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1.
ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity was shown in the soluble fraction of rat liver micochondria. Two molecular forms (ATPase 1 and 2) were isolated. ATPase 1 has already been studied. The present paper deals with the purification method of ATPase 2 which was achieved by the following steps: (NH4)2SO4 precipitation. DEAE-cellulose chromatography, hydroxyapatite chromatography, Sephadex G100 filtration and AMP-Sepharose affinity chromatography. The purified protein was characterized by bidimensional polyacrylamide gel electrophoresis. Molecular weight evaluated by SDS-polyacrylamide gel electrophoresis and Sephadex G100 gel filtration was found to be 61 500 +/- 3000.  相似文献   

2.
Although human liver contains glucosaminephosphate synthase (glucosaminephosphate isomerase (glutamine-forming), EC 5.3.1.19), its activity is rapidly lost during the course of extraction. The inactivation, however, is largely prevented if the extraction medium contains isopropanol at 1% concentration; using these "stabilized" extracts, the glucosaminephosphate synthase activity of human liver has been shown to be similar to the activity previously reported in rat liver. The enzyme precipitated from these extracts by (NH4)2SO4 is inhibited by UDP-N-acetylglucosamine, the concentration required to produce a half-maximal inhibition being 6 muM. These results seem to be sufficient to postulate that glucosaminephosphate synthase is important for UDP-N-acetylglucosamine synthesis in human liver. In contrast to the rat liver enzyme, the (NH4)2SO4-precipitated human liver enzyme is resistant to trypsin and undergoes no conversion reaction when incubated with glucose 6-phosphate.  相似文献   

3.
The hydroxymethylglutaryl-coenzyme A reductase (mevalonate:NADP+ oxidoreductase, EC 1.1.1.34) system in Fusarium oxysporum, a soil inhabiting plant pathogen, has been examined. Two forms of the enzyme catalyzing the conversion of hydroxymethylglutaryl-coenzyme A were obtained in the supernatant after precipitation at 75% (NH4)2SO4 saturation of the soluble culture extract which was previously separated from cell wall, mitochondria and microsomes. The two forms of the enzyme were separated electrophoretically. A third form, contained in the precipitate obtained at 35--75% (NH4)2SO4 saturation of the same extract, was further purified by Sephadex G-50 column chromatography. This purified form moved as a single band in sodium dodecyl sulphate electrophoresis and in immunological tests and has a molecular weight of 11 000. The apparent Michaelis constant for the substrate hydroxymethylglutaryl-coenzyme A is 21 micron at 2 micron NADP. NADPH is a more efficient reductant on a molar basis than NADH for the deacylation of the hydroxymethylglutaryl-coenzyme A substrate. Optimum activity of the enzyme was obtained at pH 7.4 and 37 degrees C. The enzyme demonstrated no cold sensitivity but rather was more stable at 4 degrees C than at 25 degrees C. The protection with dithiothreitol, though minimal compared to other systems, was more effective at the higher temperature.  相似文献   

4.
Various ammonium sulfate concentrations and reaction conditions were employed in the fractionation of sera from rabbits, sheep, horses, and goats. Precipitates and supernatant fluids were analyzed by electrophoresis to study the effects of the controlled variables. At room temperature, the third precipitate in 35% saturated (NH(4))(2)SO(4) was the best fraction from both rabbit and sheep sera; 80 to 90% of the gamma globulins were recovered. The second and third precipitates of horse sera proteins in 30% saturated (NH(4))(2)SO(4) were both satisfactory, but only 44% of the gamma globulin was recovered after three precipitations. Goat sera yielded a very satisfactory fraction; 80% of the gamma globulin was recovered after two precipitations-the first in 30% and the second in 45% saturated (NH(4))(2)SO(4). The composition of these fractions was not influenced by the pH of the sulfate solutions (pH 5.8 and 7.2), by a range of normal room temperatures (20 to 30 C), or by diluting the sera before fractionation. Crude globulins and fluorescein isothiocyanate-labeled globulins were successfully refractionated by one precipitation in the optimal sulfate concentration for the appropriate animal species. The refractionated products contained considerably less beta and alpha globulins than did the original crude fractions and little or no albumin.  相似文献   

