共查询到20条相似文献,搜索用时 15 毫秒
1.
The non-phosphorylating organophosphorus compound triisopropyl phosphate, which is known to inhibit rabbit leucocyte locomotion, can stimulate the locomotion of guinea pig leucocytes under certain conditions. Different methods of preparing guinea pig leucocyte monolayers can give preparations with different proportions of motile cells. With preparations that contain relatively slowly moving cells triisopropyl phosphate increases the number of stationary cells without significantly affecting the speed of the cells that remain motile. Most rabbit leucocytes labelled with fluorescein-labelled concanavalin A form caps within 5–10 min at 37 °C. In contrast the rate of cap formation in guinea pig leucocytes is much slower and after 20 min many cells have only random patches. Triisopropyl phosphate accelerates cap formation in guinea pig leucocytes but not in rabbit leucocytes. The local anaesthetic nupercaine inhibits cap and patch formation in rabbit and guinea pig leucocytes. Inhibition of rabbit leucocyte locomotion is induced by concanavalin A at 1 μg/ml. These results are briefly related to the known effects of triisopropyl phosphate on the isolated leucocyte plasma membrane. 相似文献
2.
A M Woodin 《Experimental cell research》1974,89(1):23-30
The cation-sensitive p-nitrophenyl phosphatase of nerve membranes is inhibited by DFP and the non-phosphorylating analogue triisopropyl phosphate. The inhibition requires 5–20 mM concentrations and is partly irreversible. The inhibition is distinct from that produced by the non-ionic detergent lubrol. Under some conditions dithiothreitol can inhibit the enzyme activity; under other conditions it can protect it against inactivation. The extent of the inactivation by DFP and triisopropyl phosphate is reduced in the presence of dithiothreitol. It is suggested that the primary action of the organophosphorus compounds is to change the reactivity of membrane sulphydryl or disulphide groups. The relevance of these findings to the blocking of conduction in the axon are discussed. 相似文献
3.
A M Woodin 《Analytical biochemistry》1975,63(2):452-456
The p-nitrophenyl phosphatase activity of leukocyte membranes is dependent on the origin of the p-nitrophenyl phosphate used as substrate. Commercial samples contain stable inhibitors and recrystalized material contains an inhibitor that is decomposed by water. The (Mg2+-K+)-p-nitrophenyl phosphatase of nerve membranes is not dependent on the origin of the substrate. 相似文献
4.
Leukocidin, Tetraethylammonium Ions, and the Membrane Acyl Phosphatases in Relation to the Leukocyte Potassium Pump 总被引:4,自引:0,他引:4
The response of the leukocyte to leukocidin and its relevance to excitable and secreting tissues are described. New data are presented on the leukocyte membrane phosphatases and the action of tetraethylammonium ion (TEA) on the leukocyte. The leukocyte surface membrane lacks a cation-sensitive ATPase but possesses a potassium- and ouabain-sensitive p-nitrophenyl phosphatase. The p-nitrophenyl phosphatase shows peak activity at three pH values and the pH dependence and potassium sensitivity depend on the state of the membranes. In the presence of magnesium, potassium can stimulate over the range pH 6 to 8. The relation of the leukocyte p-nitrophenyl phosphatase to electrolyte control in the leukocyte and to the properties of cation-sensitive phosphatases in other cells suggests that the leukocyte enzyme is a component of an electrogenic potassium pump. Leukocidin stimulates the leukocyte p-nitrophenyl phosphatase under all the conditions studied. The effect is specific and occurs under conditions that induce cytotoxic effects in the cell. It is concluded that the potassium pump is the site of action of leukocidin. TEA prevents the effects of leukocidin by inhibiting the action of leukocidin and not the responses of the cell to injury. TEA does not inhibit the p-nitrophenyl phosphatase nor prevent its stimulation by leukocidin. The enhancement of leukocidin by diisopropylphosphofluoridate (DFP) is briefly described. It is concluded that TEA acts in the opposite way to DFP and blocks the ion pathway activated by leukocidin in the leukocyte potassium pump. 相似文献
5.
