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1.
Ravendra Pal Singh 《Journal of biosciences》1982,4(4):431-439
Bacillus megaterium accumulated 3-phosphoglycerate during sporulation which was utilized during spore germination. During sporulation a protein
was synthesized before or at the start of 3-phosphoglycerate accumulation inside the developing spores about 1.5 h before
dipicolinic acid accumulation. This protein has an affinity for Mn2+ and other divalent metal ions and inhibits phosphoglycerate mutase activity which has been shown to require Mn2+ However, the levels of the inhibitor decreased considerably (75–85%) during spore germination. No appreciable amount of the
inhibitor was detected in the vegetable cell and mother cell compartment; however, the forespore compartment possesses an
activity comparable to that of dormant spores. The partially purified inhibitor has a molecular weight of 11,000 and possesses
both high and low affinity binding sites for Mn2+ and Ca2+ as determined by Scatchard plot analysis. 相似文献
2.
Wolfgang Uerkvitz Olle Karlström Agnete Munch-Petersen 《Molecular & general genetics : MGG》1973,121(4):337-346
Summary A two-step mutant lacking two periplasmic enzymes, alkaline phosphatase and 5-nucleotidase, was prepared. In extracts of this strain phosphatase activities towards several 5-nucleotides could be detected. We have partially purified a dUMP phosphatase by streptomycin precipitation and DEAE-chromatography. This preparation has significant phosphatase activity towards three substrates, viz. dUMP, dTMP and UMP, but only traces of activity towards nine other tested nucleoside monophosphates. The three activities are probably due to a single enzyme, since they decrease in parallel on long time storage at 4°C and respond in parallel to stimulatory and inhibitory influences of different buffers and metal ions. The best buffer tested is glycylglycine buffer; Mg2+ is required but 25% activity can be obtained with Co2+ and Mn2+ and 6–8% activity with Fe2+ and Ni2+. Ca2+, Cu2+ or Zn2+ inhibit the phosphatase activities in the presence of Mg2+ or Co2+. The activities towards UMP and dUMP in the DEAE—chromatography eluted identically. The enzyme has the remarkably high apparent K
m of 10-2 M with all three substrates. 相似文献
3.
A Mn2+/phospholipid-dependent protein phosphatase has been identified and characterized from brain membranes. The phosphatase contains three subunits with molecular weights of 64,000, 54,000, and 35,000 in a 1:1:1 molar ratio. On gel filtration, the enzyme has an apparent molecular weight of 180,000. The phosphatase was active on many substrates, including p-nitrophenyl phosphate, phosphotyrosine, phosphothreonine, phosphorylase a, myelin basic protein, histones, type 1 phosphatase inhibitor-2, microtubule protein, and synapsin I. To dephosphorylate phosphoproteins, the phosphatase was dependent on such acidic phospholipids as phosphatidylinositol and phosphatidylserine but not on neutral phospholipids such as phosphatidylcholine and phosphatidylethanolamine. The phospholipid-mediated activation of the phosphatase was time and dose dependent and could be reversed by Triton X-100 or gel filtration. Kinetic study further indicates that phospholipid was able to increase theV
max of the phosphatase but had no effect on theK
m value for substrates, suggesting a direct interaction of phospholipids with the phosphatase. Conversely, in order to dephosphorylate phosphoamino acids such as phosphotyrosine and phosphothreonine, this phosphatase was entirely dependent on Mn2+. Phospholipids had no effect on the dephosphorylation of phosphoamino acids, whereas Mn2+ had no effect on the dephosphorylation of phosphoproteins. It is concluded that this Mn2+/phospholipid-dependent membrane phosphatase has two distinct activation mechanisms. The enzyme requires Mn2+ to dephosphorylate micromolecules, whereas acidic phospholipids are needed to dephosphorylate macromolecules. This suggests that Mn2+ and phospholipids may play a role in regulating the substrate specificity of this multisubstrate membrane phosphatase. 相似文献
4.
