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1.
Surface-Bound Nuclease of Staphylococcus aureus: Purification and Properties of the Enzyme
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The surface-bound nuclease of Staphylococcus aureus liberated during formation of protoplasts was purified 1,000-fold by chromatography on phosphocellulose. Its properties were compared with those of the known extracellular nuclease, purified 200-fold by the same procedures. The adsorbance of the surface-bound nuclease on phosphocellulose was distinctly different from that of the extracellular nuclease, but other properties of the two enzymes were similar. Both enzymes had a pH optimum of about 10 and required Ca2+ for activity. Both enzymes hydrolyzed deoxyribonucleic acid (DNA) and ribonucleic acid, and denatured DNA was a better substrate than native DNA. Both enzymes were inhibited by the same metal ions. Nuclease-less mutants of S. aureus were isolated from S. aureus 209P by using N-methyl-N′-nitroso-N-nitrosoguanidine. These mutants contained neither surface-bound nor extracellular nuclease activity. These results suggest that the surface-bound and extracellular nucleases are expressed from the same cistron of S. aureus. 相似文献
2.
Purification, properties and mechanism of action of a staphylolytic enzyme produced by Aeromonas hydrophila
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1. An enzyme produced by Aeromonas hydrophila and capable of lysing Staphylococcus aureus cells was purified 180-fold by gel filtration and chromatography on columns of AG-50 W resin. 2. Physical measurements on the purified enzyme suggest that it is a small basic protein with an isoelectric point between pH9·0 and pH9·5. 3. Maximum lytic activity was obtained in 20mm-tris–glycine buffer, pH8·5, at 45°, with no detectable activity in the absence of a nitrogenous base. 4. The enzyme is active in the above buffer containing 1·5m-sucrose, and is useful for the preparation of protoplasts of Staphylococcus aureus. 5. Purified cell wall peptidoglycans of two strains of Staphylococcus aureus, differing in amino acid composition, were hydrolysed by the enzyme with the liberation of glycine oligopeptides, principally diglycine and triglycine. 6. Synthetic glycine oligopeptides larger than triglycine, but not polyglycine, were hydrolysed, as were a number of leucine-containing dipeptides and tripeptides, but no proteolytic activity could be demonstrated. 7. It is concluded that the enzyme is lytic towards Staphylococcus aureus because it splits the pentaglycine cross-links of the cell-wall peptidoglycan. 相似文献
3.
Valery V. Petrov Vladislav Yu. Artzatbanov Evgeny N. Ratner Anatoly I. Severin Igor S. Kulaev 《Archives of microbiology》1991,155(6):549-553
The action of the lysoamidase bacteriolytic complex on Staphylococcus aureus VKM B-209P cells has been studied to obtain protoplasts. The cells in the midlogarithmic phase were the most sensitive to lysoamidase action. It led to local destruction of cell wall due to hydrolysis of the peptidoglycan. Protoplast formation occurred in two steps in the presence of 1 M sucrose. First, osmotically fragile spheroplasts were formed. Then, the protoplasts were released from the destructed cell wall. The protoplast yield was about 80%. The protoplasts preserved the intact ultrastructure and were able to synthesize peptidoglycan fibrillae. Mainly the spheroplasts that maintained the cell-wall residues reversed into bacterial forms. The protoplasts had respiratory activity similar to cells. Respiration of cells and protoplasts was stimulated by various substrates. High rates of oxygen consumption were observed with -glycerophosphate and ethanol as substrates. 相似文献
4.