5.
Methane oxidation by cell-free extracts of Methylococcus capsulatus   总被引:8,自引:0,他引:8  
The separation of two distinct forms of cytochrome P450 from adrenal cortex mitochondria has been achieved by the following steps; (1) lyophilisation (2) iso-octane extraction, (3) (NH(4))(2)SO(4) fractionation in the presence of sodium cholate. The fraction precipitating between 25-35 percent (NH(4))(2)SO(4) gave a difference spectrum with 11-deoxycorticosterone (11-DOC) but not with 20alpha-hydroxycholesterol (20alpha-HOC). This fraction showed high 11beta-hydroxylase activity but low activity for side chain cleavage of cholesterol (S.C.C.). The fraction precipitating between 45-60 percent (NH(4))(2)SO(4) gave a difference spectrum with 20alpha-HOC but not with 11-DOC and exhibited high S.C.C. activity but low 11beta-hydroxylase activity. The absorption spectrum of the 45-60 percent fraction indicated a preponderance of high spin hemoprotein (lambda(max) 395 nm).  相似文献   

6.
The purification of cholinesterase from horse serum   总被引:2,自引:1,他引:1       下载免费PDF全文
A relatively simple method is described by which cholinesterase was purified about 19000-fold starting from horse serum. Typically 20 litres of serum were processed to yield 15-18mg of electrophoretically pure cholinesterase in the form of an active salt-free dry powder. The method included two stages: fractionation with (NH(4))(2)SO(4) and ion-exchange chromatography. The (NH(4))(2)SO(4) stage included, in principle, the acid (pH3) step of the Strelitz (1944) procedure. The step took advantage of the stabilizing effect that 33%-satd. (NH(4))(2)SO(4) has on cholinesterase activity at pH3 and it is recognized that in the absence of (NH(4))(2)SO(4) the enzyme is rapidly destroyed at pH3. Cholinesterase was significantly more stable to pH3.0 at 2 degrees C than at 24 degrees C, and the acid step was done at both temperatures. The specific activities of the final products obtained by way of acid steps were the same at either temperature, thus indicating that the step has not harmed the enzyme active sites. The product from the first two stages was purified over 18000-fold and was 85-90% cholinesterase. The remaining impurities were removed by preparative gel electrophoresis. The product was about 40% more active and contained 40% more active sites per unit weight than electrophoretically pure cholinesterase prepared from partially purified commercial starting material. Although the number of active sites per molecule was not determined with certainty, a value of at least 3 and possibly 4 was indicated. The partial specific volumes were determined with a precision density meter, on the ultracentrifuge and from the amino acid and carbohydrate composition. The values by these independent methods were 0.688, 0.71 and 0.712ml/g, respectively. The amino acid and carbohydrate composition was determined. The cholinesterase contained 17.4% carbohydrate including 3.2% N-acetylneuraminic acid.  相似文献   