A. C. Emeruwa 《Antonie van Leeuwenhoek》1979,45(1):95-101
The activities of alkaline and acid phosphatases, glucose dehydrogenase and NADH oxidase were assayed in cell-free extracts of sporogenic and asporogenic mutants of Clostridium botulinum. During growth of both mutants, the activities of alkaline and acid phosphatases were relatively constant, but during sporulation of the sporogenic mutant, the alkaline phosphatase activity rose to a maximum of 70 mol/min·mg protein whereas the acid phosphatase decreased rapidly before it increased, indicating a possible role in sporogenesis. Glucose dehydrogenase activity was detected only in cell-free extracts of the sporogenic mutant and reached a maximum of 7 mol/min·mg protein during the endospore maturation stage. The NADH oxidase activity was detected in both mutants. The NADH oxidase seems to stimulate glucose oxidation in both mutants during growth and the dehydrogenation processes of the butyric type of fermentation during spore formation in the sporogenic mutant. The findings suggest that increased glucose dehydrogenase activity in C. botulinum, as in Bacillus species, may serve as a spore event marker and that alkaline and acid phosphatases may play a regulatory role in anaerobic sporulation metablolism.This work was supported by the Aquatic Biology Research Unit of the University of Manitoba from a Federal Fisheries Research Grant. 相似文献
5.
A strain of Rhizopus stolonifer produced high levels of extracellular ribonuclease (RNase) when grown on YPG (yeast extract, peptone, glucose) medium. Influence
of various medium components on the production of extracellular RNase activity showed that divalent metal ions had a marked
effect on growth and enzyme production. Maximum enzyme activity (3000 U/ml) was obtained in 5 days when the culture was grown
in YPG medium containing Mg2+ (12 mM), Mn2+, and Fe2+ (2 ppm each). Inorganic phosphate, however, repressed enzyme production. Antibodies raised against the purified extracellular
RNase were then used to establish the relationship between intra- and extracellular enzymes. 相似文献
6.
Makiko Takagi Saburo Funahashi Keiichi Ohta Toshio Nakabayashi 《Bioscience, biotechnology, and biochemistry》2013,77(11):2417-2418
Sporulation marker enzymes, d-glucose dehydrogenase and l-alanine dehydrogenase were synthesized derepressively by vegetative cells of a Bacillus species mutant which was isolated as an improved d-ribose producer. It was also elucidated by electron microscopy that no morphological change concerning sporulation took place during the course of the enzyme syntheses in the mutant strain. The presence of Mn2+ and Ca2+ in the medium was necessary for the morphological development of sporulation even in the mutant strain. The mechanism of derepressed enzyme syntheses is discussed in relation to regulation of sporulation. 相似文献
7.
Porcel ER López JL Ferrón MA Pérez JA Sánchez JL Chisti Y 《Bioprocess and biosystems engineering》2006,29(1):1-5
The production of biomass and lovastatin by spore-initiated submerged fermentations of Aspergillus terreus ATCC 20542 was shown to depend on the age of the spores used for inoculation. Cultures started from older spores produced significantly higher titers of lovastatin. For example, the lovastatin titer increased by 52% when the spore age at inoculation rose from 9 to 16 days. The lovastatin titer for a spore age of 16 days was 186.5±20.1 mg L−1. The time to sporulation on surface cultures was sensitive to the light exposure history of the fungus and the spore inoculation concentration levels. A light exposure level of 140 μE m−2 s−1 and a spore concentration of 1,320 spore cm−2 produced the greatest extent of sporulation within about 50 h of inoculation. Sporulation was slowed in the dark and with diluted inoculants. A rigorous analysis of the data of statistically designed experiments showed the above observations to be highly reproducible. 相似文献
8.