- 1.
- 1. The participation of phospholipids in the activity of the p-nitrophenyl phosphatase of the leucocyte membrane has been studied in relation to the mode of action of leucocidin and the unique character of the leucocyte enzyme. 相似文献
6.
Carbonic anhydrase III from rabbit muscle, a newly discovered major isoenzyme of carbonic anhydrase, has been found to be also a p-nitrophenyl phosphatase, an activity which is not associated with carbonic anhydrases I and II. The p-nitrophenyl phosphatase activity has been shown to chromatograph with the CO2 hydratase activity; both activities are associated with each of its sulfhydryl oxidation subforms; and both activities follow the same pattern of pH stability. This phosphomonoesterase activity of carbonic anhydrase III has an acidic pH optimum (<5.3); its true substrate appears to be the phosphomonoanion with a Km of 2.8 mm. It is competitively inhibited by the typical acid phosphatase inhibitors phosphate (Ki = 1.22 × 10?3M), arsenate (Ki = 1.17 × 10?3M), and molybdate (Ki = 1.34 × 10?7M), with these inhibitors having no effect on the CO2 hydratase or the p-nitrophenyl acetate esterase activities of carbonic anhydrase III. The p-nitrophenyl acetate esterase activity of carbonic anhydrase III, on the other hand, has the sigmoidal pH profile with an inflection at neutral pH, typical of carbonic anhydrases for all of their substrates, and is inhibitable by acetazolamide (a highly specific carbonic anhydrase inhibitor) to the same degree as the CO2 hydratase activity. The acid phosphatase-like activity of carbonic anhydrase III is slightly inhibited by acetazolamide at acidic pH, and inhibited to nearly the same degree at neutral pH. These data are taken to suggest that the phosphatase activity follows a mechanism different from that of the CO2 hydratase and p-nitrophenyl acetate esterase activities and that there is some overlap of the binding sites. 相似文献
7.
Phosphatase synthesis in Klebsiella (Aerobacter) aerogenes growing in continuous culture 总被引:5,自引:5,他引:0
1. Phosphatase synthesis was studied in Klebsiella aerogenes grown in a wide range of continuous-culture systems. 2. Maximum acid phosphatase synthesis was associated with nutrient-limited, particularly carbohydrate-limited, growth at a relatively low rate, glucose-limited cells exhibiting the highest activity. Compared with glucose as the carbon-limiting growth material, other sugars not only altered the activity but also changed the pH–activity profile of the enzyme(s). 3. The affinity of the acid phosphatase in glucose-limited cells towards p-nitrophenyl phosphate (Km 0.25–0.43mm) was similar to that of staphylococcal acid phosphatase but was ten times greater than that of the Escherichia coli enzyme. 4. PO43−-limitation derepressed alkaline phosphatase synthesis but the amounts of activity were largely independent of the carbon source used for growth. 5. The enzymes were further differentiated by the effect of adding inhibitors (F−, PO43−) and sugars to the reaction mixture during the assays. In particular, it was shown that adding glucose, but not other sugars, stimulated the rate of hydrolysis of p-nitrophenyl phosphate by the acid phosphatase in carbohydrate-limited cells at low pH values (<4.6) but inhibited it at high pH values (>4.6). Alkaline phosphatase activity was unaffected. 6. The function of phosphatases in general is discussed and possible mechanisms for the glucose effect are outlined. 相似文献
8.