7.
1. An inhibitor of cytoplasmic guanine deaminase of rat liver was isolated from liver ;heavy mitochondrial' fraction after freezing and thawing and treatment with Triton X-100. 2. Submitochondrial fractionation revealed that the inhibitor was localized in the outer-membrane fraction. 3. The method of purification of inhibitor, involving precipitation with (NH(4))(2)SO(4) and chromatography on DEAE-cellulose, its precipitability by trichloroacetic acid and the pattern of absorption in the u.v. indicated that the inhibitor was a protein. In confirmation, tryptic digestion of the isolated material resulted in destruction of the inhibitor activity. The inhibitor was stable to acid, but labile to heat. 4. The isolated inhibitor required phosphatidylcholine (lecithin) for activity. Phosphatidylcholine also partially protected the inhibitor against heat inactivation. 5. When detergent treatment was omitted, the inhibitor activity of frozen mitochondria was precipitated by (NH(4))(2)SO(4) in a fully active form without supplementation with phosphatidylcholine, indicating that Triton X-100 ruptured the linkage between inhibitor and lipid. 6. A reconstituted sample of inhibitor-phosphatidylcholine complex was precipitated in a fully active form by dialysis against 2-mercaptoethanol, but treatment of the precipitate with NaCl yielded an extract which was inactive unless supplemented with fresh phosphatidylcholine. 7. We interpret the results as evidence that the inhibitor was present in vivo as a lipoprotein and that once the complex was dissociated by the action of detergent and the protein precipitated, there was an absolute need for exogenous phosphatidylcholine for its activity. The manner in which inhibitor associated with the outer membrane of rat liver mitochondria might regulate the activity of the enzyme in the supernatant has been suggested.  相似文献   

8.
9.
Glucosaminephosphate synthase (glucosaminephosphate isomerase (glutamine-forming), EC 5.3.1.19) prepared from rat liver by extraction in the presence of glucose 6-phosphate (Glc-6-P) followed by precipitation with (NH4)2SO4 is susceptible to digestion by trypsin. This enzyme, designated form A, can be converted to tryptic-insusceptible form B upon incubation with Glc-6-P or fructose 6-phosphate (Fru-6-P) at 37 degrees C. The two forms also differ in the degree of activation by dithiothreitol, the degree of inhibition by methyl-glyoxal and the behavior on DEAE-Sephadex and Sephadex G-200 column chromatography. During purification with DEAE-Sephadex followed by hydroxyapatite, form B is converted to form A if Fru-6-P is absent and form A to form B if Fru-6-P is present. The two forms are therefore intercovertible. Under the conditions of purification, form B is more stable than form A, since the purity and yield of the final product are greater with form B than with form A. These findings suggest that the two forms of glucosaminephosphate synthase differ conformationally and that the equilibrium position depends on the concentration of Fru-6-P. Glc-6-P is effective only when it gives rise to Fru-6-P by mediation of glucose-phosphate isomerase.  相似文献   

10.
Superoxide dismutase (SOD; EC 1.15.1.1) is an enzyme that protects against oxidative stress from superoxide radicals in living cells. This enzyme has been isolated, purified and partially characterized from chicken liver. The following steps were carried out in order to purify chicken liver SOD. Initially, the liver was homogenized and hemoglobin was removed. Subsequently protein precipitation was effected with (NH(4))(2)SO(4), methanol, (NH(4))(2)SO(4)-methanol and polyethylene glycol methods. The product from polyethylene glycol-3350 precipitation was found to have the highest SOD activity. Polyethylene glycol was removed by chromatography using a PD-10 column. After passing through an ultrafilter, the superoxide dismutase was fractionated by DEAE-ion chromatography and then Sephadex G-75 gel filtration chromatography. During this purification procedure, a specific activity of 4818.2 IU/mg was reached, corresponding to 285.8-fold purification. The purified enzyme, which was characterized as cyanide-sensitive SOD, contained two subunits having Cu and Zn elements with a molecular weight of 16000+/-500 for each. The optimum pH of purified CuZnSOD was determined to be 8.9. The enzyme was found to have good pH stability in the pH range 6.0-7.5 at 25 degrees C over a 2-h incubation period and displayed good thermal stability up to 45 degrees C at pH 7.4 over a 1-h incubation period. The SOD enzyme was not inhibited by DTT and beta-mercaptoethanol, but inhibited by CN(-) and H(2)O(2). In the presence of 2 mM iodoacetamide, the enzyme showed an approximately 40% activity loss. Finally, the inhibitory effect of ionic strength on SOD was also investigated.  相似文献   

