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1.
C E Reker M C LaPointe B Kovacic-Milivojevi? W J Chiou W V Vedeckis 《Journal of steroid biochemistry》1987,26(6):653-665
Addition of bovine intestinal alkaline phosphatase to mouse AtT-20 cell cytosol increases the rate of glucocorticoid receptor transformation, as evidenced by a change in sedimentation rate from 9.1S to 5.2S. Acid phosphatases are completely ineffective in this regard. Alkaline phosphatase-promoted receptor transformation is both time- and dose-dependent. A variety of phosphatase inhibitors are effective in inhibiting this process, the most potent being transition metal oxyanions such as molybdate, tungstate, and arsenate. The ability of the various inhibitors to suppress alkaline phosphatase-promoted receptor transformation does not correspond well with their potencies for inhibiting para-nitrophenyl phosphate hydrolysis. However, a better correspondence between the inhibition of endogenous receptor transformation and total cytosolic phosphatase activity is observed, and both sodium fluoride and glucose-1-phosphate inhibit endogenous receptor transformation. The protease inhibitors phenyl-methylsulfonyl fluoride and antipain have no effect on receptor transformation. Surprisingly, leupeptin is effective in inhibiting alkaline phosphatase-promoted receptor transformation. Although this raises the possibility of a contaminating protease activity in the alkaline phosphatase enzyme preparation, treatment of covalently affinity-labeled receptor with the enzyme shows no proteolysis of the receptor or any other non-specifically labeled cytosolic protein. Thus, it is possible that a novel action of leupeptin, unrelated to its protease-inhibitory activity, may be involved in the suppression of receptor transformation. The studies presented here suggest that dephosphorylation of some component in cytosol is involved in the destabilization of receptor subunit interactions, resulting in glucocorticoid receptor transformation. 相似文献
2.
A cytosolic phosphoprotein phosphatase of Mr = 95,000 purified from bovine cardiac muscle, which contains a catalytic subunit of Mr = 35,000, is known to be associated with a Mg2+-activated p-nitrophenyl phosphatase activity. We have found that the enzyme preparation is also active toward phosphotyrosyl-IgG and -casein phosphorylated by pp60v-src, the transforming gene product of Rous sarcoma virus. The properties of this phosphotyrosyl protein phosphatase activity closely resemble those of the p-nitrophenyl phosphatase activity but sharply differ from those of the phosphorylase phosphatase activity. Comparative studies of the activities of the Mr = 95,000 phosphatase, bovine kidney alkaline phosphatase, and ATP X Mg-dependent phosphatase toward phosphoseryl, phosphothreonyl, and phosphotyrosyl proteins and p-nitrophenyl phosphate under various conditions have been carried out. The results indicate that the Mr = 95,000 enzyme exhibits higher activity toward phosphoseryl and phosphothreonyl proteins than toward phosphotyrosyl proteins, while the kidney alkaline phosphatase preferentially dephosphorylates phosphotyrosyl proteins. ATP X Mg-dependent phosphatase is inactive toward phosphotyrosyl proteins. 相似文献
3.
Several enzymes associated with the hepatocyte cell surface, alkaline phosphatase (AP), 5'-nucleotidase (5'N), Mg++- and total Na+K+Mg++-ATpase, were assayed and localized cytochemically in order to gain insight into alterations of the plasma membrane components during reassociation of hepatocytes in primary monolayer culture. During a period of 4 days the activities of 5'nucleotidase and alkaline phosphatase increased spontaneously up to three- and four-fold, respectively. Dexamethasone reinforce the rise of alkaline phosphatase activity but retarded the increase of that of 5'nucleotidase. However, after the third day the level of 5'nucleotidase activity converged with the untreated controls. The activities of Mg++- and Na+K+Mg++-ATPase, which closely paralleled each other, remained essentially unchanged throughout cultivation and were not affected by dexamethasone. Cytochemical demonstration of alkaline phosphatase, 5'nucleotidase and Mg++-ATPase, using the lead salt method, revealed the potential presence of reaction product on the whole cell surface. However, the cells did not react uniformly, particularly on bile canalicular membranes. This heterogeneity seems to be due to different stages of canalicular development and to different functional states of the cultured hepatocytes. 相似文献
4.
