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1.
The plasma membrane of Streptococcus cremoris: isolation and partial characterization 总被引:1,自引:0,他引:1
Marja A. Rimpiläinen Kaarina Niskasaari† Katri M. S. Juutinen† Eeva-Liisa Nurmiaho-Lassila‡ Raili I. Forsean† 《Journal of applied microbiology》1986,60(5):389-394
Plasma membrane was isolated from Streptococcus cremoris using mutanolysin from a streptomycete as the cell wall-degrading enzyme and phenylmethylsulfonyl fluoride as protease inhibitor. The specific activity of membrane-bound enzyme, adenosine triphosphatase (ATPase), was 4 μmol/mg protein per min, which was 5–10 times higher than the activity found in other fractions obtained during the isolation procedure. The number of polypeptides in the plasma membrane was approximately 50 with molecular weights 13 500–100 000, minor changes in the polypeptide pattern were observed when the plasma membrane was isolated without a protease inhibitor. The chemical composition of the membrane preparation was 49.7% protein, 21.9% lipid, 5.1% aminosugars, 17.3% RNA and 0.03% DNA. Electron microscopic examination confirmed the membrane to be practically devoid of cell wall components. Our results indicate that the membrane integrity is well retained and therefore the membrane preparation is suitable for detailed studies on vectorial metabolism and its enzymes, e.g. ATPase. 相似文献
2.
J I de Vicente P del Valle F Busto D de Arriaga J Soler 《Biochimie et biologie cellulaire》1991,69(7):454-459
Mitochondrial F1-ATPase was purified from the mycelium of Phycomyces blakesleeanus NRRL 1555(-) and its kinetic characteristics were studied. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme reveals five bands (alpha, beta, gamma, delta, and epsilon) characteristic of the F1 portion with apparent molecular weights of 60,000, 53,000, 31,000, 25,000, and 21,000, respectively. The molecular weight of the native F1-ATPase from Phycomyces blakesleeanus was in agreement with the stoichiometry alpha 3 beta 3 gamma delta epsilon. The MgATP complex is the true substrate for ATPase activity which has a Km value of 0.15 mM. High concentrations of free ATP or free Mg2+ ions inhibit the ATPase activity. ADP appears to act as a negative allosteric effector with regard to MgATP hydrolysis, with the apparent Vmax remaining unchanged. 相似文献
3.
Beta-D-phosphogalactoside galactohydrolase from Streptococcus cremoris HP: purification and enzyme properties 总被引:8,自引:7,他引:1
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beta-d-phosphogalactoside galactohydrolase (beta-PGal) was isolated and purified from cell-free extracts of Streptococcus cremoris HP to apparent homogeneity to gel electrophoresis. Using the chromogenic o-nitrophenol-beta-d-galactopyranoside-6-phosphate as substrate, the purified enzyme exhibited a specific activity of 18.71 U/mg of protein and K(m) and V(max) values of 5.88 x 10(-4) M and 23.8 mumol of o-nitrophenol liberated per min per mg of protein, respectively. d-Galactose-6-phosphate was a weak competitive inhibitor of beta-PGal. Activity was relatively heat resistant and was maximal from pH 5.0 to 8.0 and over a temperature range of 45 to 52 C. Dithiothreitol, ethylenediaminetetraacetic acid, and citrate stimulated beta-PGal activity, whereas Mg(2+), Li(1+), and p-hydroxymercuribenzoate were inhibitory. Molecular weight of the enzyme was estimated at 6.76 x 10(4). Amino acid composition was similar to other beta-phosphogalactosidases previously investigated, with the exception that the S. cremoris enzyme contains a small amount of half cystine. 相似文献
4.
