Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A panel of seven alkaline phosphatase labeled lectins was used to probe nitrocellulose electroblots of SDS-PAGE separated proteins from a primary culture of normal ovarian granulosa cells and an ENU-induced Sertoli cell tumor cell line (SCTL-I). Several additional lectin binding proteins were observed in silver stained SDS-PAGE gels as well as with lectins in SCTL-I. Succinated concanavalin A (Suc. Con A), Ricin communis agglutinin (RCA-I), Ulex europaeus agglutinin (UEA 1), Soybean agglutinin (SBA), Dolichos biflorus agglutinin (DBA) and Peanut agglutinin (PNA) stained more intensely in SCTL-I than normal granulosa cells. The same lectins as above, labeled with fluorescein isothiocyanate (FITC), were used to study the distribution of specific binding sites of tissue cultured cells grown in chamber/slides. Both normal ovarian granulosa cells and SCT cells exhibited strong peninuclear cytoplasmic labeling with Con A UEA-1 and WGA exhibited predominantly a nuclear and granular cytoplasmic staining pattern. SBA and DBA exhibited a strong coarse granular cytoplasmic labeling in granulosa cells and moderate granular cytoplasmic in SCT cells. In granulosa cells, Golgi regions stained strongly with PNA but weakly in SCT cells. RCA-I staining was negative in both cultures. Labeling of tissue cultured cells with lectins provides more details than histological sections of lectins binding sites at cellular structural levels.
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PMID:Lectin binding sites of cultured ovarian Sertoli cell tumors and follicular granulosa cells. 276 15

In contrast to the situation in chronic idiopathic thrombocytopenic purpura (ITP) it has not been known whether acute childhood ITP has an autoimmune aetiology and to what extent autoantibodies directed to platelet autoantigens appear during the transient course of this disease. In the present study of 21 children with acute ITP an immunoblot technique was applied to detect serum antibodies to electrophoretically (SDS-PAGE) separated normal donor platelet membrane proteins. Platelet antigen binding antibodies detected by alkaline phosphatase conjugated protein A or anti-IgM antibody were observed in 13 out of 21 (62%) patients while controls were negative. In nine children antibodies were directed to antigens localized on the three major membrane glycoproteins. GPIb (four), GPIIb (one) and GPIIIa (five). Antibodies to a 250 kDa antigen were noted in one case and to smaller 25-52 kDa proteins in 12 patients. Four patients had IgG as well as IgM platelet antibodies while in nine only IgM was found. The reactions were eliminated after absorptions of sera with fresh platelets. In all of three tested patients the glycoprotein antigen specific antibodies could be detected in acid eluates prepared from the absorbing platelets. The presence of antibodies, predominantly IgM, to platelet surface antigens in a majority of the children with acute ITP, may suggest an autoimmune process, albeit transitory, similar to that in chronic ITP.
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PMID:IgG and IgM antibodies to platelet membrane glycoprotein antigens in acute childhood idiopathic thrombocytopenic purpura. 280 83

Isolation of two membrane-bound alkaline phosphatase (AP) species from avian growth plate cartilage matrix vesicle (MV) fractions is described. AP was first released from the membranes by phosphatidylinositol-specific phospholipase C (PIase C), followed by chromatography on DEAE-Bio-Gel A and Reactive-Red agarose. Two AP species having apparent Mr of 81.5 and 77 kDa by SDS-PAGE were purified in high yield and specific activity by this simple method. Treatment with neuraminidase to remove sialic acid residues reduced their size slightly, but did not diminish the difference in Mr between the two species. Digestion with N-glycanase, however, decreased both AP species to a common size of 59 kDa. This reveals that both enzymes are highly glycosylated and suggests that the two forms may result from differences in degree of glycation. The amino acid compositions of the two avian enzyme forms are very similar, but are markedly enriched in serine, glycine and glutamate when compared to those reported for mammalian liver-kidney-bone AP. Possible differences in amino acid sequence between the two avian forms have not been excluded. The cross-reactivity of polyclonal antibodies to these enzymes with bovine kidney, but not intestinal AP, indicate that the avian cartilage APs are of the liver-kidney-bone isozyme type.
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PMID:Isolation of two glycosylated forms of membrane-bound alkaline phosphatase from avian growth plate cartilage matrix vesicle-enriched microsomes. 280 49

Desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocytes results in a 40-65% decrease in agonist-stimulated adenylate cyclase activity and correlates with increased phosphorylation of beta-adrenergic receptors. To assess the role of phosphorylation in desensitization, membranes from isoprenaline- and dibutyryl cyclic AMP-desensitized turkey erythrocytes were incubated with alkaline phosphatase for 30 min at 37 degrees C, pH 8.0. In both preparations alkaline phosphatase treatment significantly decreased desensitization of agonist-stimulated adenylate cyclase activity by 40-75% (P less than 0.05). Similar results were obtained after alkaline phosphatase treatment of membranes from isoprenaline- and dibutyryl cyclic AMP-desensitized duck erythrocytes. Moreover, alkaline phosphatase treatment of membranes from duck erythrocytes desensitized with 12-O-tetradecanoylphorbol 13-acetate returned agonist-stimulated adenylate cyclase activity to near control values. In all experiments, inclusion of 20 mM-sodium phosphate to inhibit alkaline phosphatase during treatment of membranes attenuated the enzyme's effect on agonist-stimulated adenylate cyclase activity. In addition, alkaline phosphatase treatment of membranes from control and isoprenaline-desensitized turkey erythrocytes increased the mobility of beta-adrenergic-receptor proteins, specifically photoaffinity-labelled with [125I]iodocyanopindolol-diazirine, on SDS/polyacrylamide-gel electrophoresis. The increased mobility of the beta-adrenergic-receptor proteins after alkaline phosphatase treatment of membranes was again inhibited by 20 mM-phosphate. These results provide additional evidence for a direct role for phosphorylation in desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocytes.
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PMID:Alkaline phosphatase relieves desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocyte membranes. 284 55

The degradation of phosphorylated and dephosphorylated neurofilament proteins by the Ca2+-activated neutral proteinase calpain was studied. Neurofilaments were isolated from bovine spinal cord, dephosphorylated by alkaline phosphatase (from Escherichia coli) and radioiodinated with [125I]-Bolton-Hunter reagent. The radioiodinated neurofilament proteins (untreated and dephosphorylated) were incubated in the presence and absence of calpain from rabbit skeletal muscle, and the degradation rates of large (NF-H), mid-sized (NF-M) and small (NF-L) neurofilament polypeptides were analysed by SDS/polyacrylamide-gel electrophoresis and autoradiography. The degradation of dephosphorylated neurofilament proteins occurred at a higher rate, and to a greater extent, than did that of the phosphorylated (untreated) neurofilament proteins. The dephosphorylated high-molecular-mass neurofilament (NF-HD) was proteolyzed 6 times more quickly than the untreated NF-H. The degradation rate of the NF-M and NF-L neurofilament proteins was also enhanced after dephosphorylation, but less than that of NF-H. This indicates that the dephosphorylation of neurofilament proteins can increase their sensitivity to calpain degradation.
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PMID:Dephosphorylation of neurofilament proteins enhances their susceptibility to degradation by calpain. 285 97

An exoribonuclease has been purified nearly to homogeneity from rat liver microsomes and its mode of action and general properties were studied. The molecular weight values for the enzyme, as estimated by gel filtration and SDS-polyacrylamide gel electrophoresis, were 88 000 and 92 000, respectively. The enzyme produced, via a processive mechanism Ado5'P as the only product from poly(A). The results of the hydrolysis of 4 S (Ado5'P)n and (Ado3'P)n by the exoribonuclease with or without alkaline phosphatase and the inhibition of the enzymatic activity by oligonucleotides having a 3'-phosphate group in the 3'-terminus suggested that the degradation proceeds in the 3' to 5' direction. These findings were confirmed by the analysis of hydrolyzed products of various oligoadenylates and Ado3'PUrdPGuo and by the comparison of the rates of hydrolysis of (Ado3'P)2Ado by the enzyme in the presence of varying amounts of (Ado3'P)3. Mg2+ was required for the enzymatic activity, and Mn2+ partially substituted for Mg2+. The activity of the enzyme was stimulated by K+ and spermine.
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PMID:Purification and mode of action of a microsomal exoribonuclease from rat liver. 298 11

Prolyl hydroxylase was purified from human placentae, specific antiserum against it was prepared, and a new radioimmunoassay system employing 125I-labelled enzyme preparation was established. The molecular weight of the placental enzyme was shown to be 320,000 by gel filtration. SDS-polyacrylamide gel electrophoresis showed two bands of unequal intensity having molecular weights of 60,000 and 130,000. Their amino acid compositions were identical to each other, suggesting the polypeptide with a molecular weight of 130,000 might be a dimer of the polypeptide with a molecular weight of 60,000. The new radioimmunoassay established had a sensitivity of the order of 10 ng/ml, indicating it was more sensitive than previous radioimmunoassay employing 3H-labelling method. Clinical studies on patients with liver diseases disclosed that the concentrations of serum immunoreactive prolyl hydroxylase were elevated both in cases of hepatocellular damage and in cases of cholestasis. In cases of hepatocellular damage the enzyme behaved like cytoplasmic enzymes such as glutamic oxaloacetic transaminase, glutamic pyruvic transaminase and lactic dehydrogenase, but in cases of cholestasis it resembled biliary enzymes such as alkaline phosphatase and gamma-glutamyl transpeptidase. This result might be associated with the peculiar location of the enzyme within the cell, in the membrane of rough endoplasmic reticulum.
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PMID:Human prolyl hydroxylase. Purification, radioimmunoassay and clinical studies in liver diseases. 299 Oct 66

