Second messengers and phosphoproteins最新文献_第6页

Further studies on the phosphorylation-regulated cAMP-phosphodiesterase from the dimorphic fungus Mucor rouxii. 二态真菌柔毛霉磷酸化调控camp -磷酸二酯酶的进一步研究。

Second messengers and phosphoproteins Pub Date : 1988-01-01

C Tomes, N Kerner, S Moreno, S Passeron

{"title":"Further studies on the phosphorylation-regulated cAMP-phosphodiesterase from the dimorphic fungus Mucor rouxii.","authors":"C Tomes, N Kerner, S Moreno, S Passeron","doi":"","DOIUrl":"","url":null,"abstract":"A partially purified preparation (200-fold) of cAMP phosphodiesterase (PDE) was obtained from Mucor rouxii grown and extracted under conditions minimizing endogenous proteolysis. Four purification steps were applied: batch DEAE-Sepharose, DEAE-Sepharose chromatography, Sephadex G-150 super-fine gel filtration and sucrose gradient centrifugation. The final PDE preparation was activatable by cAMP-dependent phosphorylation and controlled trypsin treatment. A careful correlation of protein patterns with PDE activity was done throughout the whole procedure by analyzing the active fractions of each step by mini-polyacrylamide non-denaturing gel electrophoresis. The final preparation displayed four major protein bands, none of which corresponded to PDE, although PDE activity comigrated with two of them. Some properties of this preparation were studied. Vmax increased around 10-15 fold by activation of PDE by phosphorylation or proteolysis; Km values were unaffected. PDE had Stokes radius of 3.5 nm, sedimentation coefficient of 4.3 S and molecular weight of 70,000 daltons. The treatment of sucrose gradient fractions with [gamma-32P] ATP and cAMP-dependent protein kinase catalytic subunit and further analysis through minigels showed that none of the visible bands was phosphorylated, and that among the four phosphorylated bands there was one that cosedimented and comigrated with PDE activity. Trypsin treatment of the phosphorylated samples removed the label but did not modify the staining pattern.","PeriodicalId":77384,"journal":{"name":"Second messengers and phosphoproteins","volume":"12 5-6","pages":"289-99"},"PeriodicalIF":0.0,"publicationDate":"1988-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13994051","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Purification, properties and polyclonal antibodies for the particulate, low Km cAMP phosphodiesterase from bovine adipose tissue. 牛脂肪组织中颗粒状低Km cAMP磷酸二酯酶的纯化、性质及多克隆抗体。

Second messengers and phosphoproteins Pub Date : 1988-01-01

E Degerman, V C Manganiello, A H Newman, K C Rice, P Belfrage

{"title":"Purification, properties and polyclonal antibodies for the particulate, low Km cAMP phosphodiesterase from bovine adipose tissue.","authors":"E Degerman, V C Manganiello, A H Newman, K C Rice, P Belfrage","doi":"","DOIUrl":"","url":null,"abstract":"The particulate, cGMP- and cilostamide-inhibited \"low Km\" cAMP phosphodiesterase was purified greater than 100,000-fold in good yield (approximately 20%) from bovine omental fat by a procedure similar to that utilized for purification of an analogous enzyme from rat epididymal adipose tissue; ten-fold more enzyme protein (20-30 micrograms) could be prepared from bovine omentum (25 kg) than from rats (approximately 900 fat pads). Kinetic parameters (all similar to those for the rat enzyme) were: for cAMP, Km and Vmax = 0.3 microM and 2.5 mumol/min/mg, respectively; for cGMP, 0.8 microM and 1.6 mumol/min/mg. For inhibition of cAMP hydrolysis, IC50 values were: for cGMP = 0.6 microM and for OPC 3911, milrinone, CI 930, 0.1-2.0 microM. The purified enzyme, the activity of which eluted from Sephadex G-200 with Mr(app) = 110,000 and was associated with a single protein band during non-denaturing electrophoresis, exhibited on SDS-PAGE silverstained bands of 62 (probably a 61-63 doublet) and 77 kDa, perhaps due to proteolytic nicking. On Western blots, each of the polypeptides cross-reacted with a monoclonal antibody toward the bovine cardiac low Km cAMP and polyclonal rabbit antibody generated toward the purified bovine omental phosphodiesterase. Rabbit anti-phosphodiesterase IgG, which inhibited bovine and rat phosphodiesterase enzymatic activity, did not cross-react with purified bovine retina cGMP-binding or bovine cGMP-stimulated phosphodiesterase.","PeriodicalId":77384,"journal":{"name":"Second messengers and phosphoproteins","volume":"12 4","pages":"171-82"},"PeriodicalIF":0.0,"publicationDate":"1988-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13991630","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Progesterone decreases DNA binding factor activity and the expression in Xenopus oocytes of a cAMP responsive gene from rat liver. 黄体酮降低DNA结合因子活性和爪蟾卵母细胞中来自大鼠肝脏的cAMP应答基因的表达。