H. Fukushima J. Kelstrup S. Fukushima T. Umemoto H. Sagawa 《Antonie van Leeuwenhoek》1983,49(1):41-50
An extracellular bactericidal substance was isolated from the supernatant of Streptococcus mutans Rm-10 culture fluid and partially purified with 60% ammonium sulfate precipitation, differential centrifugation, and gel filtration on Sephadex G-200. There was a good correlation of the sensitivity profiles of indicator strains whether assayed on solid medium or with purified material from cell-free culture fluid, indicating that the same inhibitory substance is produced on solid medium and in broth. Vapor from organic solvents such as chloroform, acetone, ethanol, and ether as well as heat treatment at 100 degrees C for 30 min had little effect on the bactericidal factor. It was sensitive to trypsin and pronase and resistant to deoxyribonuclease, ribonuclease, lysozyme, and phospholipase C. The inhibitor was not infective, and electron microscopic studies failed to reveal phage or phage-like particles in concentrated solutions of the bactericidal material. The results indicate that the extracellular bactericidal substance is indeed a bacteriocin. Activity in broth cultures reached a maximum only after exponential growth had ceased. It was active against other streptococcal strains as well as strains of Actinomyces naeslundii, A. viscosus, Bacillus subtilis and Staphylococcus aureus, but not against strains of Fusobacterium nucleatum and Escherichia coli. 相似文献
5.
《The International journal of biochemistry》1986,18(1):93-98
- 1.1. The plasma membrane of slime-forming, encapsulated Streptococcus cremoris from “viili” was isolated in hypotonie conditions in the presence of lysozyme (EC 3.2.1.17) using density gradient centrifugation as the last purification step.
- 2.2. The membrane yield was 15.8% of wet weight cells and the preparation contained 64.4% protein. 19.1% carbohydrate, 5.8% aminosugars, 5.1% RNA and 0.07% DNA.
- 3.3. Buffered 1% (w/v) Triton X-100 solubilized 33.6% of membrane proteins. The number of polypeptides detected by SDS-polyacrylamide gel electrophoresis was 59 when the membrane was isolated without a protease inhibitor and 44 in the presence of a protease inhibitor.
- 4.4. The molecular weights of the polypeptides varied from 13,500 to 100,000.
- 5.5. Ultrathin-layer electrofocusing analysis revealed the range of protein pi values to be between 3.50 and 5.85 concerning 77.3% of proteins and between pI 5.85 and 8.15 concerning 18.2% of proteins.
- 6.6. The isoelectric point of the only basic protein component was 9.3.
6.
A large-scale purification procedure was developed to isolate the five subunits of F1-ATPase from pig heart mitochondria. The previously described procedure (Williams, N. and Pedersen, P.L. (1986) Methods Enzymol. 126, 484-489) to dissociate the rat liver F1-ATPase by cold treatment followed by warming at 37 degrees C has been adapted for the pig heart enzyme. Removal of endogenous nucleotides from that enzyme before dissociation led to the efficient separation of the alpha and gamma subunits from beta, delta and epsilon subunits. The beta subunit was purified in the hundred-milligram range by anion-exchange chromatography in the absence of any denaturing agent. This subunit was free from any bound nucleotide and almost no ATPase and adenylate kinase-like activities were detected. The delta and epsilon subunits were purified by reversed-phase chromatography (RP-HPLC) in the milligram range. As recently reported (Penin, F., Deléage, G., Gagliardi, D., Roux, B. and Gautheron, D.C. (1990) Biochemistry 29, 9358-9364), these purified subunits kept biophysical features of folded proteins and their ability to reconstitute the tight delta epsilon complex. The alpha and gamma subunits remained poorly soluble and required dissociation by 8 M guanidinium chloride prior to their purification by RP-HPLC. In addition, characterizations of the five subunits by IEF and SDS-polyacrylamide gel electrophoresis are reported, as well as ultraviolet spectra and solubility properties of the beta, delta and epsilon subunits. 相似文献
7.
