramer. The specific activity of the enzyme moiety of the conjugate in the presence of exogenous NAD is about 77% of that of the native enzyme, and this decrease is mainly due to the decrease in the Vmax value. The conjugate has the same pH-stability profile as the native enzyme and an internal activity of 0.26s−1 (as a monomer); its NAD moiety has similar coenzyme activity to poly(ethylene glycol)-bound NAD. These results indicate that GlcDH-PEG-NAD can be used as an NADH-regeneration unit for many dehydrogenase reactions. The coupled reaction of GlcDH-PEG-NAD and lactate dehydrogenase was then studied. The specific activity of the conjugate is 1.1 s−1 (as a tetramer), the recycling rate of the active NAD moiety is 0.54 s−1, and the apparent Km value for glucose is 24 mM. Kinetically, lactate dehydrogenase behaves like a substrate with an apparent Km value of 1.8 units·ml−1 in this coupled reaction system with low coenzyme concentration. l-Lactate was continuously produced from pyruvate in a reactor with a PM10 ultrafiltration membrane, and containing GlcDH-PEG-NAD and lactate dehydrogenase. GlcDH-PEG-NAD proved to be applicable in continuous enzyme reactors as an NADH-regeneration unit with a large molecular size. 相似文献
3.
《Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology》2001,1544(1-2):55-63
The α-carbonic anhydrase gene from Helicobacter pylori strain 26695 has been cloned and sequenced. The full-length protein appears to be toxic to Escherichia coli, so we prepared a modified form of the gene lacking a part that presumably encodes a cleavable signal peptide. This truncated gene could be expressed in E. coli yielding an active enzyme comprising 229 amino acid residues. The amino acid sequence shows 36% identity with that of the enzyme from Neisseria gonorrhoeae and 28% with that of human carbonic anhydrase II. The H. pylori enzyme was purified by sulfonamide affinity chromatography and its circular dichroism spectrum and denaturation profile in guanidine hydrochloride have been measured. Kinetic parameters for CO2 hydration catalyzed by the H. pylori enzyme at pH 8.9 and 25°C are kcat=2.4×105 s−1, KM=17 mM and kcat/KM=1.4×107 M−1 s−1. The pH dependence of kcat/KM fits with a simple titration curve with pKa=7.5. Thiocyanate yields an uncompetitive inhibition pattern at pH 9 indicating that the maximal rate of CO2 hydration is limited by proton transfer between a zinc-bound water molecule and the reaction medium in analogy to other forms of the enzyme. The 4-nitrophenyl acetate hydrolase activity of the H. pylori enzyme is quite low with an apparent catalytic second-order rate constant, kenz, of 24 M−1 s−1 at pH 8.8 and 25°C. However, with 2-nitrophenyl acetate as substrate a kenz value of 665 M−1 s−1 was obtained under similar conditions. 相似文献
4.
《Insect Biochemistry》1991,21(3):239-242
Alkaline phosphatase from the excretory system of the grasshopper, Poekilocerus bufonius was purified with ammonium sulphate fractionation and chromatography on Bio-Gel A-0.5 m. The specific activity of the enzyme is 152 units/mg of protein. The enzyme is a tetramer and the Mr value of the subunit is 72,000 ± 2500 as shown by gel filtration and SDS-polyacrylamide gel electrophoresis. The enzyme has a pH optimum of 9.6 and an apparent Km value of 0.28 × 10−3 M. The activity of the enzyme reached a maximum at 75°C and the enzyme showed stability at 65°C. The enzyme was inhibited by Ca2+, Na+ and Fe3+ and was stimulated by Zn2+, Mn2+ and Mg2+. 相似文献
5.
《Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology》1994,1204(2):217-224
Trihydroxybenzenes are degraded anaerobically through the phloroglucinol pathway. In Pelobacter acidigallici as well as in Pelobacter massiliensis, pyrogallol is converted to phloroglucinol in the presence of 1,2,3,5-tetrahydroxybenzene by intermolecular hydroxyl transfer. The enzyme catalyzing this reaction was purified to chromatographic and electrophoretic homogeneity. Gel filtration and electrophoresis revealed a heterodimer structure with an apparent molecular mass of 127 kDa for the native enzyme and 86 kDa and 38 kDa, respectively, for the subunits. The enzyme was not sensitive to oxygen. HgCl2, p-chloromercuribenzoic acid, and CuCl2 inhibited strongly the reaction indicating an essential function of SH-groups. Transhydroxylase had a pH-optimum of 7.0 and a pI of 4.1. The apparent temperature optimum was in the range of 53°C to 58°C. The activation energy for the conversion of pyrogallol and 1,2,3,5-tetrahydroxybenzene to phloroglucinol and tetrahydroxybenzene was 31.4 kJ per mol. Purified enzyme exhibited a specific activity of 3.1 mol. m−1 mg−1 protein and an apparent Km for pyrogallol and 1,2,3,5-tetrahydroxybenzene of 0.70 mM and 0.71 mM, respectively. The enzyme was found to contain per mol heterodimer 1.1 mol molybdenum, 12.1 mol iron and 14.5 mol acid-labile sulfur. Requirement for molybdenum for transhydroxylating enzyme activity was proven also by cultivation experiments. No hints for the presence of flavins were obtained. The results presented here support the hypothesis that a redox reaction is involved in this intermolecular hydroxyl transfer. 相似文献
6.