Isolation and Characterization of Concanavalin A-labeled Plasma Membranes of Carrot Protoplasts 总被引:12,自引:11,他引:1
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The plasma membranes of protoplasts released from carrot suspension culture cells were labeled with [14C]acetyl-concanavalin A. After homogenization a single labeled membrane fraction was isolated in a continuous isopycnic Renografin gradient. The labeled membranes peaked at an apparent density of 1.14 grams per cubic centimeter between the Golgi fraction at a density of 1.11 grams per cubic centimeter as determined by latent IDPase activity and the mitochondria at a density of 1.16 grams per cubic centimeter as determined by the cytochrome c oxidase activity. This method provided a very discrete peak of putative plasma membrane. On discontinuous Renografin gradients a relatively pure fraction of labeled plasma membranes could be readily isolated at the 1.122 to 1.146 grams per cubic centimeter interface. The labeled fraction was enriched in both an ATPase (pH 6.5) and a glucan synthetase with a pH optimum of 6.5 whose activity was promoted by magnesium and cellobiose. Enzyme activities were not altered by the membrane label. 相似文献
5.
6.
Subcellular Location of NADP-Isocitrate Dehydrogenase in Pisum sativum Leaves 总被引:1,自引:1,他引:0
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Randall DD 《Plant physiology》1981,68(1):70-73
The subcellular location of NADP+-isocitrate dehydrogenase was investigated by preparing protoplasts from leaves of pea seedlings. Washed protoplasts were gently lysed and the whole lysate separated on sucrose gradients by a rate-zonal centrifugation. Organelles were located by marker enzymes and chlorophyll analysis. Most of the NADP+-isocitrate dehydrogenase was in the soluble fraction. About 10% of the NADP+-isocitrate dehydrogenase was present in the chloroplasts as a partially latent enzyme. Less than 1% of the activity was found associated with the peroxisome fraction. NADP+-isocitrate dehydrogenase was partially characterized from highly purified chloroplasts isolated from shoot homogenates. The enzyme exhibited apparent Km values of 11 micromolar (NADP+), 35 micromolar (isocitrate), 78 micromolar (Mn2+), 0.3 millimolar (Mg2+) and showed optimum activity at pH 8 to 8.5 with Mn2+ and 8.8 to 9.2 with Mg2+. The NADP+-isocitrate dehydrogenase activity previously claimed in the peroxisomes by other workers is probably due to isolation procedures and/or nonspecific association. The NADP+-isocitrate dehydrogenase activity in the chloroplasts might help supply α-ketoglutarate for glutamate synthase action. 相似文献
7.
Susanne Maier Hans Grisebach 《Biochimica et Biophysica Acta (BBA)/General Subjects》1979,586(2):231-241
Resting cells and to a greater extent permeabilized cells of Streptomyces griseus can oxidize dihydrostreptomycin to streptomycin. The dihydrostreptomycin oxidoreductase activity was localized in the 100 000 × g particulate fraction. Sucrose density gradient centrifugation of the particulate suspension gave a band at a density of 1.09 which consisted mainly of membrane vesicles. This fraction had high dihydrostreptomycin oxidoreductase activity. S. griseus protoplasts also contain high oxidoreductase activity. These data are consistent with localization of the enzyme in the cell membrane. Dihydrostreptomycin and dihydrostreptomycin 6-phosphate can both serve as substrates for the oxidoreductase, but the phosphate was the better substrate in the cell free system. Addition of cofactors was not required for the bound dihydrostreptomycin oxidoreductase. The electron acceptor for the oxidation is unknown. Oxidation of dihydrostreptomycin 6-phosphate to streptomycin 6-phosphate very probably represents the penultimate step in the biosynthesis of streptomycin. 相似文献
8.
Subcellular Localization of Anthocyanin Methyltransferase in Flowers of Petunia hybrida 总被引:2,自引:1,他引:1
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The subcellular localization of the enzyme anthocyanin-methyltransferase was studied in cells (protoplasts) obtained from the upper epidermis of petals of Petunia hybrida Hort. Vacuoles were isolated from protoplasts to ascertain the possible presence of the enzyme in these organelles. The recovery of methyltransferase activity in vacuole-enriched fractions equalled that of the cytosolic marker enzyme glucose-6-phosphate dehydrogenase. The relative activity of methyltransferase in the vacuole fraction was one tenth of that in the protoplast. Neither whole protoplasts nor isolated vacuoles contained inhibitors of methyltransferase activity. Examination of fractions obtained by differential centrifugation of a protoplast lysate showed that the major part of the methyltransferase activity was cytosolic. Activity found in a 130,000g pellet was due to nonspecific adhesion to membranes. The results indicate that terminal steps of anthocyanin biosynthesis take place in the cytosol. They do not lend support to the notion that the vacuole might be involved in (part of) this process. 相似文献
9.
