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)

The effect of vitamin D status on the topography of intestinal cell membranes was studied in isolated brush borders, as well as their purified membranes, by limited proteolysis. Addition of papain to brush borders isolated from vitamin D3-treated and deficient chicks resulted in a differential solubilization of leucine aminopeptidase, maltase, and sucrase activities (114, 195, and 79%, respectively, of appropriate control levels) but not alkaline phosphatase activity. In comparison, proteolysis of purified membranes exhibited vitamin D3- and 1,25-dihydroxycholecalciferol [1,25(OH)2D3]-dependent differences in release of all four marker hydrolases monitored. Calcium uptake studies revealed that preincubation with papain yielded vesicles with a calcium content that was 125% of corresponding native vesicles, in preparations from vitamin D3-treated, as well as deficient birds. Membrane vesicles prepared from 1,25(OH)2D3-treated chicks initially accumulated calcium to a greater extent than those from rachitic birds, but thereafter exhibited a decline in calcium content to basal levels. Preincubation with papain, however, abolished this loss of calcium. The combined results indicate that vitamin D mediates alterations in brush border protein topography and raise the possibility that this action of the seco-steroid might be involved in calcium absorption. However, if vitamin D-stimulated calcium transport across the brush border is dependent on a protein carrier, the molecular entity is not sensitive to inactivation by papain.
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PMID:Vitamin D-mediated alterations in the topography of intestinal brush border proteins: effect of papain on hydrolase release and calcium uptake. 684 6

Urinary high molecular mass proteins (fraction P) solubilized in Triton X-100 and by papain have been compared with the solubilized human renal brush border membrane proteins. Crossed immunoelectrophoresis of Triton X-100 fraction P extract, by means of two polyspecific antisera directed against either renal membrane or fraction P, revealed eleven immunoprecipitates antigenically identical with detergent renal membrane antigens. Among them, five hydrolases were identified by zymogram staining: microvillus aminopeptidase, maltase, trehalase, gamma-glutamyltransferase and alkaline phosphatase. Eight papain-solubilized fraction P proteins and Triton X-100-solubilized membrane extract presented 'identity' patterns in tandem crossed immunoelectrophoresis, but differed in their amphiphilicity, as demonstrated by the change of precipitation pattern on charge-shift caused immunoelectrophoresis. Among the eleven detergent-solubilized fraction P antigens, nine were proved to be amphiphilic proteins and six presented bidirectional charge shifting properties similar to those of renal membrane antigens. Quantitatively, five detergent fraction P proteins were found in the same amounts as in renal membrane extract, two in lesser amounts and four in greater. Moreover, the same two plasma proteins were identified in fraction P as in the renal membrane. Thus important similarities exist between the urinary fraction P and the native renal membrane.
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PMID:Immunochemical analysis of high molecular mass urinary proteins. 712 88

Using immunoelectrophoresis and other techniques, we have demonstrated an association between lipoprotein-X and (a) alkaline phosphatase and (b) other enzymes originating from the hepatocyte membrane, namely gamma-glutamyltransferase and leucine aminopeptidase. The high-molecular-mass forms of these enzymes, in both serum and bile, were precipitated by lipoprotein-X antiserum but not by antisera to other plasma proteins. The activity of high-molecular-mass alkaline phosphatase in serum was positively correlated with lipoprotein-X and with lipoprotein-X-associated alkaline phosphatase, both assessed semi-quantitatively. On the other hand, many sera possessed high activities of high-molecular-mass alkaline phosphatase but no detectable lipoprotein-X. Incubation of serum with conjugated bile salts and with synthetic detergents, at concentrations which did not dissociate the high-molecular-mass enzymes, caused parallel alterations in the electrophoretic mobility of serum lipoprotein-X and its associated enzyme activity. Incubation of normal dialyzed hepatic bile with normal, lipoprotein-X-negative serum produced an alteration in electrophoretic mobility of biliary lipoprotein and its associated enzyme activity from anodal to cathodal in agar gel. Digestion with papain had a variable effect on the different enzymes in the complex, without affecting the lipoprotein moiety. Leucine aminopeptidase was removed most readily from the complex to give the low-molecular-mass form present in normal serum; gamma-glutamyltransferase dissociated somewhat less readily, and alkaline phosphatase was completely resistant to dissociation from the complex. These results are discussed in the light of current knowledge, and a hypothesis is proposed for the nature of the high-molecular-mass enzymes in serum and bile.
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PMID:High-molecular-mass alkaline phosphatase in serum and bile: nature and relationship with lipoprotein-X. 723 66

Papain treatment of renal brush border vesicles was carried out as a successful first step towards the purification of the membrane components involved in dipeptide transport. The treated vesicles exhibited increased specific transport activity of glycyl-L-proline. In contrast, the specific transport activity of L-alanine in the treated vesicles was less than that in the control vesicles. Papain treatment resulted in the solubilization of 38% of protein, 55% of alkaline phosphatase, 90% of gamma-glutamyltransferase and 95% of leucine aminopeptidase. There was no change in the intravesicular volume nor was there any increase in vesicular permeability. Glycyl-L-proline transport was Na+-independent in the control and papain-treated vesicles. Diamide reduced the Na+-dependent L-alanine transport while glycyl-L-proline transport remained unaffected in the presence of Na+. Many dipeptides inhibited glycyl-L-proline transport both in the presence and absence of Na+. The inhibition by dipeptides was greater than the inhibition by equivalent concentrations of free amino acids. These data demonstrate that renal brush border vesicles can efficiently handle dipeptides by a mechanism completely different from that of amino acid transport.
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PMID:Evidence for a dipeptide transport system in renal brush border membranes from rabbit. 728 63