《Process Biochemistry》2007,42(8):1237-1243
The pectinolytic enzyme obtained from Penicillium viridicatum RFC by solid-state fermentation was purified to homogeneity by pretreatment with kaolin (40 mg mL−1) and ultrafiltration, followed by chromatography on a Sephadex G50 column. The apparent molecular weight of the enzyme was 24 kDa. Maximal activity occurred at pH 6.0 and at 60 °C. The enzyme proved to be an exo-polygalacturonase, releasing galacturonic acid by hydrolysis of highly esterified pectin. The presence of 10 mM Ba2+ increased the enzyme activity by 96% and its thermal stability by 30%, besides increasing its stability at acid pH. The apparent Km with apple pectin as substrate was 1.82 mg mL−1 and the Vmax was 81 μmol min−1 mg−1. 相似文献
7.
Eco-physiological responses of the declining population Spartina anglica to N and P fertilizer addition 总被引:2,自引:0,他引:2
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Nitrogen and phosphorus are both important life elements. N, P and combined N-P fertilizers were added to the declining population Spartina anglica Hubbard in coastal China. Some growth parameters and eco-physiological responses of S. anglica to different fertilizer treatments (N, P and combined N-P fertilizer addition with high, medium and low levels, respectively) were measured. The fertilizer addition had a highly significant effect on the dynamics of its height-growth, number of leaves, number of roots and total biomass. Only N addition had a significant effect on leaf area and leaf thickness in all fertilizer treatments. On the dynamics of its height-growth, the effect of N addition was the most apparent, and the effect of N-P addition was not greater than those of N and P addition separately. The photosynthesis rate was enhanced and the yield was the highest with the highest N, the highest N-P and the medium P addition. The rates were higher than those of CK by 19.08 μmol·m?2·s?1, 15.47 μmol·m?2·s?1 and 11.23 μmol·m?2·s?1, respectively. The activity of SOD and POD increased with the treatments after freshwater stress for 14 days. Effects of medium N and P addition were significant for SOD activity. However, POD activity was significantly higher with the treatment of higher N and higher N-P addition. In a word, fertilizer addition improved the growth of the declining population S. anglica. The results indicated that the decline of S. anglica was correlated with the nutriment deficiency in soil, especially with the lack of N. 相似文献
8.
Copper is necessary for all organisms since it acts as a cofactor in different enzymes, although toxic at high concentrations. ATP7B is one of two copper-transporting ATPases in humans, its vital role being manifested in Wilson disease due to a mutation in the gene that encodes this pump. Our objective has been to determine whether pathways involving protein kinase C (PKC) modulate ATP7B activity. Different isoforms of PKC (α, ɛ, ζ) were found in Golgi-enriched membrane fractions obtained from porcine liver. Cu(I)–ATPase activity was assessed in the presence of different activators and inhibitors of PKC signaling pathways. PMA (10−8 M), a PKC activator, increased Cu(I)–ATPase activity by 60%, whereas calphostin C and U73122 (PKC and PLC inhibitors, respectively) decreased the activity by 40%. Addition of phosphatase λ decreased activity by 60%, irrespective of pre-incubation with PMA. No changes were detected with 2 μM Ca2+, whereas PMA plus EGTA increased activity. This enhanced activity elicited by PMA decreased with a specific inhibitor of PKCɛ to levels comparable with those found after phosphatase λ treatment, showing that the ɛ isoform is essential for activation of the enzyme. This regulatory phosphorylation enhanced Vmax without modifying affinities for ATP and copper. It can be concluded that signaling pathways leading to DAG formation and PKCɛ activation stimulate the active transport of copper by ATP7B, thus evidencing a central role for this specific kinase-mediated mechanism in hepatic copper handling. 相似文献
9.