L. V. Trilisenko J. Novotná V. Erban V. Běhal Z. Hoštálek I. S. Kulaev 《Folia microbiologica》1987,32(5):402-410
Mycelia of a low- and a high production strain ofStreptomyces aureofaciens were converted into protoplasts and divided into five subcellular fractions in order to localize exopolyphosphatases (EC
3.6.1.11), triphosphatase (EC 3.6.1.25), inorganic diphosphatase (EC 3.6.1.1), apyrase (EC 3.6.1.5) and glucokinase (EC 2.7.1.2).
The highest specific activity of enzymes hydrolyzing polyphosphates was found in cytoplasmic vesicles and membranes. Triphosphatase
was detected in the periplasmic fraction. Periplasmic vesicles and cytoplasm exhibited a high activity of diphosphatase. Apyrase
was found mainly in the fractions of membranes and cytoplasmic vesicles. Glucokinase was a cytoplasmic enzyme. The enzymes
were released from membrane structures into cytoplasm or periplasmic space if benzyl thiocyanate (10 μm) was present in the
growth medium. 相似文献
10.
Cellular site in Bacillus subtilis of a nuclease which preferentially degrades single-stranded nucleic acids 总被引:8,自引:6,他引:2
H C Birnboim 《Journal of bacteriology》1966,91(3):1004-1011
Birnboim, H. C. (Albert Einstein College of Medicine, New York, N.Y.). Cellular site in Bacillus subtilis of a nuclease which preferentially degrades single-stranded nucleic acids. J. Bacteriol. 91:1004-1011. 1966.-A nuclease, identified by a marked preference for single-stranded nucleic acids, has been demonstrated in extracts of Bacillus subtilis. The enzyme was associated with the cell wall-membrane fraction of mechanically disrupted cells and was released from cells which had been converted to protoplasts by lysozyme. The nuclease activity prepared by the latter procedure was found to be activated and solubilized by treatment with trypsin. The enzyme had about 2% activity on native deoxyribonucleic acid (DNA) as compared with denatured DNA. By use of CsCl analytical density gradient ultracentrifugation, this preparation was shown to degrade denatured DNA selectively in mixtures of native and denatured DNA. 相似文献
11.
Amylolytic lactic acid bacteria in fish silage 总被引:1,自引:1,他引:1
An ∝aL-amylase activity has been observed in lactic acid bacteria occurring initially in fermented fish silage. The organisms belong to the genus Leuconostoc . The main fraction of the amylolytic enzyme produced by one of the isolated bacteria is cell-bound and is released into the medium at a late stage of growth. Treating cells with ultrasound or Triton X-100 increases enzyme activity in the culture filtrate. The pH range for enzyme activity is 5.0–7.0, with an optimum at pH 6.0. The enzyme is extremely labile at pH 8.0 and is inactivated at temperatures above 50°C at pH 5.8. Two enzyme fractions were found by isoelectric focusing, the main one at pH 5.00 and another at pH 4.5. Chromatography on DEAE cellulose gave two active peaks. 相似文献
12.