Summary A polygalacturonase from culture filtrates of a strain ofRhizopus
stolonifer was purified about 80 fold by ethanol precipitation, followed by ion exchange chromatography (CM-Sepharose 6B) and gel filtration (Sephadex G-100). The purified preparation was homogeneous when examined by PAGE. The enzyme is an endopolygalacturonase with an optimum catalytic activity at pH 5.0 and 45°C, and a molecular weight of 57,000±500 daltons. The activity was stimulated by Fe+++, Mg++, Co++, and inhibited by Mn++ and Zn++. The enzyme was stable in the pH range of 3.0 to 5.0. The purified enzyme was specific for nonmethoxylate polygalacturonic acid, with Km and Vmax values respectively of 0.19 mg/ml and 1.3 mol/g/min. In addition, this enzymatic preparation degraded pectic substances in organge peel. 相似文献
9.
Ramesh C. Gupta Ramji L. Khandelwal Prakash V. Sulakhe 《Molecular and cellular biochemistry》1990,97(1):53-66
Summary The effects of divalent metals, metal chelators (EDTA, EGTA) and sodium dodecyl sulfate were investigated on the phosphatase activity of isolated bovine brain calcineurin assayed in the absence (called intrinsic) and presence of calmodulin. Intrinsic phosphatase was increased by Mn2+, was unaffected by Mg2+, Ca2–, and Ba+, and was markedly inhibited by Ni2–, Fe2+, Zn2+ and Cu2–. When assayed in the presence of calmodulin, many divalent metals (Ni2–, Zn2+, Pb2+, Cd2+), besides Mn2+, increased modestly the phosphatase activity at low concentrations (10–100 M) and inhibited it markedly at high concentrations. Ca2–-calmodulin stimulated phosphatase activity was antagonized by Ni2+, Zn2+, Fe2+, Cu2+, Pb2+, at low concentrations (50 M), and by Ba2+, Cd2+ at slightly higher concentrations (> 100 M); Mn2+ and Co2– (50 M to 1 mM) in fact augmented it. EDTA and EGTA in a concentration and time dependent fashion inhibited the intrinsic phosphatase activity, particularly that of trypsinized calcineurin. SDS in low concentrations (0.005%) augmented the phosphatase activity and inhibited it at high concentrations. Mn2+ (± calmodulin) and Ca2+ only with calmodulin present increased the phosphatase activity assayed with low concentrations of SDS. The EDTA dependent inhibition of intrinsic phosphatase was almost abolished in assays containing SDS. Prior exposure of calcineurin to Mn2+ led to a high activity conformation state of calcineurin that was long-lived or pseudo-irreversible. Such Mn2+-activated state of calcineurin exhibited no discerbible change in the affinity towards myelin basic protein or its inhibition by trifluoperazine. At alkaline pH, Mg2+ supported the intrinsic phosphatase activity, although to a lesser degree than Mn2+. The latter cation, compared to Mg2+ and Ni2+, was also a more powerful stimulator of the calcineurin phosphatase assayed with histone (III-S) and myosin light chain as substrates. 相似文献
10.
A soluble protein phosphatase from the promastigote form of the parasitic protozoanLeishmania donovani was partially purified using Sephadex G-100, DEAE-cellulose and again Sephadex G-100 columns. The partially purified enzyme showed a native molecular weight of about 42, 000 in both Sephadex G-100 and sucrose density gradient centrifugation. The sedimentation constant, stokes radius and frictional ratio were found to be 3.43S, 2.8 nm and 1.20 respectively. The enzyme preferentially utilized phosphohistone as the best exogenous substrate. Mg2+ ions were essential for enzyme activity; among other metal ions Mn2+ can replace Mg2+ to a certain extent whereas Ca2+, Co2+ and Zn2+ could not substitute for Mg2+. The pH optimum of the enzyme was 6.5–7.5 and the temperature optimum 37°C. The apparent Km for phosphohistone was 7.14 M. ATP, ADP, inorganic phosphate and pyrophosphate had inhibitory effect on the enzyme activity whereas no inhibition was observed with sodium tartrate and okadaic acid. These results suggest thatL. donovani promastigotes possess a protein phosphatase which has similar characteristics with the mammalian protein phosphatase 2C.Abbreviations PMSF
phenylmethylsulfonyl fluoride
- DTT
dithiothreitol
- TCA
trichloroacetic acid
- BSA
bovine serum albumin
- EDTA
ethylenediamine tetraacetic acid
- ATP
adenosine triphosphate
- ADP
adenosine diphosphate
- AMP
adenosine monophosphate
- EGTA
Ethyleneglycol-bis-(-aminoethyl ether) N,N,N,N-tetraacetic acid 相似文献
11.