The K+-dependent p-nitrophenylphosphatase activity catalyzed by purified (Na+ + K+)-ATPase from pig kidney shows substrate inhibition (Ki about 9.5 mM at 2.1 mM Mg2+). Potassium antagonizes and sodium favours this inhibition. In addition, K+ reduces the apparent affinity for substrate activation, whereas p-nitrophenyl phosphate reduces the apparent affinity for K+ activation. In the absence of Mg2+, p-nitrophenyl phosphate, as well as ATP, accelerates the release of Rb+ from the Rb+ occluded unphosphorylated enzyme. With no Mg2+ and with 0.5 mM KCl, trypsin inactivation of (Na+ + K+)-ATPase as a function of time follows a single exponential but is transformed into a double exponential when 1 mM ATP or 5 mM p-nitrophenyl phosphate are also present. In the presence of 3 mM MgCl2, 5 mM p-nitrophenyl phosphate and without KCl the trypsin inactivation pattern is that described for the E1 enzyme form; the addition of 10 mM KCl changes the pattern which, after about 6 min delay, follows a single exponential. These results suggest that (i) the shifting of the enzyme toward the E1 state is the basis for substrate inhibition of the p-nitrophenulphosphatase acitivy of (Na+ + K+)-ATPase, and (ii) the substrate site during phosphatase activity is distinct from the low-affinity ATP site. 相似文献
9.
A low-molecular-weight human liver acid phosphatase was purified 2580-fold to homogenity by a procedure involving ammonium sulfate fractionation, acid treatment, and SP-Sephadex ion-exchange chromatography with ion-affinity elution. The purified enzyme contains a single polypeptide chain and has a molecular weight of 14,400 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition of this enzyme (E) is reported. A pH dependence study using p-nitrophenyl phosphate as a substrate (S) revealed the effect of substrate ionization (pKa 5.2) and the participation of a group in the ES complex having a pKa value of 7.8. The enzyme is readily inactivated by sulfhydryl reagents such as heavy metal ions. Alkylation of the enzyme with iodoacetic acid and iodoacetamide causes complete inactivation of the enzyme and this inactivation is prevented by the presence of phosphate ion. The enzyme is also inactivated by treatment with diethyl pyrocarbonate; protection against this reagent is afforded by phosphate ion. The substrate specificity of this enzyme is unusual for an acid phosphatase. Of the many alkyl and aryl phosphomonoesters tested, the only possibly physiological substrate hydrolyzed by this enzyme was flavin mononucleotide, which exhibits a V which is 3-fold larger at pH 5.0 and 6-fold larger at pH 7.0 than that for p-nitrophenyl phosphate. However, the enzyme also catalyzes the hydrolysis of acetyl phosphate at pH 5.0 with a velocity eight times larger than that reported for an acyl phosphatase from human erythrocytes. 相似文献
10.
A specific alkaline phosphatase from Saccharomyces cerevisiae with protein phosphatase activity 总被引:1,自引:0,他引:1
Boriyana Tuleva Evgenia Vasileva-Tonkova Danka Galabova 《FEMS microbiology letters》1998,161(1):139-144
In this paper, specific PHO13 alkaline phosphatase from Saccharomyces cerevisiae was demonstrated to possess phosphoprotein phosphatase activity on the phosphoseryl proteins histone II-A and casein. The enzyme is a monomeric protein with molecular mass of 60 kDa and hydrolyzes p-nitrophenyl phosphate with maximal activity at pH 8.2 with strong dependence on Mg2+ ions and an apparent Km of 3.6×10−5 M. No other substrates tested except phosphorylated histone II-A and casein were hydrolyzed at any significant rate. These data suggest that the physiological role of the p-nitrophenyl phosphate-specific phosphatase may involve participation in reversible protein phosphorylation. 相似文献
11.
Properties of a Membrane-bound Phosphatase from the Thylakoids of Spinach Chloroplasts 总被引:3,自引:2,他引:1
下载免费PDF全文
![点击此处可从《Plant physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A 3-phosphoglycerate phosphatase activity of about 2 micromoles per minute per milligram chlorophyll is associated with the thylakoid membranes of spinach chloroplasts. The Km for 3-phosphoglycerate is 3 millimolar. The enzyme can be solubilized from thylakoid membranes by treatment with 0.33 molar MgCl2 or sodium deoxycholate. The activity is not stimulated by sulfhydryl reagents or the addition of 10 millimolar MgCl2. The enzymic activity is insensitive to ethylenediaminetetraacetate. The pH optimum is broad, between 5.5 to 7.5. Although the substrate specificity is broad, 3-phosphoglycerate is the best substrate of those tested at neutral pH. However, p-nitrophenyl phosphate was a more effective substrate at pH 5.5. The enzyme exhibits the general characteristics of an acid phosphatase. 相似文献
12.