Isolation and characterization of infected and uninfected cells from soybean nodules : role of uninfected cells in ureide synthesis 总被引:18,自引:13,他引:5
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The distribution of organelles and associated enzymes between cells containing bacteroids and uninfected cells from nodules of Glycine max L. Merr. cv Amsoy 71 was investigated by separation of protoplasts on a sucrose step-gradient. Infected protoplasts were much larger, irregular in shape, and more dense than uninfected protoplasts. The peroxisomal enzymes, uricase and catalase, were present at much higher specific activity in the uninfected cell fraction. Allantoinase, an enzyme of the endoplasmic reticulum, had a greater specific activity in the uninfected cell fraction. Several enzymes whose products are required for purine biosynthesis, including phosphoglycerate dehydrogenase, aspartate aminotransferase, 6-phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase, exhibited a higher specific activity in the uninfected cell fraction. Isozymes of aspartate aminotransferase were separated on native gels and located by an activity stain. The soluble isozyme was predominantly found in the uninfected cell fraction. These data suggest that peroxisomes, containing uricase and catalase for conversion of uric acid to allantoin, are present only in the uninfected cells of soybean nodules. The uninfected cells also appear to be the site of the allantoinase reaction. 相似文献
13.
Characteristics of Penicillinase Secretion by Growing Cells and Protoplasts of Bacillus licheniformis 总被引:11,自引:4,他引:7
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Cultures of the inducible penicillinase-producing strain 749 of Bacillus licheniformis, induced with small amounts of benzylpenicillin, synthesized penicillinase at a high rate for a short period, after which the rate of synthesis slowly declined. During the period of active synthesis, the rate of secretion, as a fraction of the level of cell-bound penicillinase (which is originally high), gradually decreased to a constant level. Chloramphenicol, at a concentration (40 mug/ml) which completely inhibited synthesis of penicillinase, partially inhibited secretion if added during the period of active synthesis. During the phase of reduced synthesis, chloramphenicol was without effect on secretion. Penicillinase secretion, by actively growing cultures of the constitutive penicillinase-producing mutant 749/C, was inhibited by 75% immediately after addition of chloramphenicol. The secretion of part of the penicillinase released during active growth is probably dependent on synthesis of penicillinase, but part of the secreted penicillinase can be released in the absence of synthesis. Protoplasts were obtained from which periplasmic penicillinase has been removed, and these protoplasts were capable of substantial growth and penicillinase synthesis without lysis. At pH 7.5, there was no net incorporation of penicillinase into the cell membrane; the enzyme released was almost entirely of the exo form and was roughly equivalent to the amount of new enzyme formed. At pH 6.0, there was some incorporation of penicillinase into the plasma membrane, and approximately half of the extracellular penicillinase was in the exo form; the remainder perhaps represented membrane fragments. In the presence of chloramphenicol, a small amount of penicillinase was released at pH 7.5 as the exo form; at pH 6.0, practically none was released. We suggest that, with the removal from protoplasts of the periplasmic penicillinase-containing particles, a restriction on secretion has been lifted. 相似文献
14.
A cell-wall fraction of the mycorrhizal fungus Amanita muscaria increased the chitinase activity in suspension-cultured cells of spruce (Picea abies (L.) Karst.) which is a frequent host of Amanita muscaria in nature. Chitinase activity was also increased in roots of spruce trees upon incubation with the fungal elicitor. Non-induced levels of chitinase activity in spruce were higher in suspension cells than in roots whereas the elicitorinduced increase of chitinase activity was higher in roots. Treatment of cells with hormones (auxins and cytokinin) resulted in a severalfold depression of enzyme activity. However, the chitinase activity of hormone-treated as well as hormone-free cells showed an elicitor-induced increase. Suspension cells of spruce secreted a large amount of enzyme into the medium. It is postulated that chitinases released from the host cells in an ectomycorrhizal system partly degrade the fungal cell walls, thus possibly facilitating the exchange of metabolites between the symbionts. 相似文献
15.