Rapid purification of bovine kidney branched-chain 2-oxoacid dehydrogenase complex containing endogenous kinase activity 总被引:7,自引:0,他引:7
Acid and alkaline phosphatase activity, determined by the hydrolysis of p-nitrophenyl phosphate, was found in preparations of microtubules purified from bovine brain by temperature-dependent assembly-disassembly and ion-exchange chromatography. Phosphocellulose-purified tubulin contained an associated acid phosphatase activity, stimulated by Mg2+ and by Zn2+. Alkaline phosphatase activity with a pH optimum of 10.4 was measured in a fraction of microtubule-associated proteins (MAPs). Kinetics and the effects of sodium fluoride, sodium tartrate, sulfhydryl-blocking agents, EDTA and Zn2+ are reported. 相似文献
5.
A partially purified bovine cortical bone acid phosphatase, which shared similar characteristics with a class of acid phosphatase known as tartrate-resistant acid phosphatase, was found to dephosphorylate phosphotyrosine and phosphotyrosyl proteins, with little activity toward other phosphoamino acids or phosphoseryl histones. The pH optimum was about 5.5 with p-nitrophenyl phosphate as substrate but was about 6.0 with phosphotyrosine and about 7.0 with phosphotyrosyl histones. The apparent Km values for phosphotyrosyl histones (at pH 7.0) and phosphotyrosine (at pH 5.5) were about 300 nM phosphate group and 0.6 mM, respectively, The p-nitrophenyl phosphatase, phosphotyrosine phosphatase, and phosphotyrosyl protein phosphatase activities appear to be a single protein since these activities could not be separated by Sephacryl S-200, CM-Sepharose, or cellulose phosphate chromatographies, he ratio of these activities remained relatively constant throughout the purification procedure, each of these activities exhibited similar thermal stabilities and similar sensitivities to various effectors, and phosphotyrosine and p-nitrophenyl phosphate appeared to be alternative substrates for the acid phosphatase. Skeletal alkaline phosphatase was also capable of dephosphorylating phosphotyrosyl histones at pH 7.0, but the activity of that enzyme was about 20 times greater at pH 9.0 than at pH 7.0. Furthermore, the affinity of skeletal alkaline phosphatase for phosphotyrosyl proteins was low (estimated to be 0.2-0.4 mM), and its protein phosphatase activity was not specific for phosphotyrosyl proteins, since it also dephosphorylated phosphoseryl histones. In summary, these data suggested that skeletal acid phosphatase, rather than skeletal alkaline phosphatase, may act as phosphotyrosyl protein phosphatase under physiologically relevant conditions. 相似文献
6.
Alkaline inorganic pyrophosphatase activity of mammalian-cell alkaline phosphatase 总被引:4,自引:1,他引:3
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Alkaline phosphatase prepared from mammalian cell cultures was found to have alkaline inorganic pyrophosphatase activity. Both of these activities appear to be associated with a single protein, as demonstrated by: (1) concomitant purification of alkaline phosphatase and alkaline inorganic pyrophosphatase; (2) proportional precipitation of alkaline phosphatase and inorganic pyrophosphatase activities by titrating constant amounts of an enzyme preparation with increasing concentration of antibody; (3) immune electrophoresis, which showed that precipitin bands that have alkaline phosphatase activity also have pyrophosphatase activity; (4) inhibition of pyrophosphatase activity by cysteine, an inhibitor of alkaline phosphatase activity; (5) similar subcellular localization of the two enzyme activities as demonstrated by histochemical methods; (6) hormonal and substrate induction of alkaline phosphatase activity in mammalian cell cultures, which produced a nearly parallel rise in inorganic pyrophosphatase activity. 相似文献
7.
