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Alkaline phosphatase, an enzyme secreted byBacillus intermedius S3-19 cells to the medium, was also detected in the cell wall, membrane, and cytoplasm. The relative content of alkaline
phosphatase in these cell compartments depended on the culture age and cultivation medium. The vegetative growth ofB. intermedius on 0.3% lactate was characterized by increased activity of extracellular and membrane-bound phosphatases. The increase in
lactate concentration to 3% did not affect the activity of membrane-bound phosphatase but led to a decrease in the activity
of the extracellular enzyme. Na2HPO4 at a concentration of 0.01 % diminished the activity of membrane-bound and extracellular phosphatases. CoCl2 at a concentration of 0.1 mM released membrane-bound phosphatase into the medium. By the onset of sporulation, phosphatase
was predominantly localized in the medium and in the cell wall. As is evident from zymograms, the multiple molecular forms
of phosphatase varied depending on its cellular localization and growth phase. 相似文献
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Sharipova MR Shakirov EV Balaban NP Gabdrakhmanova LA Shilova MA Rudenskaia GN Leshchinskaia IB 《Mikrobiologiia》2000,69(5):660-667
The activities of proteinases in the culture fluid and cellular fractions of Bacillus intermedius 3-19 grown under various conditions were studied. Thiol-dependent serine proteinase was the prevalent enzyme in the total pool of extracellular proteinases (70%); its catalytically active form was also detected in the cell membrane and, during active enzyme production, in the cell wall. Another enzyme, glutamyl endopeptidase (10% of the total pool), was detected in the cell membrane; it was also found in the cell wall and cytoplasm during active enzyme secretion into the growth medium. Production of these enzymes was maximal on medium containing inorganic phosphate and gelatin and decreased 2- to 4-fold on medium with glucose and lactate. The level of activity of extracellular enzymes correlated with that of corresponding membrane-bound proteins. The addition of CoCl2 (2 mM) into the medium caused an essential increase in extracellular glutamyl endopeptidase activity and promoted the release of the membrane-bound enzyme into the culture fluid. Proteolytic activity towards casein was also detected in the cytoplasm. The proteinases localized in the cytoplasm were shown to differ in their properties from those secreted. 相似文献
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The biosynthesis of extracellular alkaline phosphatase in the streptomycin-resistant strains Bacillus intermedius S3-19 and S7 in the presence in the medium of 5'-nucleoside monophosphates and different sources of carbon--glucose, sodium pyruvate, sodium lactate, or glycerol--was studied. It was established that, in the presence of mononucleotides, the content of extracellular alkaline phosphatase in both strains increased; the maximal effect was caused by 5'-AMP at a concentration of 20 micrograms/ml. In medium with a low orthophosphate content, where active biosynthesis of alkaline phosphatase occurred, 1% glucose and 0.5% pyruvate stimulated this process 2.5-4 times, and 2% sodium lactate and sodium pyruvate, on the contrary, inhibited it by 20-40%. Analysis of the dynamics of growth and accumulation of extracellular phosphatase in the presence of different sources of carbon in the medium gives evidence of an interrelationship between the biosynthesis of alkaline phosphatase and carbon metabolism in Bacillus intermedius. 相似文献
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I B Grishina I A Bolotina N G Esipova A G Pavlovski? A A Makarov 《Molekuliarnaia biologiia》1989,23(5):1455-1468
Two independently melting regions (energetic domains) were localized in Bacillus intermedius 7P ribonuclease by methods of circular dichroism and high resolution X-ray analysis: the lov-temperature melting domain, containing C-terminal region of the molecule with five strands in antiparallel beta-structure and the high-temperature melting alpha-helical domain in the N-terminal region. The contact between these domains is stabilized mainly by ionic interaction Asp-22 - Lys+-48. At pH 2.4 and 30.5 0 C, when the low-temperature domain melts, half of the beta-structure content in binase is destroyed though the alpha-helical structure content is conserved. It has been shown that in pH interval 2.4-4.8 at 15 0 C no changes in secondary structure and local surrounding of aromatic amino acid residues could be identified. Thus, the changes in ionic interactions in the binase molecule due to protonation of Asp side chain groups does not effect the secondary or tertiary structure, though it changes the energetical state of the binase molecule, revealing a change of number and size of energetic domains. 相似文献