Second messengers and phosphoproteins Pub Date : 1988-01-01

J A Williams, D Schlichter, W D Wicks

{"title":"Progesterone decreases DNA binding factor activity and the expression in Xenopus oocytes of a cAMP responsive gene from rat liver.","authors":"J A Williams, D Schlichter, W D Wicks","doi":"","DOIUrl":"","url":null,"abstract":"Stage VI Xenopus oocytes were injected with a plasmid (pBB0.6-CAT) which contains the cAMP regulatory element (CRE) from the rat liver phosphoenolpyruvate carboxykinase (PEPCK) gene fused upstream from a reporter gene [chloramphenicol acetyltransferase (CAT)]. Inhibition of the expression of the reporter gene (average = 51%) was observed in the presence of 10 microM progesterone, which is known to lead to inactivation of the oocyte cAMP dependent protein kinase (A kinase). In contrast, oocytes injected with a control plasmid (pSV2CAT), which contains no CRE, exhibited a variable increase (average = 31%) in CAT activity after progesterone treatment. Injection of the purified bovine cardiac A kinase catalytic subunit prior to exposure of oocytes injected with pBB0.6 CAT to progesterone prevents the loss of CAT activity generated by incubation with the steroid. Gel retardation analyses with oocyte lysates and a labeled synthetic oligonucleotide fragment containing the CRE from the PEPCK gene showed the existence of a complex with the same Rf and specificity as that formed with rat liver extracts. Subsequent exposure to progesterone, however, led to a rapid and extensive decrease in this binding activity. Taken together, these results are consistent with but do not prove the hypothesis that progesterone treatment and A kinase inactivation lead to a decrease in pBB0.6 CAT expression by virtue of a decline in the binding activity of an oocyte factor(s) to the CRE of the PEPCK fragment in pBB0.6-CAT, thereby decreasing transcription of the CAT gene.","PeriodicalId":77384,"journal":{"name":"Second messengers and phosphoproteins","volume":"12 5-6","pages":"261-70"},"PeriodicalIF":0.0,"publicationDate":"1988-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"14112874","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Rapid purification of iodinated ligands for cyclic nucleotide radioimmunoassays. 环核苷酸放射免疫测定用碘化配体的快速纯化。

Second messengers and phosphoproteins Pub Date : 1988-01-01

S P Wilson

引用次数: 0

Correlation of cell-free brain cyclic nucleotide phosphodiesterase activities to cyclic AMP decay in intact brain slices. 完整脑切片中无细胞脑环核苷酸磷酸二酯酶活性与环AMP衰减的相关性。

Second messengers and phosphoproteins Pub Date : 1988-01-01

M E Whalin, R L Garrett, W J Thompson, S J Strada

{"title":"Correlation of cell-free brain cyclic nucleotide phosphodiesterase activities to cyclic AMP decay in intact brain slices.","authors":"M E Whalin, R L Garrett, W J Thompson, S J Strada","doi":"","DOIUrl":"","url":null,"abstract":"Differential and gradient centrifugation of rat brain cerebral cortical homogenates show three cyclic nucleotide phosphodiesterase (CN PDE) activities localized to different subcellular fractions with varying relative specific activities and responsiveness to pharmacologic agents. Type I (calcium/calmodulin-activatable) CN PDE is found primarily in the cytosolic fraction, Type II (cGMP-activatable) CN PDE is predominately membrane associated, and Type IV (cGMP-insensitive) cAMP PDE is distributed equally between soluble and particulate fractions. Fractionation of cerebral cortical membranes shows that Type II and Type IV CN PDE activities reside in synaptosomes. Type II CN PDE is the predominate hydrolytic activity in synaptosomes whereas Type IV cAMP PDE contributes only a small percentage of the total cAMP hydrolysis and Type I CN PDE is not detected in this fraction. The contribution of CN PDE isozymes to the regulation of intracellular cAMP levels was studied using rat brain cortical slices. The rate of cAMP decay in the absence and presence of selective CN PDE inhibitors after adenosine or beta-adrenergic agonist stimulation was determined using an adenine prelabeling technique. These studies show that a rolipram-sensitive, high affinity cAMP PDE (Type IV) is principally responsible for cyclic AMP decay in intact cortical tissue following elevation of cyclic AMP levels by either adenosine or beta-adrenergic receptor agonists. However, this isozyme, which is sensitive to inhibition by rolipram, RO 20-1724 and SQ 65442 contributes only a small percentage of the total cAMP hydrolytic activity in cell-free preparations of cortex.","PeriodicalId":77384,"journal":{"name":"Second messengers and phosphoproteins","volume":"12 5-6","pages":"311-25"},"PeriodicalIF":0.0,"publicationDate":"1988-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13994054","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Structure and evolution of bacterial adenylate cyclase: comparison between Escherichia coli and Erwinia chrysanthemi. 细菌腺苷酸环化酶的结构和进化:大肠杆菌和菊花的比较。