《The International journal of biochemistry》1993,25(1):7-12
- 1.1. Alkaline p-nitrophenylphosphate phosphatase of Halobacterium halobiium, either purified or in crude extracts, was progressively inactivated by treatment with several metal chelators.
- 2.2. The activity of treated crude extracts was fully restored in the presence of 25–50 μM Mn2+ or 1 mM Co2+, and partially restored in the presence of 1 mM Cd2+.
- 3.3. Zn2+ ions, as well as other divalent cations tested, were without effect.
- 4.4. In the presence of a saturating concentration of Mn2+, but not Co2+ or Cd2+, the activity of the metal-depleted enzyme reached values well over the native control activity.
- 5.5. Activation of the metal-depleted enzyme by Mn2+ showed cooperative kinetics, whereas activation by Co2+ showed Lineweaver-Burk kinetics.
- 6.6. The results suggest that the enzyme contains two different types of metal-binding sites: essential site(s), occupied by endogenous Mn2+ ions, and regulatory site(s), that can be occupied by exogenous Mn2+ with an activating effect.
12.
A soluble extracellular antigen produced byPasteurella haemolytica serotype 1 possesses neuraminidase activity. The metal ions Mn2+, Mg2+, and Ca2+ stimulate enzyme activity, while the heavy metal ions Hg2+ and Fe3+ inhibit neuraminidase activity. Extensive dialysis of the enzyme with 50 mM ethylenediaminetetraacetic acid does not result in loss of enzyme activity. Antiserum prepared againstPasteurella haemolytica serotype 1 inhibits neuraminidase activity 2.0- to 2.7-fold, suggesting that neuraminidase is part of the antigenic complex. Initial rate kinetics of neuraminidase reveals that the Michaelis constant for fetuin is 3.84 mg/ml (96 M) and the maximal velocity (V
max) is 0.53 mmol/min/mg of protein. 相似文献
13.
Ponnambalam Subhashini Neelamegam Annamalai Ayyappan Saravanakumar Thangavel Balasubramanian 《Biologia》2012,67(4):629-635
An extracellular alkaline protease-producing Vibrio sp. was isolated from mangrove sediments of Vellar estuary. A 9.36-fold purification was achieved by a three-step purification procedure and the molecular weight of the enzyme was determined as 33 kDa by SDS-PAGE. The enzyme was active in a broad range of pH (6.0–11.0) and temperature (30–70°C), the optimum being at pH 9.0 and temperature 55°C. The enzyme was stable at alkaline pH range of 9–11 and up to a temperature of 60°C, after incubation for 1 h. Metals like Co2+, Hg2+, Ni2+ and Cu2+ inhibited the enzyme activity, whereas Fe2+, Ca2+ and Mn2+ were found to enhance the activity. The protease was found to be highly stable in the presence of oxidizing agents like H2O2, detergents such as SDS and Triton-X-100 and also some of the commonly used commercial detergents. The organic solvents like xylene, isopropanol, hexane and benzene were found to enhance as well as stabilize the enzyme activity. The extracellular production of the enzyme, the pH and thermal stability, and the stability in presence of oxidants, surfactants, commercial detergents and organic solvents, altogether suggest that it can be used as a laundry additive. 相似文献
14.