A rabbit anti-lymphotoxin serum produced against partially purified, antigeninduced, guinea pig lymphotoxin, was used to study the role of lymphotoxin in lymphocyte-mediated cytotoxicity in vitro. The anti-lymphotoxin serum inhibited cytolysis resulting from incubation of ovalbumin-immune guinea pig spleen cells with either mouse (P815 mastocytoma) or guinea pig (line 10 hepatoma) target cells in the presence of soluble ovalbumin. The antiserum also inhibited the cytolysis of ovalbumin-coupled target cells by ovalbumin-immune guinea pig spleen cells. In contrast, the anti-lymphotoxin serum did not inhibit: (a) the lysis of line 10 (strain 2) hepatoma cells by spleen cells from alloimmunized Hartley or strain 13 animals; (b) the lysis of line 10 hepatoma cells by spleen cells from tumor-bearing syngeneic animals; or (c) the lysis of P815-mastocytoma cells by spleen cells from P815-immune guinea pigs. These results support the hypothesis that there are at least two distinct pathways by which immune lymphocytes can destroy target cells in vitro, one which involves secretion of a nonspecific soluble factor, i.e., lymphotoxin, and another which probably requires intimate contact between the plasma membranes of the target and killer cells. 相似文献
13.
Alkaline Phosphatase Assay for Freshwater Sediments: Application to Perturbed Sediment Systems 总被引:10,自引:5,他引:5
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The p-nitrophenyl phosphate hydrolysis-phosphatase assay was modified for use in freshwater sediment. Laboratory studies indicated that the recovery of purified alkaline phosphatase activity was 100% efficient in sterile freshwater sediments when optimized incubation and sonication conditions were used. Field studies of diverse freshwater sediments demonstrated the potential use of this assay for determining stream perturbation. Significant correlations between phosphatase and total viable cell counts, as well as adenosine triphosphate biomass, suggested that alkaline phosphatase activity has utility as an indicator of microbial population density and biomass in freshwater sediments. 相似文献
14.
Purification and Characterization of a Phosphoenolpyruvate Phosphatase from Brassica nigra Suspension Cells 总被引:11,自引:9,他引:2
下载免费PDF全文
![点击此处可从《Plant physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Phosphoenolpyruvate phosphatase from Brassica nigra leaf petiole suspension cells has been purified 1700-fold to apparent homogeneity and a final specific activity of 380 micromole pyruvate produced per minute per milligram protein. Purification steps included: ammonium sulfate fractionation, S-Sepharose, chelating Sepharose, concanavalin A Sepharose, and Superose 12 chromatography. The native protein was monomeric with a molecular mass of 56 kilodaltons as estimated by analytical gel filtration. The enzyme displayed a broad pH optimum of about pH 5.6 and was relatively heat stable. Western blots of microgram quantities of the final preparation showed no cross-reactivity when probed with rabbit polyclonal antibodies prepared against either castor bean endosperm cytosolic pyruvate kinase, or sorghum leaf phosphoenolpyruvate carboxylase. The final preparation exhibited a broad substrate selectivity, showing high activity toward p-nitrophenyl phosphate, adenosine diphosphate, adenosine triphosphate, gluconate 6-phosphate, and phosphoenolpyruvate, and moderate activity toward several other organic phosphates. Phosphoenolpyruvate phosphatase possessed at least a fivefold and sixfold greater affinity and specificity constant, respectively, for phosphoenolpyruvate (apparent Michaelis constant = 50 micromolar) than for any other nonartificial substrate. The enzyme was activated 1.7-fold by 4 millimolar magnesium, but was strongly inhibited by molybdate, fluoride, zinc, copper, iron, and lead ions, as well as by orthophosphate, ascorbate, glutamate, aspartate, and various organic phosphate compounds. It is postulated that phosphoenolpyruvate phosphatase functions to bypass the adenosine diphosphate dependent pyruvate kinase reaction during extended periods of orthophosphate starvation. 相似文献
15.