Second messengers and phosphoproteins Pub Date : 1988-01-01

A Danchin, G Lenzen

引用次数: 0

Cyclic AMP turnover in response to prostaglandins in intact platelets: evidence for separate stimulatory and inhibitory prostaglandin receptors. 完整血小板中对前列腺素的循环AMP转换反应:单独刺激和抑制前列腺素受体的证据。

Second messengers and phosphoproteins Pub Date : 1988-01-01

B Ashby

{"title":"Cyclic AMP turnover in response to prostaglandins in intact platelets: evidence for separate stimulatory and inhibitory prostaglandin receptors.","authors":"B Ashby","doi":"","DOIUrl":"","url":null,"abstract":"The shape of the time-course of cyclic AMP formation by intact human platelets in response to the stable prostaglandin I2 analogue iloprost varied with the concentration of the prostaglandin. At low concentrations of iloprost, the time-course showed a rise to a plateau with little subsequent decrease in cyclic AMP level. At high concentrations of iloprost, the initial rate of cyclic AMP formation was more rapid than at low concentrations, but the curves showed a marked time-dependent fall in cyclic AMP level to values below those observed at lower prostaglandin concentration. By contrast, PGE1 gave a rise and marked fall in cyclic AMP level at all concentrations of the prostaglandin and the curves did not cross. The time- and concentration-dependent fall in cyclic AMP level in response to iloprost was still apparent in the presence of phosphodiesterase inhibitors, indicating that inhibition of adenylate cyclase, rather than activation of cyclic AMP phosphodiesterases, was responsible for the fall in cyclic AMP level. Activators of protein kinase C, which phosphorylates platelet Ni and impairs its function, abolished the time-dependent fall in cyclic AMP level, indicating that Ni may be involved in prostaglandin-induced inhibition of adenylate cyclase. Time-courses were analyzed using an equation derived by Barber et al. (Adv. Cyc. Nuc. Res. 9, 507-516 (1978)) to yield rate constants for activation and inhibition of adenylate cyclase. Because of the difference in prostaglandin dependence of the activation and inhibition rate constants we propose that activation of adenylate cyclase in platelets is mediated by a rapid-acting stimulatory receptor, while time-dependent inhibition (desensitization) is mediated through a separate, slow-acting inhibitory receptor. The stimulatory receptor has an affinity for prostaglandin greater than the putative inhibitory receptor in the case of iloprost (as well as PGI2 and PGD2), and a lower affinity than the inhibitory receptor in the case of PGE1 (and PGE2). Prostaglandin-induced inhibition may be mediated through Ni.","PeriodicalId":77384,"journal":{"name":"Second messengers and phosphoproteins","volume":"12 1","pages":"45-57"},"PeriodicalIF":0.0,"publicationDate":"1988-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13986220","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

The kinetics of Ins(1,4)P2 dephosphorylation by Ins(1,4)P2 1-phosphatase in bovine brain. 牛脑Ins(1,4)P2 1-磷酸酶对Ins(1,4)P2去磷酸化的动力学研究。

Second messengers and phosphoproteins Pub Date : 1988-01-01

A Delvaux, J E Dumont, C Erneux

引用次数: 0

Substrate specificity of ribosomal protein S6 kinase II from Xenopus eggs. 非洲爪蟾卵核糖体蛋白S6激酶II的底物特异性。

Second messengers and phosphoproteins Pub Date : 1988-01-01

E Erikson, J L Maller

引用次数: 0

Species specificity of antibodies to regulatory subunits of cyclic AMP-dependent protein kinases. 抗体对环amp依赖性蛋白激酶调控亚基的物种特异性。

Second messengers and phosphoproteins Pub Date : 1988-01-01

A M Maddox, A L Steiner, S Shenolikar

{"title":"Species specificity of antibodies to regulatory subunits of cyclic AMP-dependent protein kinases.","authors":"A M Maddox, A L Steiner, S Shenolikar","doi":"","DOIUrl":"","url":null,"abstract":"Polyclonal antibodies were generated against regulatory subunits (RI and RII) of type-I and type-II cAMP-dependent protein kinases from rat skeletal muscle. Western immunoblot analyses showed specific cross-reactivity of rat and bovine RI with anti-RI. Similarly, RII from both species was specifically recognized by anti-RII. Quantitative immunoassays, using antisera against proteins from either species, indicated selectivity towards regulatory subunits from the same species. Molecular basis for this selectivity was examined by comparison of peptide maps of 32P-8-azido-cAMP-labelled or autophosphorylated peptides. Detailed analysis of two-dimensional peptide fingerprints demonstrated extensive homology between either RI or RII from the two species. The data suggests that the overall protein-chemical and functional determinants characterizing type-I and type-II regulatory subunits of cyclic AMP dependent protein kinase from different species are substantially similar. However, minor differences in structure, also predicted by amino-acid sequences for RI and RII obtained by molecular cloning, may account for the distinct immunological properties of the proteins from rat and bovine tissues.","PeriodicalId":77384,"journal":{"name":"Second messengers and phosphoproteins","volume":"12 2-3","pages":"83-94"},"PeriodicalIF":0.0,"publicationDate":"1988-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"14372741","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0