11.
Phosphofructokinase from the epithelial cells of rat small intestine was characterized with respect to isoenzyme type in a comparison of its properties with those of the skeletal-muscle, brain and major liver isoenzymes by using five different techniques, namely electrophoresis on cellulose acetate and in polyacrylamide gels, chromatography on DEAE-cellulose, (NH4)2SO4 precipitation and immunotitration. When precautions were taken to inhibit the formation of active proteolytic artifacts by the action of endogenous proteinases, each technique revealed that rat intestinal mucosa contains only a single form of phosphofructokinase. The mucosal isoenzyme was found to be very similar to, although not identical with, the major liver isoenzyme and to be quite distinct from the skeletal-muscle isoenzyme when studied by the techniques of cellulose acetate electrophoresis, chromatography on DEAE-cellulose and immunotitration, whereas the converse was true when studied by the techniques of (NH4)2SO4 precipitation and polyacrylamide-gel electrophoresis. The mucosal isoenzyme was distinct from the brain isoenzyme when studied by each of the five techniques. Tsai & Kemp [(1973) J. Biol. Chem. 248, 785-792] reported that animal tissues contain three principal isoenzymes of phosphofructokinase, type A found as the sole isoenzyme in skeletal muscle, type B found as the major isoenzyme in liver and type C found as a significant isoenzyme in brain. Phosphofructokinase from mucosa is distinct from each of these isoenzymes. Following the nomenclature of Tsai & Kemp (1973), the isoenzyme from the mucosa of rat intestinal epithelial cells is designated phosphofructokinase D. The mucosal and liver isoenzymes behave so similarly with respect to their charge and immunological characteristics, on which the typing of isoenzymes is conventionally based, that it is likely that some tissues reported to contain the liver isoenzyme contain instead the mucosal isoenzyme.  相似文献   

12.
The Cl(-)/anion exchanger pendrin (SLC26A4) is expressed on the apical side of renal non-type A intercalated cells. The abundance of pendrin is reduced during metabolic acidosis induced by oral NH(4)Cl loading. More recently, it has been shown that pendrin expression is increased during conditions associated with decreased urinary Cl(-) excretion and decreased upon Cl(-) loading. Hence, it is unclear if pendrin regulation during NH(4)Cl-induced acidosis is primarily due the Cl(-) load or acidosis. Therefore, we treated mice to increase urinary acidification, induce metabolic acidosis, or provide an oral Cl(-) load and examined the systemic acid-base status, urinary acidification, urinary Cl(-) excretion, and pendrin abundance in the kidney. NaCl or NH(4)Cl increased urinary Cl(-) excretion, whereas (NH(4))(2)SO(4), Na(2)SO(4), and acetazolamide treatments decreased urinary Cl(-) excretion. NH(4)Cl, (NH(4))(2)SO(4), and acetazolamide caused metabolic acidosis and stimulated urinary net acid excretion. Pendrin expression was reduced under NaCl, NH(4)Cl, and (NH(4))(2)SO(4) loading and increased with the other treatments. (NH(4))(2)SO(4) and acetazolamide treatments reduced the relative number of pendrin-expressing cells in the collecting duct. In a second series, animals were kept for 1 and 2 wk on a low-protein (20%) diet or a high-protein (50%) diet. The high-protein diet slightly increased urinary Cl(-) excretion and strongly stimulated net acid excretion but did not alter pendrin expression. Thus, pendrin expression is primarily correlated with urinary Cl(-) excretion but not blood Cl(-). However, metabolic acidosis caused by acetazolamide or (NH(4))(2)SO(4) loading prevented the increase or even reduced pendrin expression despite low urinary Cl(-) excretion, suggesting an independent regulation by acid-base status.  相似文献   