A purification procedure, which included ethanol treatment as a step for dissociating the large molecular forms of type I phosphoprotein phosphatase, was employed for the studies of the alkaline phosphatase and phosphoprotein phosphatase activities in bovine brain, heart, spleen, kidney, and uterus, rabbit skeletal muscle and liver, and lobster tail muscle. The results indicate that the major phosphoprotein phosphatase (phosphorylase a as a substrate) and alkaline phosphatase (p-nitrophenyl phosphate as a substrate; Mg2+ and dithiothreitol as activators) activities in the extracts of all tissues studied were copurified as an entity of Mr = 35,000. The purified enzymes from different tissues exhibit similar physical and catalytic properties with respect to either the phosphoprotein phosphatase or the alkaline phosphatase activity. The present findings indicate that (a) the Mr = 35,000 species, which represents a catalytic entity of the large molecular forms of type I phosphoprotein phosphatase, is widespread in animal tissues, indicating that it is a multifunctional phosphatase; (b) the association of type I alkaline phosphatase activity with type I phosphoprotein phosphatase is a general phenomenon. 相似文献
16.
The role of immunoglobulin receptors on lymphocytes in production of MIF in guinea pigs 总被引:1,自引:0,他引:1
Teresa Gościcka 《Cellular immunology》1974,13(2):207-215
The effect of anti-guinea pig IgG sera and anti-rabbit light kappa chain serum on the capacity of sensitized lymphocytes of guinea pigs to production of migration inhibitor factor (MIF) was investigated. The lymph node cells, thymocytes and circulating lymphocytes taken from dinitrophenyl- (DNP) sensitized guinea pigs were preincubated with antisera against gamma1 + gamma2 globulins, gamma1 globulins, gamma2 globulin, light kappa chains or normal rabbit serum as control and stimulated with antigen in vitro to production of MIF. The inhibitory effect of lymphocyte culture supernates on the migration of guinea pig normal macrophages was determined by capillary tube test. It was found that all the anti-immunoglobulin sera used suppressed, in varied degree, the release of MIF by sensitized lymphocytes. It is suggested that the suppressive influence of anti-IgG sera reflects their blocking effect on surface receptors specific for antigen. 相似文献
17.
Isolation and characterization of a tartrate-sensitive splenic acid phosphatase in Gaucher's disease
The acid phosphatase activity that is increased in the spleens of patients with Gaucher's disease can be separated into two principal isoenzymes by chromatography on sulphopropyl-Sephadex. The acid phosphatase species that is resistant to inhibition by l-(+)-tartrate is retained by the cation-exchange resin while the tartrate-sensitive species passes through. We have isolated and characterized the tartrate-sensitive acid phosphatase (designated SPI) from the spleen of a patient with the adult (type 1) form of Gaucher's disease. SPI acid phosphatase, representing approximately 30 to 50% of the total acid phosphatase activity in a detergent (Triton X-100) extract of spleen tissue, has been purified approximately 400-fold to a specific activity of 48 units/mg of protein (substrate, 4-methylumbelliferyl phosphate). The final preparation of acid phosphatase contains at least two protein components—each with phosphatase activity—when analyzed by polyacrylamide gel electrophoresis at pH 8.9 or isoelectric focusing. SPI acid phosphatase exhibits a broad substrate specificity and catalyzes the hydrolysis of a variety of artificial and natural phosphate-containing compounds including p-nitrophenyl phosphate, α-naphthyl phosphate, phosphoenolpyruvate, and CMP. The enzyme is inhibited by l-(+)-tartrate, sodium fluoride, and ammonium molybdate and has the following properties: pH optimum, 4.5; Km on 4-methylumbelliferyl phosphate, 44 μm; pI, 3.8–4.1; Mr, 177,400; s20,w, 6.8. 相似文献
18.