A simple and high yield purification procedure for the isolation of F0F1-ATPase from spinach leaf mitochondria has been developed. This is the first report concerning purification and composition of the plant mitochondrial F0F1-ATPase. The enzyme is selectively extracted from inner membrane vesicles with the zwitterionic detergent, 3-[(3-cholamidopropyl) dimethyl ammonio]-1- propane sulfonate (CHAPS). The purified enzyme exhibits a high oligomycin-sensitive ATPase activity (3,6 mumol.min-1.mg-1). SDS-PAGE of the purified F0F1-ATPase complex reveals protein bands of molecular masses of 54 kDa (F1 alpha,beta), 33 kDa (F1 gamma), 28 kDa, 23 kDa, 21 kDa (F1 delta), 18.5 kDa, 15 kDa, 10.5 kDa, 9.5 kDa (F1 epsilon) and 8.5 kDa. All polypeptides migrate as one complex in a polyacrylamide gradient gel under non-denaturing conditions in the presence of 0.1% Triton X-100. Five polypeptides could be identified as subunits of F1. Polypeptides of molecular masses 28 kDa, 23 kDa, 18.5 kDa, 15 kDa, 10.5 kDa, 9.5 kDa and 8.5 kDa constitute the F0 part of the complex. Our results show that polypeptide composition of the plant mitochondrial F0 differs from other eukaryotic F0 of yeast, mammals and chloroplasts. 相似文献
8.
Reiner Gromes Harry Schwartz Martin Heinrich Walther Johannssen 《Applied microbiology and biotechnology》1991,35(4):491-495
Summary Several yeast strains were assayed for occurence of nitrate reductase after growth in a defined medium with nitrate as the sole nitrogen source, Candida boidinii DSM 70026, showing the highest specific activity, was further investigated. The procedures for yeast fermentation and nitrate reductase purfication are described in detail. Nitrate reductase from this yeast was characterized as NAD(P)H: nitrate oxidoreductase (E.C.1.6.6.2). The enzyme activity with NADH (NADPH) was highest at pH 7.0 (7.1) and 30° C (25° C). The values of K
m determinations with NADH/NADPH were both 4 × 10–4 mol/l; values for the substrate inhibition constant (K
i) were 6 × 10–4 mol/l. The molecular mass of the native enzyme was estimated by gel permeation chromatography to be approximately 350 kDa.
Offprint requests to: R. Gromes 相似文献
9.
Cereolysin: production, purification and partial characterization 总被引:21,自引:0,他引:21
10.
A method is described for purification of (Na+, K+)-ATPase which yielded approximately 60 mg of enzyme from 800 g of cardiac muscle with specific activities ranging from 340 to 400 mumol inorganic phosphate/mg protein per h (units/mg). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated the presence of a major 94 000 dalton polypeptide and four or five lesser components, one of which was a glycoprotein with an apparent molecular weight of 58 000. The enzyme preparation bound 600-700 pmol of [3H]ouabain/mg protein when incubated in the presence of either Mg2+ plus Pi, or Mg2+ plus ATP plus Na+, and incorporated more than 600 pmol 32P/mg protein when incubated with gamma-32P-labelled ATP in the presence of Mg2+ and Na+. The preparation is approximately 35% pure. 相似文献
11.
Purification and characterization of the F1-ATPase from Clostridium thermoaceticum. 总被引:6,自引:6,他引:0
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The F1 portion of the H+-ATPase from Clostridium thermoaceticum was purified to homogeneity by solubilization at low ionic strength, ion-exchange chromatography, and gel filtration. The last indicated the Mr to be 370,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the pure enzyme revealed four bands with Mr corresponding to 60,000, 55,000, 37,000, and 17,000 in an apparent molar ratio of 3:3:1:1. The purified enzyme would bind to stripped membranes to reconstitute dicyclohexylcarbodiimide-sensitive ATPase activity. Phosphohydrolase activity, measured at 58 degrees C, was optimal at pH 8.5. In the presence of a 1 mM excess of Mg2+ over the concentration of ATP, the Km for ATP was 0.4 mM, and the Vmax was 6.7 mumol min-1 mg-1. Unlike the membrane-bound F1F0 complex, the F1-ATPase was relatively insensitive to the inhibitors dicyclohexylcarbodiimide and tributyltin chloride. Both the complex and the F1-ATPase were inhibited by quercetin, azide, 7-chloro-4-nitro-benz-2-oxa-1,3-diazole, and free magnesium, and both were stimulated by primary alcohols and sulfite. In whole cells, the F1F0-ATPase catalyzed the synthesis of ATP in response to a pH gradient. 相似文献
12.