13.
The potential of immunoaffinity chromatography as a means of purifying legumin from a wide range of Pisum (pea) types was assessed. The method required small amounts of highly purified legumin from a single Pisum type, and this was obtained by salting out with (NH4)2SO4 followed by zonal isoelectric precipitation, ion-exchange chromatography on DEAE-cellulose and sucrose-density-gradient centrifugation. Some physiocochemical properties of purified legumin were determined, a number of which (Strokes radius, subunit molecular weights, subunit N-terminal residues and subunit molar ratios) have not previously been reported for Pisum legumin. Examination of Pisum legumin by two-dimensional gel isoelectric focusing/electrophoresis indicated the existence of extensive subunit heterogeneity, and polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate showed apparent variation in the nature of this heterogeneity from one Pisum variety to another. Despite this variation, immunoaffinity chromatography on immobilized anti-legumin (which was prepared by affinity chromatography on the immubolized purified legumin from the single Pisum type) was shown to be a generally applicable method for the purification of undegraded legumin from a range of pisum types, including two primate lines.  相似文献   

14.
Alpha1-Antitrypsin was isolated from rabbit plasma by salting out with (NH4)2SO4 followed by ion-exchange chromatography either on DEAE-Sephadex or DEAE-cellulose (each at pH8.8 and 6.5), and affinity chromatography on Sepharose-Cibacron Blue and Sepharose-concanavalin A. The protein thus obtained was homogeneous during crossed immunoelectrophoresis by using an antiserum to whole rabbit plasma, but it migrated as two broad bands when electrophoresed in alkaline polyacrylamide gels. Under optimal loading conditions, two or three subcomponents could be distinguished in each band. The two major forms of rabbit alpha1-antitrypsin, designated components F and S, were separated by preparative polyacrylamide-gel electrophoresis, and some of their physico-chemical properties were established. Both forms reacted with trypsin at a molar ratio of 1:1. Their elution volumes from a Sephadex G-200 column were identical, corresponding to a mol.wt. of 58000; however, some heterogeneity was observed after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Isoelectric focusing in polyacrylamide gel in a pH 4-6 gradient revealed a multiple-band pattern for each form in the range of pH4.4-4.9. The two forms of rabbit alpha1-antitrypsin possessed the same N-terminal amino acid (glutamic acid) and had very similar amino acid and carbohydrate compositions.  相似文献   

15.
1. Enzymes catalysing the reaction between GSH and methylparathion (dimethyl p-nitrophenyl phosphorothionate), 1-chloro-2,4-dinitrobenzene and S-crotonyl-N-acetylcysteamine were separated by (NH(4))(2)SO(4) precipitation from homogenates of sheep, rat and mouse livers and from homogenates of cockroaches, houseflies and grass grubs. 2. Electrofocusing of the preparations from each of these species separated a number of zones, each of which catalysed the reaction of GSH with all three substrates. 3. Ion-exchange chromatography on CM-cellulose also separated a number of fractions in which activity towards the three substrates coincided. 4. In both separation methods patterns of the activities were consistent with the presence in all species of several GSH transferases each having a degree of cross specificity towards the three substrates.  相似文献   

16.
1. Gel-filtration of an extract from the liver of the local Hausa goat Capra hircus indicated the presence of two molecular forms of alkaline phosphatase (orthophosphoric monoester phosphohydrolase, E.C. 3.1.3.1.). 2. Cellulose acetate electrophoresis showed that the lower-molecular-weight form had a similar electrophoretic mobility to alpha 2-globulin from goat serum, whereas the higher-molecular-weight form had a similar electrophoretic mobility to gamma-globulin. 3. Only the lower-molecular-weight form was detected on electrophoresis of a liver extract which contained some residual n-butanol used in the extraction procedure, whereas dialysed acetone powder obtained from the liver extract contained both molecular-weight forms. 4. The partially purified enzyme showed maximum activity at pH 9.8, and was stimulated by Mg2+. 5. The enzyme was heat-labile, and was competitively inhibited by phosphate ions but uncompetitively inhibited by L-phenylalanine. 6. These results are discussed in terms of the properties of the enzyme from other sources.  相似文献   