Reuber rat hepatoma cells (R-Y121B) cultured at 0.5% serum accumulated apoalkaline phosphatase in intact cells. When R-Y121B cells were cultured in the presence of bovine serum albumin, alkaline phosphatase activity increased in the cells, and the associated increase in enzyme activity differed amongst bovine serum albumin preparations. The treatment of bovine serum albumin with activated charcoal not only enhanced the effect of serum albumin on alkaline phosphatase activity, but also cancelled the differences due to different preparations of serum albumin. In contrast, no effect from serum albumin was observed in the increase of alkaline phosphatase activity in R-Y121B cell homogenates incubated at 37 degrees C. The activated-charcoal treatment of bovine serum albumin increased the amount of Zn2+ bound to the protein. When R-Y121B cells were cultured with bovine serum albumin, the concentration of Zn2+ in the cytosol fraction slightly increased. However, the effect of serum albumin on Zn2+ concentration in the cytosol fractions was independent of charcoal treatment. It was concluded that serum albumin with Zn2+ induces the activation of apoalkaline phosphatase due to Zn2+ binding. 相似文献
8.
The hydrolysis of p-Nitrophenylphosphate has been studied in vitro in a tubulin preparation from bovine brain. The activity at pH 6.8 was 16.4 ± 2.2 nmol/mg protein, h. At least two phosphatases were responsible for this activity. They were found to have pH-optima at 5.1 and 10.4. respectively, and their apparent KM values were 1.23 ± 0.10 mm and 0.17 ± 0.03 mm . respectively. Mg2+ was found to stimulate activity at both pHs while Zn2+ inhibited at pH 5.1 and stimulated activity at pH 10.4. All of the alkaline and part of the acid phosphatase activity were found to be closely associated with microtubules/tubulin. Tubulin purified by phosphocellulose chromatography contained phosphatase activity, and it is suggested that such activity is an intrinsic property of tubulin itself. Phosphatase activity was also found in association with the microtubule-associated proteins that co-purify with tubulin. Two proteins of high molecular weight constituted the major part of the associated material. The results indicate an association of phosphatase activity with the larger of these two proteins. 相似文献
9.
E W Bingham E L Malin 《Comparative biochemistry and physiology. B, Comparative biochemistry》1992,102(2):213-218
1. Alkaline phosphatase is covalently bound to bovine mammary microsomal membranes and milk fat globule membranes through linkage to phosphatidylinositol as demonstrated by the release of alkaline phosphatase following treatment with phosphatidylinositol-specific phospholipase C. 2. The release of alkaline phosphatase from the pellet to the supernatant was demonstrated by enzyme assays and electrophoresis. 3. Electrophoresis of the solubilized enzymes showed that the alkaline phosphatase of the microsomal membranes contained several isozymes, while only one band with alkaline phosphatase activity was seen in the fat globule membrane. 4. Levamisole and homoarginine were potent inhibitors of the alkaline phosphatase activities in both membrane preparations and in bovine liver alkaline phosphatase, but not in calf intestinal alkaline phosphatase. 相似文献
10.
N N Chuang B C Yang 《Comparative biochemistry and physiology. B, Comparative biochemistry》1990,96(4):787-789
1. Alkaline phosphatases were purified from human placenta, bovine milk, shrimp and clam with a final spec. act. of 67,000, 32,000, 22,000 and 15,000 U/mg of protein respectively. 2. The alkaline phosphatase from Meretrix lusoria is unique with its thermostability at 65 degrees C for 30 min; whereas the remaining enzymes studied, including the human placental alkaline phosphatase, are inactivated and have negligible activities. 3. The alkaline phosphatase from Penaeus monodon can be differentiated by its pH optimum at 9.0; the remaining enzymes studied have their optimal pH at 10.0. 4. The alkaline phosphatases from shrimp and clam are proposed to be applied as "reporters" in the study of mammalian cells. 相似文献
11.