P. Khanna S. Sivakami Sundari N. Jothi Kumar 《World journal of microbiology & biotechnology》1995,11(2):242-243
AnAspergillus sp., isolated from a rubbish dump, produced 10.6 IU ml-1 xylanase activity. Two xylanases were recognized and each was purified to homogeneity by two-stage chromatography on DEAE-and CM-Sepharose. Xylanase I had a pI of 7.2 and anM
r of 26 kDa whereas xylanase II had a pI of 4.7 and anM
r of 21 kDa. At 50°C, xylanase I was stable for 2.5 h but xylanase II was only stable for 1 h.P. Khanna is with the National Environmental Engineering Research Institute, Nehru Marg, Nagpur 440 020, India. S. Sivakami Sundari and N. Jothi Kumar are with the National Environmental Engineering Research Institute, Madras Zonal Laboratory, CSIR Madras Complex, Taramani 600 113, India. 相似文献
13.
14.
Previously identified mutations in subunits a and b of the F0 sector of the F1F0-ATPase from Escherichia coli are further characterized by isolating detergent-solubilized, partially purified F1F0 complexes from cells bearing these mutations. The composition of the various F1F0 complexes was judged by quantitating the amount of each subunit present in the detergent-solubilized preparations. The composition of the F0 sectors containing altered polypeptides was determined by quantitating the F0 subunits that were immunoprecipitated by antibodies directed against the F1 portion. In this way, the relative amounts of F0 subunits (a, b, c) which survived the isolation procedure bound to F1 were determined for each mutation. This analysis indicates that both missense mutations in subunit a (aser206----leu and ahis245----tyr) resulted in the isolation of F1F0 complexes with normal subunit composition. The nonsense mutation in subunit a (atyr235----end) resulted in isolation of a complex containing the b and c subunits. The bgly131----asp mutation in the b subunit results in an F0 complex which does not assemble or survive the isolation. The isolated F1F0 complex containing the mutation bgly9----asp in the b subunit was defective in two regards: first, a reduction in F1 content relative to F0 and second, the absence of the a subunit. Immunoprecipitations of this preparation demonstrated that F1 interacts with both c and mutant b subunits. A strain carrying the mutation, bgly9----asp, and the compensating suppressor mutation apro240----leu (previously shown to be partially unc+) yielded an F1F0 ++ complex that remained partially defective in F1 binding to F0 but normal in the subunit composition of the F0 sector. The assembly, structure, and function of the F1F0-ATPase is discussed. 相似文献
15.
Cell surface proteins of two slime-forming, encapsulated Streptococcus cremoris strains, MLS96 and T5 from the fermented milk product viili, were extracted with the non-ionic detergent Triton X-100. The isolated protein antigens were characterized by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immuno-blotting with antisera produced against whole Strep. cremoris cells. When protein profiles of these strains were compared, seven prominent polypeptides were found common to both and were recognized by both antisera. Five of these polypeptides with molecular weights of 70000, 54000, 50000, 47000 and 40000 were identified as cell wall components. The remaining two polypeptides with molecular weights of 42000 and 26000 are being studied further in connection with slime formation for which modified Triton X-100 extraction provides a suitable method for isolation of the surface-associated antigens of lactic streptococci. 相似文献
16.
17.