《The International journal of biochemistry》1994,26(7):885-890
- 1.1. The acid phosphatase (AcPase, EC 3.1.3.2) IV from rat testicular tissue was purified to apparent homogeneity.
- 2.2. The enzyme displays a native molecular weight of 70 kDa determined on gel permeation chromatography on a Sephadex G-100 column and 68 kDa using linear 5–20% sucrose density gradient centrifugation. The subunit molecular weight on SDS-PAGE analysis is 67 kDa, suggesting that the enzyme is a monomeric protein.
- 3.3. The enzyme does not bind to Concanavaline A-Sepharose 4B column, indicating that it is not a glycoprotein.
- 4.4. The rat testis AcPase IV is a metal activated enzyme in which Mg2+ is the metal activating agent with a Ka, = 0.88 × 10−3 M. The Michaelis constant for p-nitrophenylphosphate, in the presence of saturating concentrations of Mg2+ ions, is 0.23 × 10−3 M.
- 5.5. The enzyme preferentially hydrolizes p-nitrophenylphosphate, phenylphosphate and ATP.
10.
《Plant science》1987,50(2):111-115
NADH-Nitrate reductase (EC 1.6.6.1) from spinach (Spinacea oleracea L. v. Noorman) has been purified to apparent homogeneity by immunoaffinity chromatography using a monoclonal antibody linked covalently to Sepharose 4B followed by affinity chromatography. A pre-column of covalently linked non-immune rat γ globulin prevented non-specific binding. The enzyme, released with 1 M KNO3, was purified 1550-fold to a specific activity of 24.8 μmol NO2− produced min−1, mg protein−1 with a recovery of 60% of applied NADH-NR activity. Proteolytically ‘nicked’ subunits, detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) were removed by 5′-AMP Sepharose chromatography (Fido and Notton, Plant Sci. Lett., 37 (1984) 87). 相似文献
11.
《Biochimica et Biophysica Acta (BBA)/General Subjects》1986,884(3):599-601
Effects of novel taurolipid A and B localized in Tetrahymena lysosomes on the activities of lysosomal enzymes purified from Tetrahymena were investigated. Both taurolipids activated acid phosphatase, while they did not affect α-glucosidase and β-hexosaminidase. The acid phosphatase activity was activated approximately 3-fold by both taurolipids A and B, with the half-maximum activations for taurolipid A and B being at approximately 1.03·10−4 and 0.72·10−4 M, respectively. When the purified acid phosphatase was incubated at 37°C in citrate-phosphate buffer (pH 5.0) its activity was rapidly inactivated, but the inactivation was prevented to a remarkable extent by the addition of taurolipids to the incubation medium. These results thus suggest that the taurolipids may be involved in activating and stabilizing acid phosphatase in Tetrahymena lysosomes. 相似文献
12.
Michael J. Wilson Khalil Ahmed Thomas J. Fischbach 《Biochimica et Biophysica Acta (BBA)/General Subjects》1978,542(1):12-20
A protein phosphatase activity has been demonstrated in nuclei of rat ventral prostate utilizing 32P-labelled phosvitin as a model acidic phosphoprotein substrate. This phosphoprotein phosphatase has a pH optimum of 6.7, is unaffected by the sulphydryl protecting agent 2-mercaptoethanol, and requires a divalent cation for maximal activity. Of the various divalent cations tested, Mg2+ is the most effective in reactivating the EDTA-inhibited enzyme. The phosphatase is inhibited by sodium fluoride, sodium oxalate, N-ethylmaleimide, ATP and ADP but is relatively insensitive to ammonium molybdate. Increased ionic strength of the reaction medium also causes a reduction in the enzyme activity, e.g., by 48% at 200 mM sodium chloride. The activity of the acidic phosphoprotein phosphatase did not change significantly at 48 h or 96 h postorchiectomy when expressed per unit of nuclear protein. However, it is reduced by approx. 30% at these times after castration if based on DNA content. The decline in activity per nucleus reflects the decrease in the realtive nuclear protein content observed at 48 h or 96 h post-orchiectomy. This suggests that the decline in the phosphorylation of prostatic nuclear acidic proteins which occurs upon androgen withdrawal is not due to increased nuclear phosphatase activity. 相似文献
13.
《The International journal of biochemistry》1990,22(5):525-532
- 1.1. An ld-dipeptidase (EC 3.4.13.-) that hydrolyzes the unrelated dipeptides l-Ala-d-Glu (sp. act. 0.85 μmol·min−1·mg−1) and l-Lys-d-Ala (sp. act. 11 μmol · min−1·mg−1) has been purified 250-fold from the sporulation medium of Bacillus sphaericus with a 4% recovery of lytic activity.