Renal brush border membrane vesicles (BBMV) of the dog possess at least two ATPase activities. In the present study, we have examined the effect of pH, ions, and inhibitors on the activity of ATPase in BBMV. Two different sets of conditions were identified that produced stimulation of ATPase activity. A unique stimulation of BBMV ATPase activity occurred at acidic pH in the presence of 1 mM ZnCl2. In the absence of Zn2+, a second ATPase activity was stimulated by alkaline pH values with peak stimulation occurring between pH 8.5 and 9.0. The results suggest that the alkaline pH-stimulated hydrolysis of ATP probably represents the activity of BBMV alkaline phosphatase. The unique acidic pH + Zn2(+)-stimulated ATPase activity must represent the activity of a second protein other than the alkaline phosphatase, since purified alkaline phosphatase did not show this activity. The biochemical identity and physiological function of this renal BBMV ATPase activity remain to be determined, but it may be an ecto-ATPase. 相似文献
12.
Modification of opioid receptor activity by acid phosphatase in neuroblastoma x glioma NG108-15 hybrid cells 总被引:1,自引:0,他引:1
A K Louie J N Zhan P Y Law H H Loh 《Biochemical and biophysical research communications》1988,152(3):1369-1375
Opioid receptor activity in neuroblastoma x glioma NG108-15 hybrid cell membranes was attenuated by acid phosphatase purified by high performance liquid chromatography and devoid of protease activity. Treatment of membranes with this phosphatase decreased opioid inhibition of adenylate cyclase and this effect was potentiated by the presence of the opioid agonist during the phosphatase treatment. Phosphatase treatment did not affect the number of opioid receptors but it did alter the distribution of receptors among affinity states, by increasing the percentage of receptors in the low affinity state. The similarities between these effects and desensitization of the opioid receptor, during chronic opioid treatment, are discussed. 相似文献
13.
H C Li 《European journal of biochemistry》1979,102(2):363-374
A metal-ion-independent, nonspecific phosphoprotein phosphatase (Mr = 35000) which represents the major phosphorylase phosphatase activity in bovine adrenal cortex has been purified to apparent homogeneity. An alkaline phosphatase activity (p-nitrophenyl phosphate as a substrate) of the same molecular weight, which requires both a metal ion (Mg2+ greater than Mn2+ greater than Co2+) and a sulfhydryl compound for activity, has been found to co-purify with the phosphoprotein phosphatase throughout the purification procedures. Characterization of the phosphoprotein and the alkaline phosphatase activities with respect to their catalytic properties, substrate and metal ion specificities, relationship with large molecular forms of the enzymes and responses to various effectors has been carried out. The results indicate that the phosphoprotein phosphatase can be converted by pyrophosphoryl compounds (e.g. PPi and ATP) to a metal-ion-dependent form which, subsequently, can be reactivated by Co2+ greater than Mn2+ but not by Mg2+ or Zn2+. The results also indicate that, although the phosphoprotein and the alkaline phosphatase activities are closely associated, they exhibit distinct physical and catalytic properties. Discussions concerning whether these two activities represent two different forms of the same protein or two different yet very similar polypeptide chains have been presented. 相似文献
14.
A procedure for the purification of cholesterol ester hydrolase from bovine adrenal cortical 105000 x g supernatant is described. Preincubation of a crude enzyme extract with [gamma-32P]ATP followed by purification resulted in the isolation of a phosphorylated preparation of cholesterol ester hydrolase. The phosphorylated cholesterol ester hydrolase appeared to be composed of 4 subunits, each having a molecular weight of 41000 +/- 280, only one of which may be phosphorylated. Preincubation of the crude enzyme preparation with [alpha-32P]ATP followed by purification did not produce a phosphorylated preparation of cholesterol ester hydrolase. Cyclic-AMP-dependent protein kinase, cyclic AMP, ATP and magnesium ions were required for activation of purified cholesterol ester hydrolase in vitro and the time course of activation closely paralleled the time course of phosphorylation of the enzyme. The addition of ATP, cyclic AMP and magnesium ions to the bovine adrenal cortical 105000 x g supernatant produced a 2.5-fold stimulation in cholesterol ester hydrolase activity. This stimulation was abolished if protein kinase inhibitor was added prior to the addition of ATP cyclic AMP and magensium ions. The addition of magnesium ions or calcium ions to a crude preparation of cholesterol ester hydrolase was found to inhibit activity; however the same additions made to a purified preparation of cholesterol ester hydrolase were not inhibitory. The decrease in cholesterol ester hydrolase activity on incubation with magnesium ion was accompanied by a loss of 32P radioactivity from the protein. Preincubation of a crude preparation of cholesterol ester hydrolase with alkaline phosphatase resulted in a deactivation of cholesterol ester hydrolase. It is suggested that bovine adrenal cortex cholesterol ester hydrolase is activated by a phosphorylation catalysed by a cyclic-AMP-dependent protein kinase. Deactivation of cholesterol ester hydrolase is accomplished by dephosphorylation catalysed by a phosphoprotein phosphatase, dependent on magnesium or calcium ions. 相似文献
15.