Using molecular dynamics, we study the unbinding of ATP in F(1)-ATPase from its tight binding state to its weak binding state. The calculations are made feasible through use of interpolated atomic structures from Wang and Oster [Nature 1998, 396: 279-282]. These structures are applied to atoms distant from the catalytic site. The forces from these distant atoms gradually drive a large primary region through a series of sixteen equilibrated steps that trace the hinge bending conformational change in the beta-subunit that drives rotation of gamma-subunit. As the rotation progresses, we find a sequential weakening and breaking of the hydrogen bonds between the ATP molecule and the alpha- and beta-subunits of the ATPase. This finding agrees with the "binding-zipper" model [Oster and Wang, BIOCHIM: Biophys. Acta 2000, 1458: 482-510.] In this model, the progressive formation of the hydrogen bonds is the energy source driving the rotation of the gamma-shaft during hydrolysis. Conversely, the corresponding sequential breaking of these bonds is driven by rotation of the shaft during ATP synthesis. Our results for the energetics during rotation suggest that the nucleotide's coordination with Mg(2+) during binding and release is necessary to account for the observed high efficiency of the motor. 相似文献
18.
A facile and rapid purification procedure, based upon the heat denaturation of extraneous proteins and GMP-Sepharose affinity chromatography, has been used to purify hypoxanthine phosphoribosyltransferase from human brain. A homogeneous enzyme preparation, as judged by sodium dodecyl sulfate and gradient polyacrylamide gel electrophoresis, was obtained. The subunit molecular weight of the enzyme was estimated as 24,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The native molecular weight, determined by gradient gel electrophoresis, was approximately 100,000. These results suggest human brain hypoxanthine phosphoribosyltransferase is a tetramer, consistent with recent results reported for the human erythrocyte enzyme. At least three charge variant forms of the human brain enzyme were distinguished by nondenaturing polyacrylamide gel electrophoresis, electrofocusing, and chromatofocusing. Acidic pI values of approximately 5.7, 5.5, and 5.0 were estimated for the three major species. 相似文献
19.
Inhibitory activity directed against metalloenzymes has been highly purified from extracts of red kidney beans (Phaseolus vulgaris). The inhibitor is a substance of small molecular weight and appears to be a chelator of Zn2+. One milligram of the preparation inhibited 23 milligrams carboxypeptidase A. The inhibitor also strongly inhibited carboxypeptidase B and alkaline phosphatase and could activate phosphoglucomutase that had previously been inactivated with Zn2+. The isoelectric point of the inhibitor is 4.7. The inhibitor activity was abolished by preincubation with Zn2+, Ni2+, Co2+, or Cu2+. The mechanism of inhibition of carboxypeptidases and alkaline phosphatase by the bean inhibitor is apparently due to the complexing and complete removal of Zn2+ from the enzymes. 相似文献
20.
N-acetyl-beta-D-hexosaminidase A from rat urine: partial purification and characterization 总被引:1,自引:0,他引:1
C Sánchez-Bernal J Martín-Barrientos J A Rodríguez-Hernández J A Cabezas 《Biochimie》1988,70(2):227-236
N-Acetyl-beta-D-hexosaminidase A was purified from rat urine by ion-exchange chromatography on DEAE-cellulose, followed by concanavalin A chromatography, and finally by chromatography on 2-acetamido-N-(epsilon-aminocaproyl)-2-deoxy-beta-glucosylamine-Se pharose 4B. The enzyme was purified 482-fold with a yield of about 7%. The optimal pH was 4.5 for N-acetyl-glucosaminidase activity and 4.0-4.5 for N-acetylgalactosaminidase activity. The enzyme was heat-labile and stable from pH 4.5 to pH 7.0 but it was very unstable at lower pH values. Km values were 0.55 mM and 0.059 mM, respectively. The glycoprotein nature of the enzyme was deduced from its behavior on concanavalin A. The effect of some carbohydrates and ionic compounds on the activities of the enzyme was studied. When N-acetyl-D-glucosaminolactone and N-acetyl-D-galactosaminolactone were used as inhibitors, Ki values were also calculated. 相似文献