- 2.2. Throughout the purification steps, followed with both substrates, the enzyme peaks of activities were congruent and the ratios of activities were constant. Both activities were activated 50-fold by cobalt. Polyacrylamide gel electrophoresis of the final preparation showed the two enzyme activities to be coincident. The data are consistent with those activities being due to a single enzyme.
- 3.3. Sodium dodecylsulfate polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band (Mr 38,000).
- 4.4. This dipeptidase hydrolyzes some other ld-dipeptides with a free amino and carboxyl group. Although dipeptides having a di-amino acid as the amino terminus are the best of the substrates tested, the hydrolysis occurs also when neutral amino acids are N-terminal. The activity is higher with neutral C-terminal residues such as Gly or d-Ala than with a di-acid residue such as d-Glu.
- 5.5. This enzyme may have a function in peptidoglycan metabolism.
14.
Plasma membrane extracts from Herpes simplex virus type 1 transformed hamster embryo fibroblasts were chromatographed on Lens culinaris lectin coupled to Sepharose (LcH-Sepharose) and analysed by dodecyl sulphate polyacrylamide gel electrophoresis. Coomassie blue-staining revealed two major protein bands with apparent molecular weights of 125 000 and of about 75 000–90 000. In plasma membranes isolated from these tumor cells prior labeled with [3H]fucose or [3H]glucosamine these bands contained the highest amounts of incorporated radioactivity. Separation by LeH-Sepharose-affinity chromatography as well as metabolic labeling clearly demonstrates their glycoprotein character. The 125 000 protein coincides with alkaline phosphodiesterase I activity with a Km of 6 · 10?4 M for TMP p-nitrophenyl ester and is competitively inhibited by UDP-N-acetylglucosamine. This enzymatic activity is also present in normal hamster embryo fibroblasts. Gel electrophoresis of the Lens culinaris lectin-binding glycoproteins from plasma membranes of normal hamster embryo fibroblasts additionally revealed a strong alkaline phosphatase activity represented by an apparent molecular weight of 150 000, while HSV1 hamster tumor cells contain only a very weak activity of this enzyme activity. HSV-lytically infected cells, however, have unchanged levels of alkaline phosphatase activity, whereas alkaline phosphodiesterase activity increases slightly. 相似文献
15.
Cyclic GMP-dependent protein kinase has been purified to apparent homogeneity from bovine adrenal cortex and its presence in the rat adrenal cortex has been demonstrated. Sucrose density sedimentation studies indicated that the Mr of the enzyme was 145,000. This protein was composed to two identical subunits each with Mr of 75,000. The enzyme molecule was asymmetric with a frictional coefficient of 1.54, Stokes radius of 53.5 Å and a sedimentation coefficient of 6.5. The enzyme self-phosphorylated and the stoichiometry of cyclic GMP binding was two molecules per holoenzyme. Calmodulin or troponin C markedly stimulated the apparent maximal velocity of cyclic GMP-dependent protein kinase without affecting its basal activity. This effect of protein modulators was independent of calcium. Sucrose density gradient studies indicated that the stimulatory effect of calmodulin was due to its interaction with histones. An interaction of calmodulin with the enzyme was not observed. The steroidogenic potential of cyclic GMP and its analogs correlated closely with their ability to stimulate cyclic GMP-dependent protein kinase; the order of potency for both activities was 8-bromocylic GMP > cyclic GMP > N2-monobutyryl cyclic GMP > N2, O2-dibutyryl cyclic GMP. In each case, calmodulin enhanced the cyclic GMP-dependent protein kinase activity for histone phosphorylation. These results indicate that although cyclic GMP is the primary regulator of cyclic GMP-dependent protein kinase, other modulator proteins such as calmodulin could act as additional regulators of the phosphorylation of substrate proteins. In addition, the demonstration of cyclic GMP-dependent protein kinase in rat adrenal glands, and the results with cyclic GMP and its analogs relating to their activation of protein kinase and steroidogenesis are consistant with the concept that cyclic GMP is one of the mediators of adrenal steroidogenesis. 相似文献
16.