Nicole D’Souza Prajakta Joshi Snehal Kaginkar Subhojit Sen 《Plant Molecular Biology Reporter》2018,36(2):363-371
Chromatin maps by enzymatic digestion using micrococcal nuclease (MNase) have served as popular substrates for molecular epigenetic studies. Such analyses have been limited to cell wall minus mutants of Chlamydomonas reinhardtii because of the complications in nucleus isolation due to cell wall removal. Here, we describe an endogenous endonuclease in Chlamydomonas that is specifically released from the cells upon abrasion with beads (glass or zirconia), in the presence of detergents. The resulting in vivo digests obtained from both cell wall containing cc124/cc125 and mutant strains (cc400) are typical of repetitive nucleosomal fragments (MNase-like ladders) ranging from mono- to poly-nucleosomes, indicating that the nuclease acts preferentially on inter-nucleosomal linker DNA. We demonstrate that the endonuclease activity is strictly dependent on divalent cations (Ca+2?>?Mg+2) and can be inhibited by both Cu+2 and Zn+2 (5 mM) or by divalent cation chelators like EDTA. Detailed standardization reveals that the nuclease is released only when the cells are treated with detergents, implying that it might be membrane bound or vesicular and intracellular in nature. This endonuclease seems to accumulate in late log-phase cultures and probably has a role in generation of apoptotic ladders in Chlamydomonas. Further activity-based in-gel DNase analyses of whole cell extracts, using denaturing SDS-PAGE, revealed a single major ~?30-kDa band upon renaturation. We further characterized this endonuclease by partial purification using ammonium sulfate precipitation. Activity-based analysis revealed partial enrichment in the 50% (NH4)2SO4 peak fraction and also confirmed Ca dependence. Finally, adding back the released endonuclease from a detergent-treated supernatant of cc124 cells to intact cc400 cw ? cells as substrate resulted in a typical ladder formation in the latter, confirming the intracellular release of the nuclease and its activity. In summary, by controlled release of calcium-dependent endogenous endonucleases (altering vortex and detergent conditions), we can generate dose-dependent nucleosomal ladders for epigenetic analyses in Chlamydomonas, as an alternate to MNase-derived substrates. 相似文献
16.
Chitin synthesis in Candida albicans: comparison of yeast and hyphal forms. 总被引:14,自引:0,他引:14
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Chitin synthesis was studied in both yeast and hyphae of the dimorphic fungus Candida albicans. Incorporation of N-acetyl-d-[1-(3)H]glucosamine ([(3)H]GluNAc) into an acid-alkali-insoluble fraction was 10 times greater in hyphal-phase cells. A crude preparation of chitin synthetase was obtained from sonically treated protoplasts of both forms of Candida. Enzyme activity, which was determined by using [(14)C]UDP-GLuNAc as a substrate, was exclusively associated with the 80,000 x g pellet from sonically treated protoplasts of both forms. It was determined that enzyme activity (nanomoles of [(14)C]UDP-GluNAc incorporated per milligram of protein) was approximately 2 times greater in hyphae versus yeast cells. Enzyme activity in both yeast and hyphae increased six- to sevenfold when the enzyme preparations were preincubated with trypsin. A vacuolar fraction, obtained from yeast cells but not from hyphae, stimulated enzyme activity when incubated with either yeast or hyphal enzyme preparations. Membrane fractions from protoplasts coated with [(3)H]concanavalin A before disruption were isolated by Renografin density gradient centrifugation. Chitin synthetase activity was preferentially associated with the concanavalin A-labeled fraction, suggesting that the enzyme was located on the plasma membrane. In addition, enzyme activity in protoplasts treated with cold glutaraldehyde before disruption was significantly greater than in protoplasts that were sonically disrupted and then treated with cold glutaraldehyde, indicating that the enzyme resides on the inner side of the plasma membrane. 相似文献
17.