Gen-ichi Danno 《Bioscience, biotechnology, and biochemistry》2013,77(7):997-1006
A functional role of Co2+ and Mn2+ in the d-glucose- and d-xylose-isomerizing reactions by d-glucose-isomerizing enzyme obtained from the cells of Bacillus coagulans, strain HN–68 was investigated. (1) The enzyme required Co2+ and Mn2+ for d-glucose- and d-xylose-isomerizing activities, respectively. (2) The enzyme which bound the metal, Co2+- or Mn2+-enzyme, was active form. Co2+ was bound to the enzyme in a molar ratio of 4:1. (3) The rate of activation by metal ion varied with incubation pH. (4) The binding of substrate to the enzyme was completely independent in the presence of metal ions. (5) However, it seemed unlikely that the Co2+ and Mn2+ acted as catalyzer on the reaction. (6) The binding sites for Co2+ and Mn2+ were different from each other. (7) The experimental data obtained might be successfully explained in terms of the suitable conformational changes for d-glucose and d-xylose isomerization, which were induced in the catalytic sites of the enzyme by binding Co2+ and Mn2+, respectively. 相似文献
15.
Alex Ferrandi Federica Castani Mauro Pitaro Sara Tagliaferri Claire Bouthier de la Tour Rosa Alduina Suzanne Sommer Mauro Fasano Paola Barbieri Monica Mancini Ian Marc Bonapace 《Biochimica et Biophysica Acta (BBA)/General Subjects》2019,1863(1):118-129
Background
Deinococcus radiodurans R1 (DR) survives conditions of extreme desiccation, irradiation and exposure to genotoxic chemicals, due to efficient DNA breaks repair, also through Mn2+ protection of DNA repair enzymes.Methods
Possible annotated domains of the DR1533 locus protein (Shp) were searched by bioinformatic analysis. The gene was cloned and expressed as fusion protein. Band-shift assays of Shp or the SRA and HNH domains were performed on oligonucleotides, genomic DNA from E. coli and DR. shp knock-out mutant was generated by homologous recombination with a kanamycin resistance cassette.Results
DR1533 contains an N-terminal SRA domain and a C-terminal HNH motif (SRA-HNH Protein, Shp). Through its SRA domain, Shp binds double-strand oligonucleotides containing 5mC and 5hmC, but also unmethylated and mismatched cytosines in presence of Mn2+. Shp also binds to Escherichia coli dcm+ genomic DNA, and to cytosine unmethylated DR and E. coli dcm? genomic DNAs, but only in presence of Mn2+. Under these binding conditions, Shp displays DNAse activity through its HNH domain. Shp KO enhanced >100 fold the number of spontaneous mutants, whilst the treatment with DNA double strand break inducing agents enhanced up to 3-log the number of survivors.Conclusions
The SRA-HNH containing protein Shp binds to and cuts 5mC DNA, and unmethylated DNA in a Mn2+ dependent manner, and might be involved in faithful genome inheritance maintenance following DNA damage.General significance
Our results provide evidence for a potential role of DR Shp protein for genome integrity maintenance, following DNA double strand breaks induced by genotoxic agents. 相似文献16.
P. S. Apte M. V. Deshpande V. Shankar 《World journal of microbiology & biotechnology》1993,9(2):205-209
A strain of Rhizopus stolonifer produced a high activity of extracellular DNAase when grown on YPG (yeast extract peptone glucose) medium. The source of peptone had a marked effect on the enzyme yield and only one peptone (i.e. from Sarabhai M. Chemicals Ltd, India) supported enzyme production. Maximum enzyme activity (88 U/ml) was obtained after 4 days' growth under submerged conditions in YPG medium containing 100 M Mn2+, Co2+ or Mg2+, and glucose as the sole carbon source. The unpurified enzyme was optimally active at pH 7.5 and 45°C. It had a higher activity with sonicated and heat-denatured DNA than native DNA. 相似文献
17.