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Excretion of alkaline phosphatase of Bacillus subtilis 总被引:10,自引:0,他引:10
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Localization of alkaline phosphatase in three gram-negative rumen bacteria 总被引:17,自引:5,他引:12
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Of the three species (Bacteroides ruminicola, B. succinogenes, and Megasphaera elsdenii) of anaerobic gram-negative rumen bacteria studied, only B. ruminicola produced significant amounts of alkaline phosphatase. This enzyme, which is constitutive, showed a greater affinity for p-nitrophenylphosphate than for sodium-beta-glycerophosphate and was shown to be located exclusively in the periplasmic space of log-phase cells. Small amounts of this enzyme were released from these cells in stationary-phase cultures, but washing in 0.01 M MgCl(2) and the production of spheroplasts by using lysozyme in 0.01 M MgCl(2) did not release significant amounts of the enzyme. Exposure to 0.2 M MgCl(2) did not release significant amounts of the periplasmic alkaline phosphatase of the cell, and when these cells were spheroplasted with lysozyme in 0.2 M MgCl(2) only 25% of the enzyme was released. Spheroplasts were formed spontaneously in aging cultures of B. ruminicola, but even these cells retained most of their periplasmic alkaline phosphatase. It was concluded that the alkaline phosphatase of B. ruminicola is firmly bound to a structural component within the periplasmic area of the cell wall and that the enzyme is released in large amounts only when the cells break down. The behavior of alkaline phosphatase in this bacterium contrasts with that of conventional periplasmic enzymes of aerobic bacteria, which are released upon conversion into spheroplasts by lysozyme and ethylenediaminetetraacetic acid and by other types of cell wall damage. All three species of bacteria studied here, as well as bacteria found in mixed populations in the rumen, have thick, complex layers external to the double-track layer of their cell walls. In addition, B. ruminicola produces a loose extracellular material. 相似文献
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Sporulation in Bacillus subtilis 168. Comparison of alkaline phosphatase from sporulating and vegetative cells 总被引:18,自引:5,他引:13
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1. The purification of the `vegetative' alkaline phosphatase of Bacillus subtilis 168 was simplified by ionic elution of the enzyme from intact cells. 2. The enzyme has a molecular weight of about 70000 and treatment of the enzyme with 10mm-hydrochloric acid or 6.0m-guanidine hydrochloride, β-mercaptoethanol (0.1m) gives rise to enzymically inactive subunits. 3. The amino acid composition of the enzyme was determined. The N-terminal residue determined by the DNS chloride method is glycine. 4. The properties of this enzyme were compared with the `sporulation' alkaline phosphatase of the same strain. 5. Although the `sporulation' enzyme differs from the `vegetative' enzyme in its physiology of appearance and apparent mRNA stability, an examination of properties of the enzymes revealed no differences. 6. The enzyme from both cell forms is bound to the particulate fraction of cell extracts, but can be solubilized by high concentrations of magnesium chloride; removal of the magnesium chloride, by dialysis, results in precipitation of both enzymes. Both enzymes can be removed from intact cells by ionic elution. 7. The `vegetative' and `sporulation' enzymes have identical pH optima, Km and Ki values and electrophoretic mobilities in cellulose acetate. 8. Their half-life is 28min at 65°C and their Q10 is 1.25. 9. The molecular size determined by gel filtration on Sephadex G-100 is about 69000. 10. `Vegetative' and `sporulation' forms gave precipitin lines that were continuous and non-spurred when tested against antiserum prepared against the `vegetative' enzyme. 11. The `sporulation' alkaline phosphatase appears to be associated with stage II of sporulation and appears to be induced by something specifically concerned in sporulation and not by phosphate starvation. 相似文献
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C S Adams 《Acta anatomica》1983,116(2):146-151