μ型阿片受体在阿片类药物镇痛与成瘾中发挥重要作用 .从人脑组织总RNA通过一次反转录和两次PCR法扩增获得 μ型阿片受体的cDNA ,将其克隆至pcDNA3 1 (+)中 ,转染CHO细胞后 ,筛选单克隆细胞株并制备膜受体 ,检测重组细胞株表达的 μ型阿片受体与特异性配体的结合能力 .通过饱和性结合和竞争性结合试验证实 ,重组细胞株表达的 μ型阿片受体与天然的 μ型阿片受体具有基本一致的生物学特性 ,为进一步研究阿片受体与配体相互作用的分子机制打下了基础 相似文献
16.
Optimal activity was recorded at pH 4.5–5 and pH 9.0–9.5 and specific activity was seen to be 0.013 μmoles of p-nitrophenyl phosphate/min/mg protein at 37 C at pH 4.5 and 0.00169 μmoles at pH 9.0. The ratio of acid to alkaline phosphatase was 7.7:1.0. The Km for acid phosphatase (EC 3.1.3.2) was 0.5 mM with a Vmax of 0.0128 units/mg protein and 0.2mM for alkaline phosphatase (EC 3.1.3.1) with a Vmax of 0.00175 units/mg protein. Acid phosphatase activity was optimal at 60 C and alkaline at 37 C. Linearity of enzyme activity was observed with time after the first 15 min of incubation and with homogenate concentration. KCN at 20 mM inhibited 82% of activity at pH 9.0 but also 91.5% activity at pH 4.5. NaF at 10?2M inhibited 92% of activity at pH 4.5 but had no effect at pH 9.0. The two flukicides rafoxanide and nitroxynil at 20mM had little effect on activity at pH 9.0 and pH 4.5. Enzyme activity at pH 4.5 was found to be greatest in the microsomal fraction with high activity in the lysosomal and soluble fractions. Histochemically, alkaline phosphatase was restricted to the excretory system, vitellaria, and uterus while acid phosphatase was found in the integument and gastrodermis. 相似文献
17.
The successful preparation of an active remnant of Cu,Zn-superoxide dismutase from mummified brain tissue stimulated the isolation of both biochemically and immunologically active alkaline Zn2Mg-phosphatase from antique bone samples of different archaeological sites and age. In particular, specimens from pharaonic Egypt being up to 4000 years of age were used. Gel filtration, ion exchange and affinity chromatographies were employed to optimise the preparation of the ancient enzyme. Compared to the specific activity of alkaline phosphatase from modern autopsy some 50% for a Ptolemaic and 10% for the Old Kingdom enzyme was detectable. The possibility of microbial contamination was checked by employing specific monoclonal antibodies directed against the human bone enzyme. Fortunately, ubiquitously present specified microorganisms on the respective ancient bones did not cross-react with these antibodies while the ancient metalloenzyme reacted with high specificity. Alkaline phosphatase mimicks could be excluded as in the presence of the inhibitors 1,10-phenanthroline and L-homoarginine the enzyme activity was diminished. The presence of ortho-vanadate as a substrate analogon abolished the catalytic function of the enzyme. Likewise, heating to 100 degrees C and replacement of zinc(II) by cadmium(II) resulted in a dramatic loss of activity. In conclusion, alkaline phosphatase appears to be a useful marker enzyme in molecular archaeology. 相似文献
18.