Shunya Takahashi Takayuki Oritani Kyohei Yamashita 《Bioscience, biotechnology, and biochemistry》2013,77(12):3205-3206
Phosphodiesterase production with bis-p-nitrophenyl phosphate as a substrate by alkalophilic Bacillus No. A-40-2 increased with increasing Mn2+ concentration, showing maximum productivity at 10 mm. The enzyme production was negligible in the medium without Mn2+. The simultaneous addition of 10 mm Mn2+ and one of the several cations Mg2+, Co2+, Mo6+, and Pb2+ at suitable concentrations stimulated the enzyme production 1.8-fold at most over that with only 10 mm Mn2+. Inorganic phosphate hardly repressed the enzyme production. The enzyme was purified homogeneously. The purified enzyme had the optimum pH of 7.5 and was fairly stable from pH 7–11. The enzyme hydrolyzed 2′,3′-cyclic-nucleotides and 3′-nucleotides, but did not hydrolyze 3′,5′-cyclic-nucleotides or 5′-nucleotides, indicating it to be a 2′,3′-cyclic-nucleotide 2′-phosphodiesterase (EC 3.1.4.16). The enzyme had activity without metals, but Mg2+, Ca2+, Ba2+, and Mo6+ activated the enzyme reaction. 相似文献
18.
A mycelial suspension of the crayfish plague fungus, Aphanomyces astaci, was able to produce large numbers of zoospores, when transferred to redistilled water, at 20°C, even after storage for months at 2°C. Spore production was greater in redistilled water than in tap water and heavier under shake conditions than under stationary ones. In buffered redistilled water sporulation occurred between pH 5 and 8 and the optimal range was about pH 5 to 7. Of the tested aliphatic alcohols, aldehydes, and carboxylic acids, the long analogues were more toxic to spore formation than the shorter ones. Ethylenediamine-tetraacetic acid (EDTA) prevented sporulation probably by removing some essential metal (s) with an affinity for EDTA near that of calcium. Calcium protected against the toxic effect of lithium, sodium, and potassium. Magnesium, only tested against lithium, had no such protecting effect. Cu2+, Ni2+, Zn2+, Co2+, K+ Mn2+, NH4+, Li+, Na+, Ca2+, Mg2+ was the approximate order among tested cations in their ability to stop the swimming stage of the zoospores, the first mentioned being the most effective ones. Nitrate and acetate were more active in the same respect than sulphate, chloride, phosphate, or bicarbonate. The optimal pH range for swimming seemed to be pH 6–7.5, and the maximal range 4.5–9.0. The zoospores showed no chemotactic response to tested substances. The germination ability was as high in horse blood as in crayfish blood. A spore suspension stored for 2 months at 2°C still contained viable spores. 相似文献
19.
Christian Larroche Chantal Desfarges Jean-Bernard Gros 《Applied microbiology and biotechnology》1988,28(1):85-92
Summary The effect of substrate (buckwheat seeds) pretreatment on the growth and the sporulation behaviour of Penicillium roqueforti is presented. When a saccharifying enzyme (-amylase) is added to a medium which exhibits a low water content (0.46 g water/g initial dry matter, IDM), a more rapid internal colonization of the seeds occurs, but the final spore production does not increase and remains close to 8.109 spores/g dry matter (DM) at 500 h. No carbon source limitation is then observed. The addition of casein hydrolysate to this medium gives rise to a great increase of the sporulation, since 14.5 109 spores/g DM are obtained after 600 h. This result is attained by a better spore yield from the mycelium, the substrate colonization being unchanged. High water content (0.60 g water/g IDM) of buckwheat seeds induces a shorter cultivation time along with a higher biomass production. However, the spore content of the medium remains close to the low water content one, but 60% total spores are external against 30% to 35% in the other media. 相似文献
20.
William M. Fogarty Catherine T. Kelly Anne C. Bourke Evelyn M. Doyle 《Biotechnology letters》1994,16(5):473-478
Summary
Pseudomonas sp. IMD 353 produced an extracellular consititutive maltotetraose-forming amylase in a medium containing glucose (or fructose), yeatex and mineral salts. Km values on starch, amylopectin and short chain amylose were 4.0, 2.8 and 3.0 mg/ml, repectively. Sulphydryl reducing agents activated the enzyme considerably, as did Co2+ and Mn2+. 相似文献