Phosphoinositide breakdown in response to thrombin stimulation of human platelets results in the formation of the calcium-mobilizing messenger molecules inositol 1,4,5-trisphosphate and inositol 1,2-cyclic-4,5-trisphosphate and of diglyceride, which activates protein kinase C. We find that protein kinase C phosphorylates and thereby increases the activity of inositol 1,4,5-trisphosphate 5'-phosphomonoesterase, a phosphatase that hydrolyzes these molecules to inert compounds. The 5'-phosphomonoesterase phosphorylated using [gamma-32P]ATP comigrates on SDS-polyacrylamide gels with a protein (40 kd) phosphorylated rapidly in response to thrombin stimulation of 32PO4-labeled platelets. Peptide maps of proteolytic digests of these two phosphorylated proteins indicate that they are the same. We propose that platelet Ca2+ mobilization is regulated by protein kinase C phosphorylation of the inositol 1,4,5-trisphosphate 5'-phosphomonoesterase. These results explain the observation that phorbol ester treatment of intact human platelets results in decreased levels of inositol trisphosphate and decreased Ca2+ mobilization upon subsequent thrombin addition.
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PMID:Protein kinase C phosphorylates human platelet inositol trisphosphate 5'-phosphomonoesterase, increasing the phosphatase activity. 301 58

Two kinds of membrane (luminal and abluminal membrane domains) fractions have been isolated from bovine aortic endothelial cells by fractionation of whole cell homogenate on discontinuous sucrose density gradients. The luminal membrane domain was enriched 12-16-fold for angiotensin-converting enzyme activity and 8-10-fold in alkaline phosphatase activity. The abluminal membrane domain displayed an enrichment of 8-fold in (Na+ + K+)-ATPase activity. Both of the membrane domains were minimally contaminated with mitochondria, microsomes and Golgi bodies, as assessed by their corresponding marker enzyme activities. 125I-labeling of endothelial cell monolayers by the Enzymo-Bead lactoperoxidase-catalyzed iodination procedure, followed by isolation of membranes, revealed that the radioactivity was predominantly associated with membranes enriched in angiotensin-converting enzyme activity, corresponding to the luminal membrane domain. However, when cells were radioiodinated in suspension culture, radioactivity was found equally associated in both the luminal and abluminal membrane fractions. Electron microscopy of freeze-fractured and sectioned material showed both luminal and abluminal membrane domains to be in the form of vesicles varying in size from 100 to 400 nm in diameter. To characterize the separation of endothelial cell membrane domains, we have attempted to prepare monoclonal antibodies specific for endothelial cells. Several clones were obtained, producing antibodies which bound to endothelial cells of arterial, venous and capillary origin. Two antibodies of these clones, XIVC6 and XVD2, were studied in more detail. In the ELISA assay, these antibodies reacted with bovine vascular endothelial cells, but not with human umbilical cord endothelial cells, nor with bovine corneal endothelial cells, smooth muscle cells or fibroblasts. Both of these antibodies are directed against an antigen of approximately 130 kDa, under reducing and non-reducing conditions, as assayed by the immunoprecipitation method. Western blot analysis of luminal and abluminal membrane fractions revealed that only MAb XVD2 reacted with an antigen, indicating that the antibody XIVC6 is directed against an epitope which is denatured by SDS. Moreover, MAb XVD2 preferentially reacted with the luminal membrane compared to the abluminal membrane domain of the endothelial cell. These monoclonal antibodies do not react with platelet membrane proteins, indicating that this 130 kDa membrane antigen is not common to both endothelial cells and platelets.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Separation of luminal and abluminal membrane enriched domains from cultured bovine aortic endothelial cells: monoclonal antibodies specific for endothelial cell plasma membranes. 303 Apr 21

A major glycoprotein of rat hepatoma plasma membranes was selectively released as a soluble form by incubating the membrane with phosphatidylinositol-specific phospholipase C. The soluble form corresponding to the glycoprotein was also prepared by butan-1-ol extraction of microsomal membranes at pH 5.5, whereas extraction at pH 8.5 yielded an electrophoretically different form with a hydrophobic nature. The soluble glycoprotein extracted at pH 5.5 was purified by sequential chromatography on concanavalin A-Sepharose, Sephacryl S-300 and anti-(alkaline phosphatase) IgG-Sepharose, the last step being used to remove a contaminating alkaline phosphatase. The glycoprotein thus purified was a single protein with Mr 130,000 in SDS/polyacrylamide-gel electrophoresis, although it behaved as a dimer in gel filtration on Sephacryl S-300. The glycoprotein was analysed for amino acid and carbohydrate composition. The composition of the carbohydrate moiety, which amounted to 64% by weight, suggested that the glycoprotein contained much larger numbers of N-linked oligosaccharide chains than those with O-linkage. It was confirmed that the purified glycoprotein was immunologically identical not only with that released by the phospholipase C but also with the hydrophobic form extracted with butan-1-ol at pH 8.5. The results indicate that the glycoprotein of rat hepatoma plasma membranes, which has an unusually high content of carbohydrate, is another membrane protein released by phosphatidylinositol-specific phospholipase C, as documented for alkaline phosphatase, acetylcholinesterase and Thy-1 antigen.
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PMID:Purification and characterization of a major glycoprotein in rat hepatoma plasma membranes. One of the membrane proteins released by phosphatidylinositol-specific phospholipase C. 303 62


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