The activities of alkaline phosphatase and reduced nicotinamide adenine dinucleotide (NADH) diaphorase in the principal cells of the guinea pig epididymis were studied histochemically. Alkaline phosphatase activity was absent from the principal cells but was present in the basement membrane of the epididymal epithelium. NADH diaphorase activity was distributed throughout the cytoplasm of the principal cells in each epididymal segment. There was a gradual increase in NADH diaphorase activity from segments 1 through 7. Possible functions of alkaline phosphatase and NADH diaphorase in the epididymis are discussed. 相似文献
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Dr. Marilynn E. Etzler Edward H. Birkenmeier Florence Moog 《Histochemistry and cell biology》1969,20(2):99-104
Summary Recent studies on the alkaline phosphatases of the mouse duodenum have revealed the presence of two classes of isozymes, differing in immunochemical, electrophoretic, chromatographic, and kinetic properties. We have now examined the localization of these two types of phosphatases by the immunofluorescence technique. The use of antisera prepared against both types reveals that the isozymes of both classes are localized in the microvilli of the epithelial cells, and both are found from the villi tips to their bases. No significant intracellular localization of either class of phosphatase was observed.Supported by research grant HD03490 from the National Institute of Child Health and Human Development, U.S. Public Health Service. 相似文献
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Alkaline phosphatase activity appears to be altered when chondrocyte cultures are incubated with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). This study examined whether the hormone-responsive enzyme activity is associated with alkaline phosphatase-enriched extracellular membrane organelles called matrix vesicles. Confluent, third passage cultures of rat costochondral growth cartilage (GC) or resting zone chondrocytes (RC) were incubated with 1,25-(OH)2D3 or 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3) and enzyme specific activity was assayed in the cell layer or in isolated matrix vesicle and plasma membrane fractions. Alkaline phosphatase-specific activity in the matrix vesicles was enriched at least 2-fold over that of the plasma membrane and 10-fold over that of the cell layer. Matrix vesicle alkaline phosphatase was stimulated by 1,25-(OH)2D3 in GC cultures and by 24,25-(OH)2D3 in RC cultures. The cell layer failed to reveal these subtle differences. 1,25-(OH)2D3 increased GC enzyme activity but the effect was one-half that observed in the matrix vesicles alone. No effect of 1,25-(OH)2D3 on enzyme activity of the RC cell layer or of 24,25-(OH)2D3 on either GC or RC cell layers was detected. Thus, response to the metabolites is dependent on chondrocytic differentiation and is site specific: the matrix vesicle fraction is targeted and not the cells per se. 相似文献
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S Githens 《The journal of histochemistry and cytochemistry》1983,31(5):697-705
The light microscopic histochemical localization of alkaline phosphatase (APase) and adenosine triphosphatase (ATPase) in the mammalian pancreas is reviewed. Capillary endothelial cells usually exhibit both enzymes. ATPase is usually present in endocrine and acinar cells and absent in duct cells. APase is often found in islet cells but is almost always absent in exocrine cells. These enzymes should therefore be used with caution as markers for specific cells and organelles of the pancreas, and for monitoring diseases that might lead to the release of these enzymes from the pancreas. 相似文献
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Znamenskaia LV Vershinina OA Vershinina VI Krasnov SI Kostrov SV Akimkina TV Leshchinskaia IB Hartley RW 《Molekuliarnaia genetika, mikrobiologiia i virusologiia》1999,(1):12-17
Plasmids with whole genes for ribonucleases from B. intermedius (binase) and B. pumilis (RNase Bp) assembled with the whole gene of barstar, a specific intracellular inhibitor, are constructed. The resultant plasmids pMZ55 and pMZ56 effectively express binase and RNase Bp genes in B. subtilis cells. A medium for maximum expression of RNase genes by recombinant strains is developed. The expression of binase and RNase Bp genes in B. subtilis cells is negatively regulated by exogenic inorganic phosphate. 相似文献
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