《Enzyme and microbial technology》2005,36(2-3):168-175
Paenibacillus sp. A11 produced an intracellular cyclodextrinase (CDase), its presence was confirmed by activity detection on an agar plate with specific screening medium containing β-cyclodextrin (β-CD) and phenolphthalein. The CDase was purified up to 22-fold with a 28% yield. The enzyme was a single polypeptide with a molecular weight of 80 kDa. Optimum activity was at pH 7.0 and 40 °C. The enzyme had an isoelectric point of 5.4 and N-terminal sequence was M F L E A V Y H R P R K N W S. When relative hydrolytic activities of the CDase on different substrates were compared, it was found that high specificity was exerted by β-CD while maltoheptaose, its linear counterpart, was only 40% as active. The enzyme recognized α-1,4-glucose units and the hydrolysis depended on the size of oligosaccharides. Highly branched carbohydrates such as glycogen or dextran or other heteropolymers as glucomannan could not be hydrolyzed. This enzyme was different from other CDases in its ability to hydrolyze maltose and trehalose, though with very low hydrolytic activity. The major product from all substrates was maltose. The kcat/Km value for β-CD was 8.28 × 105 M−1min−1. The enzyme activity was completely inactivated by 1 mM N-bromosuccinimide and diethylpyrocarbonate suggesting the crucial importance of Trp and His for its catalytic activity. Essential Trp was confirmed to be at enzyme active site by substrate protection experiment. Partial inactivation by 5 mM phenylglyoxal suggests the involvement of Arg, which has never been reported in other CDases. 相似文献
17.
18.
《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2002,131(1):55-61
Nucleotide-metabolizing enzymes play important roles in the regulation of intracellular and extracellular nucleotide levels. We studied ATPase activity in the nervous ganglia of Phyllocaulis soleiformis, a terrestrial slug. The ATPase was divalent cation-dependent, with a maximal rate for ATP hydrolysis at pH 6.0 and 7.2 in the presence of Ca2+ (5 mM). Mg2+-ATPase activity was only 26% of the activity observed in the presence of Ca2+ (5 mM). ZnCl2 (10 mM) produced a significant inhibition of 70%. Ca2+-ATPase activity was insensitive to the classical ATPase inhibitors ouabain, N-ethylmaleimide, orthovanadate and sodium azide. Levamisole, an inhibitor of alkaline phosphatase, was ineffective. Among nucleotides, ATP was the best substrate. The apparent Km (ATP) for Ca2+-ATPase was 348±84 μM ATP and the Vmax was 829±114 nmol Pi min−1 mg−1 protein. The P. soleiformis ganglial ATPase does not appear to fit clearly into any of the previously described types of Ca2+-ATPases. 相似文献
19.
Inulin has been widely used as a cheap bioresource for producing many valuable products by enzymatic hydrolysis or microbial fermentation, such as high-fructose syrup and fructooligosaccharides. In this work, a one-pot two-enzyme reaction system was developed to produce d-allulose from inulin using A. piperis exoinulinase and Dorea sp. d-Allulose 3-epimerase. The exoinulinase that was identified from Aspergillus piperis CBS 112811 was cloned and intracellularly expressed in Escherichia coli. The enzyme displayed the maximal activity as 3750 U mg−1 at pH 6.0 and 55 °C. For the effects of different cations, Mn2+ simulated the enzyme activity by 41 %. When 10 g L−1 inulin was hydrolyzed by A. piperis exoinulinase, the conversion rate reached 98 % within 6 h. Furthermore, the optimum pH, temperature and the ratio of the two enzymes loaded for one-pot reaction were measured to be pH 6.0, 60 °C and 15/15 U mL−1, respectively. The conversion rate of inulin to d-allulose reached 23.3 % after reaction for 4 h with 10 g L−1 inulin. When adding 100 g L−1 as a substrate, 21.4 g L−1 d-allulose was produced using the two-enzyme system. 相似文献
20.
Ribulose 1,5-diphosphate-dependent oxygenase activity was demonstrated for crystallized Fraction 1 protein (RuDP2 carboxylase EC 4.1.1.39) from tobacco. The kinetic properties of this oxygenase function were examined polarographically in air-equilibrated medium. Optimum activity was obtained at pH 8.4–8.6, and required 4–8 mm MgCl2. Higher Mg2+ concentrations decreased activity and slightly shifted the pH optimum to 8.2–8.3. The apparent Km (RuDP) and Km (Mg2+) were 22 μm and 0.5 mm, respectively. Oxygenase activity was inhibited by bicarbonate and indirectly by KCN. Kinetic studies suggest that the active inhibitory substance is the cyanohydrin derivative formed from the reaction of KCN with RuDP.Changes in oxygenase kinetics were observed upon addition of RuDP, as previously reported for the carboxylase function of this enzyme. Oxygenase activity required preincubation of the enzyme with both Mg2+ and low concentrations of bicarbonate. Activities were enhanced about 20 and 70% when FDP (0.1 mm) and NADPH (0.5 mm), respectively, were included during preincubation. 相似文献