Tyrosine phosphorylation of the insulin receptor beta subunit activates the receptor-associated tyrosine kinase activity 总被引:32,自引:0,他引:32
The regulation of kinase activity associated with insulin receptor by phosphorylation and dephosphorylation has been examined using partially purified receptor immobilized on insulin-agarose. The immobilized receptor preparation exhibits predominately tyrosine but also serine and threonine kinase activities toward insulin receptor beta subunit and exogenous histone. Phosphorylation of the insulin receptor preparation with increasing concentrations of unlabeled ATP, followed by washing to remove the unreacted ATP, results in a progressive activation of the receptor kinase activity when assayed in the presence of histone and [gamma-32P]ATP. A maximal 4-fold activation is achieved by prior incubation of receptor with concentrations of ATP approaching 1 mM. High pressure liquid chromatographic analysis of tryptic hydrolysates of the 32P-labeled insulin receptor beta subunit reveals three domains of phosphorylation (designated peaks 1, 2, and 3). Phosphotyrosine and phosphoserine residues are present in these three domains while peak 2 contains phosphothreonine as well. Thus, at least seven sites are available for phosphorylation on the beta subunit of the insulin receptor. Incubation of the phosphorylated insulin receptor with alkaline phosphatase at 15 degrees C results in the selective dephosphorylation of the phosphotyrosine residues on the beta subunit of the receptor while the phosphoserine and phosphothreonine contents are not affected. The dephosphorylation of the receptor is accompanied by a marked 65% inhibition of the receptor kinase activity. Almost 90% of the decrease in [32P]phosphate content of the receptor after alkaline phosphatase treatment is accounted for by a decrease in phosphotyrosine content in peak 2, while very small decreases are observed in peaks 1 and 3, respectively. These results demonstrate that the extent of phosphorylation of tyrosine residues in receptor domain 2 closely parallels the receptor kinase activity state, suggesting phosphorylation of this domain may play a key role in regulating the insulin receptor tyrosine kinase. 相似文献
19.
Differential expression of alkaline phosphatase and ATPase activities in human colon carcinoma cell line HT-29.18 during differentiation 总被引:2,自引:0,他引:2
A Le Bivic J P Arsanto 《Biology of the cell / under the auspices of the European Cell Biology Organization》1987,60(1):41-47
The expression and cytochemical localization of alkaline phosphatase and Na+-pump sites were investigated in the human adenocarcinoma cell line HT-29.18 during differentiation. In the undifferentiated state, HT-29.18 cells expressed ATPase activity on plasma membrane whereas they displayed no alkaline phosphatase activity. In differentiated HT-29.18 cells, strong alkaline phosphatase activity was present on the apical membrane, whereas ATPase activity was restricted to the basolateral membrane. Intra- and intercellular lumina (cysts) observed in undifferentiated cells were devoid of both enzyme activities. In differentiated cells, cysts bearing well developed microvilli were strongly positive for alkaline phosphatase activity, while this activity seemed to be lacking in cysts without microvilli. ATPase activity was not found in either type of structure. Finally, HT-29.18 differentiated cells expressed, at pH 9.0, a p-nitrophenylphosphatase activity six-fold greater than that of undifferentiated cells. 相似文献
20.
Both spermidine and spermine are acetylated in chicken brain and retina. From spermidine, more N1-acetylspermidine than N8-acetylspermidine is formed by both the brain and the retinal cytosol. Km for spermidine is similar with the enzyme preparation of the two tissues, but that for spermine is lower with the retinal preparation. Both tissues contain an activity able to reduce spermidine acetyltransferase activity. Both alkaline phosphatase and cAMP-dependent protein kinase (catalytic subunit) are able to inactivate the spermidine acetyltransferase activity of both tissues. Spermidine acetyltransferase activity and polyamine levels have been measured in both brain and retina during embryonic life. Only in the last part of the development can enzyme activity be correlated with the retina spermidine and spermine concentration. 相似文献