Separation of mesophyll protoplasts and bundle sheath cells from maize leaves for photosynthetic studies 总被引:6,自引:34,他引:6
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Mesophyll protoplasts and bundle sheath strands of maize (Zea mays L.) leaves have been isolated by enzymatic digestion with cellulase. Mesophyll protoplasts, enzymatically released from maize leaf segments, were further purified by use of a polyethylene glycol-dextran liquid-liquid two phase system. Bundle sheath strands released from the leaf segments were isolated using filtration techniques. Light and electron microscopy show separation of the mesophyll cell protoplasts from bundle sheath strands. Two varieties of maize isolated mesophyll protoplasts had chlorophyll a/b ratios of 3.1 and 3.3, whereas isolated bundle sheath strands had chlorophyll a/b ratios of 6.2 and 6.6. Based on the chlorophyll a/b ratios in mesophyll protoplasts, bundle sheath cells, and whole leaf extracts, approximately 60% of the chlorophyll in the maize leaves would be in mesophyll cells and 40% in bundle sheath cells. The purity of the preparations was also evident from the exclusive localization of phosphopyruvate carboxylase (EC 4.1.1.31) and NADP-dependent malate dehydrogenase (EC 1.1.1) in mesophyll cells and ribulose 1,5-diphosphate carboxylase (EC 4.1.1.39), phosphoribulokinase (EC 2.7.1.19), and “malic enzyme” (EC 1.1.1.40) in bundle sheath cells. NADP-glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) was found in both mesophyll and bundle sheath cells, while ribose 5-phosphate isomerase (EC 5.3.1.6) was primarily found in bundle sheath cells. In comparison to the enzyme activities in the whole leaf extract, there was about 90% recovery of the mesophyll enzymes and 65% recovery of the bundle sheath enzymes in the cellular preparations. 相似文献
18.
Vacuoles isolated from Nicotiana rustica var brasilia have been shown to contain significant levels of glycosidase activity when assayed using p-nitrophenyl-glycosides as substrates. The substrate specificity for the glycosidases in the vacuolar fraction closely paralleled that found in the protoplasts, and the leaf tissue from which the vacuoles were isolated. The substrate specificity of the vacuolar enzyme(s) was different from glycosidic activity found in the commercial digestive enzyme preparations used to isolate the protoplasts from leaf tissue. It was demonstrated that 70 to 90% of the glycosidases that were found in the protoplasts appeared to be localized within the vacuole, when the p-nitrophenyl substrates α- and β-;d-galactose, β-d-glucose, and α-d-mannose were used. Neither the vacuolar nor the protoplast enzymes were active towards the naturally occurring phenolic glycoside, rutin. α-Mannosidase appears to be a valuable marker enzyme for vacuoles isolated from mesophyll leaf cells of tobacco. 相似文献
19.
A cell-bound staphylocoagulase could be detected in chemostat cultures of Staphylococcus aureus 104 under magnesium- and oxygen-limited growth conditions. A distribution study revealed that 81% of the enzyme was membrane-bound and could be optimally released by Triton X-100. The remaining part was located in the periplasmic space and was released during protoplasting of the organism. From inhibition studies with cerulenin, quinacrine, lincomycin and chloramphenicol, it was concluded that the cell-bound form was a precursor in the secretion of extracellular staphylocoagulase. The involvement of a lipid intermediate/exoprotein- releasing protease system in the secretion of staphylocoagulase, and of exoproteins in general, is discussed. 相似文献
20.
Formation of Protoplasts from Cultured Tobacco Cells and Arabidopsis thaliana by the Action of Cellulosomes and Pectate Lyase from Clostridium cellulovorans
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Yutaka Tamaru Sadaharu Ui Koichiro Murashima Akihiko Kosugi Helen Chan Roy H. Doi Bo Liu 《Applied microbiology》2002,68(5):2614-2618
The crude culture supernatants from Clostridium cellulovorans were tested for their ability to convert plant cells to protoplasts. The supernatants readily released protoplasts from cultured tobacco cells and Arabidopsis thaliana. The crude culture supernatant from pectin-grown cells was more active than supernatants from glucose-, cellobiose-, xylan-, and locust bean gum-grown cells. After removal of cellulosomes, the crude culture supernatant lost its protoplast formation activity. The protoplast formation activity of the crude culture supernatant from C. cellulovorans was more effective than those of commercial enzymes based on protein content. 相似文献