A procedure for preparing highly purified brush border membranes from rabbit kidney cortex using differential and density gradient centrifugation is described. Brush border membranes prepared by this procedure were substantially free of basal-lateral membranes, mitochondria, endoplasmic reticulum and nuclear material as evidenced by an enrichment factor of less than 0.3 for (Na+ + K+)-ATPase, succinate dehydrogenase, NADPH-cytochrome c reductase and DNA. Alkaline phosphatase was enriched ten fold indicating that the membranes were enriched at least 30 fold with respect to other cellular organelles. The yield of brush border membranes was 20%.Transport of d-glucose by the membranes was identical to that previously reported except that the Arrhenius plot for temperature dependence of transport was curvilinear (EA = 11.3–37.6 kcal/mol) rather than biphasic. Transport of p-aminohippuric acid and uric acid were increased by the presence of NaCl, either gradient or preequilibrated. However, no overshoot was obtained in the presence of a NaCl gradient, and KCl and LiCl also produced equivalent stimulation of transport suggesting a nonspecific ionic strength effect. Uptakes of p-aminohippuric acid and uric acid were not saturable, and were increased markedly by reducing the pH from 7.5 to 5.6. Probenecid (1 mM) reduced p-aminohippuric acid and uric acid (50 μM) uptake by 49% and 21%, respectively. We conclude that the uptake of uric acid and p-aminohippuric acid by renal brush border membranes of the rabbit occurs primarily by a simple solubility-diffusion mechanism. 相似文献
19.
Abstract A strontium capture method, using p-nitrophenyl phosphate as substrate, was used to determine the subcellular localization of (Na+ + K+)-ATPase activity in Malpighian tubules of Locusta migratoria L. Ultrastructural studies revealed that (Na+ + K+)-ATPase activity was restricted to the basolateral plasma membranes with little evidence of activity associated with the apical microvilli. In contrast, alkaline phosphatase activity was specifically associated with the apical cell membrane. Biochemical assays of fixed and non-fixed tubule homogenates were used to evaluate the p-nitrophenyl phosphate-strontium procedure for localization of the phosphatase component of (Na+ + K+)-ATPase. No significant potassium-dependent, ouabain-sensitive p-nitrophenyl phosphatase activity was demonstrated in homogenates under conditions necessary for the cytochemical procedure, viz fixation, pH 9.0 and the presence of strontium. The significance of the biochemical results are discussed in relation to the validity of such cytochemical techniques for (Na+ + K+)-ATPase localization. 相似文献
20.
Mamoru Noda Ken-ichiro Shibata Yoshihiko Sawa Hirokata Shimokoube Tsuguo Watanabe 《Microbiology and immunology》1994,38(2):103-107
An acid phosphatase associated with the cell membranes of Mycoplasma fermentans was released from the membranes with Triton X-100, then purified by ion-exchange chromatography on DEAE-Sephacel and CM-Sepharose, followed by affinity chromatography on Con A-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed a single band with a molecular mass of 31.2 kilodaltons. The enzyme activity toward p-nitrophenyl phosphate was enhanced remarkably by Cu2+, Co2+ and Mg2+, but the activity was not inhibited by EDTA. The enzyme dephosphorylated O-phospho-l -tyrosine as well as p-nitrophenyl phosphate, but not O-phospho-l -threonine, O-phospho-l -serine, glucose-1-phosphate, phosphoryl choline and adenosine triphosphate. The level of the O-phospho-l -tyrosine phosphatase activity was the highest in Mycoplasma faucium and the second highest in Mycoplasma fermentans of all tested human mycoplasmas. 相似文献