17.
A procedure is described for the purification of bacterial flagella in the form of a filament-hook-basal body complex (intact flagella) free from detectable cell wall, membrane, or cytoplasmic material. Spheroplasts produced with lysozyme and ethylenediaminetetraacetic acid were lysed with Triton X-100, and the flagella were purified by (NH(4))(2)SO(4) precipitation, differential centrifugation, and CsCl gradient centrifugation. As much as 40% of the flagella were recovered, and they contained about one basal body per 4 to 6 mum of flagella. The same procedure developed for Escherichia coli was also successful for purifying intact flagella from Bacillus subtilis.  相似文献   

18.
1. Washed suspensions of two Achromobacter species (G2 and 2L), capable of growth upon 2- and 3-hydroxypyridine respectively as sources of C and N, rapidly oxidized their growth substrate pyridine-2,5-diol (2,5-dihydroxypyridine) and the putative ring-cleavage product maleamate without a lag. Suspensions derived from fumarate plus (NH(4))(2)SO(4) cultures were unable to do so. 2. Extracts of both bacteria oxidized pyridine-2,5-diol with the stoicheiometry of an oxygenase forming 1mol of NH(3)/mol of substrate. 3. Heat-treated extracts, however, formed maleamate and formate with little free NH(3). 4. The conversion of maleamate into maleate plus NH(3) by extracts of strain 2L, fractionated with (NH(4))(2)SO(4), and the metabolism of maleamate and maleate to fumarate by extracts of both strains demonstrated the existence of the enzymes catalysing each reaction of the maleamate pathway in these bacteria. 5. The pyridine-2,5-diol dioxygenase (mol.wt. approx. 340000) in extracts of these Achromobacter species required Fe(2+) (1.7mum) to restore full activity after dialysis or treatment with chelating agents; the enzyme from strain 2L also had a specific requirement for l-cysteine (6.7mm), which could not be replaced by GSH or dithiothreitol. 6. The oxygenase was strongly inhibited in a competitive manner by the isomeric pyridine-2,3- and -3,4-diols.  相似文献   

19.
The glycoprotein alpha-1-proteinase inhibitor (alpha-1-PI) is a member of the serpin super family that causes rapid and irreversible inhibition of redundant serine protease activity. A homogenous preparation of ovine alpha-1-PI, a 60 kDa protein was obtained by serially subjecting ovine serum to 40-70% (NH(4))(2)SO(4) precipitation, Blue Sepharose, size-exclusion, and concanavalin-A chromatography. Extensive insights into the trypsin, chymotrypsin, and elastase interaction with ovine alpha-1-PI, point towards the involvement of Phe(350) besides the largely conserved Met(356) in serine protease recognition and consequent inhibition. The N-terminal of C-terminal peptides cleaved on interaction with elastase, trypsin, and chymotrypsin prove the presence of diffused sub-sites in the vicinity of Met(356) and the strategically positioned Pro anchored peptide stretch. Further, human alpha-1-PI is more thermolabile compared to ovine alpha-1-PI, higher thermolability is mainly attributed to poorer glycosylation. The enzymatic deglycosylation of human and ovine alpha-1-PI results in diminished thermostability of the inhibitors, with sharp decrease in thermal transition temperatures but retaining their inhibitory potency. Homology modeling of the deduced amino acid sequence of ovine alpha-1-PI using the human alpha-1-PI template has been used to explain the observed inhibitor-protease interactions.  相似文献   

20.
Purification of pig synovial collagenase to high specific activity.   总被引:5,自引:0,他引:5       下载免费PDF全文
1. Pig synovium in tissue culture secretes a specific collagenase in a latent form. 2. The latent enzyme was concentrated by (NH4)2SO4 precipitation and activated with 4-aminophenylmercuric acetate, and the active enzyme was purified by chromatography on Ultrogel AcA44, DEAE-cellulose, heparin-Sepharose and a zinc-chelate medium to a specific activity of 53 400 units/mg. of protein. 3. The enzyme was shown to be essentially homogeneous by polyacrylamide-gel electrophoresis. 4. The purified collagenase digested collagen to give the characteristic three-quarter and one-quarter pieces.  相似文献   

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