The interaction of alkaline phosphatase (EC 3.1.3.1) with bismuth was studied. Among the tested alkaline phosphatases, bismuth was found to be the most effective inhibitor of the placental enzyme. Partial denaturation of the placental enzyme by papain digestion had little effect, if any, on the inhibition. Bismuth inhibition of the placental enzyme activity was more progressive with mixed glycosidase treatment than with sialidase treatment. The pH activity profile of the mixed glycosidase-treated placental enzyme was clearly shifted in the presence of bismuth. The mixed glycosidase-treated placental enzyme/bismuth mixture was more heat labile than the non-treated placental enzyme. Based on the results of kinetic studies, the inhibition mechanism of the placental enzyme by bismuth was shown to be of the competitive type, and the Ki value and Hill coefficient of the mixed glycosidase-treated placental enzyme was found to be 92 mu mol/l and 2.25, respectively. L-Phenylalanine does not interfere with the inhibitory effect of bismuth on alkaline phosphatase. Inorganic phosphate, on the other hand, appears to disturb bismuth bindings.
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PMID:Inhibition of alkaline phosphatase by bismuth. 729 84

We developed a non-radioactive method of ligand western blotting for specific detection of active forms of serine proteases. The method consists of three steps: (i) separation of proteins by electrophoresis in sodium dodecyl sulfate-polyacrylamide gel, followed by blotting of proteins to nitrocellulose membrane; (ii) binding of a specific ligand, such as soybean trypsin inhibitor labeled with biotin, to protease on the membrane; and (iii) detection of the protease-inhibitor complex by color reaction (or chemiluminescence) developed by streptavidin-conjugated peroxidase (or alkaline phosphatase). By using this method, plasmin and trypsin (serine proteases) were detected, but papain (thiol protease) or pepsin (acidic protease) was not. Plasmin was detectable up to less than 4 ng. Inactive precursors of serine protease, i.e. plasminogen and trypsinogen, did not exhibit visible bands until they were activated by treatment with streptokinase or trypsin, respectively. We applied this method to clinical samples, and succeeded in detecting plasminogen, after conversion to plasmin with streptokinase treatment, in as little as 5 microliters of serum or trypsin, as it was in 10 microliters of pancreatic juice.
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PMID:Ligand western blotting for specific detection of active forms of proteases. 766 77

A rapid, cheap, and sensitive method has been developed for determining proteolytic activity of different classes of endoproteinases. The method is based on a solid-phase assay employing as substrate biotinylated gelatin adsorbed onto microtiter plates. Enzymatic activity is measured by incubating proteinase with the immobilized biotin-protein. Any remaining, undigested substrate bound to the microtiter plate is assayed with streptavidin-alkaline phosphatase. It was established that papain, pepsin, thermolysin, and trypsin all hydrolyzed the biotinylated substrate to varying degrees. Furthermore, the activity of these proteinases was blocked by their respective inhibitors. The assay presented is quick, highly reproducible, inexpensive, and useful for detecting all classes of endoproteolytic enzymes. By using different biotinylated proteins or peptides as substrates, and employing specific buffers and inhibitors, this assay may be utilized for detecting other and more specific endoproteinases.
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PMID:An assay for detecting nanogram levels of proteolytic enzymes. 766 84

Intact PAM212 keratinocytes were found to preferentially degrade exogenous phosphatidic acids (PA) containing short fatty acid chains. The product of this degradation was inorganic phosphate (Pi), suggesting that the enzyme was a phosphatase. Systematic studies using enzymatically synthesized PA and lysophosphatidic acid (lysoPA) demonstrated that the sn-2 fatty acid chain length was the determining factor for suitability of PA as substrate for this cell-associated enzyme. Thus 1-acyl-2-lyso-PA provided the best substrate for this enzyme while long-chain PA were poor substrates. The enzyme was effectively inhibited by NaF and Na3VO4, but was insensitive to inhibitors of alkaline phosphatase or other nonspecific phosphatases. The enzyme activity was solubilized from intact cells by proteinases, such as trypsin and papain, and the reaction product Pi was distributed exclusively in the extracellular medium, suggesting that this (lyso)PA phosphatase is an ectoenzyme. These results unequivocally demonstrated the presence of a (lyso)PA phosphatase located at the cell surface. This novel ectoenzyme may provide a mechanism for the inactivation of the potent bioactive phospholipid, lysoPA.
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PMID:Identification and characterization of an ecto-(lyso)phosphatidic acid phosphatase in PAM212 keratinocytes. 803 Nov 35

High-molecular-mass alkaline phosphatase (H-Mr AP) was detected in sera from children with solid tumors without liver metastases. H-Mr AP activities were determined by a liquid chromatographic and an electrophoretic method. In 5 out of 10 cases with solid tumors--Ewing sarcoma (n = 2), neuroblastoma (n = 2), and rhabdoid tumor (n = 1)--H-Mr AP activities ranged from 3.1-40.4 U/L and 3.1-16% of total serum AP activity. In sera of patients with leukemia (n = 18) H-Mr AP was not detectable. After the treatment of the sera with papain and phosphatidylinositol-specific phospholipase C, which release membrane-associated AP from membrane particles, H-Mr AP was no longer detectable. These results indicate that H-Mr AP in the sera of patients with solid tumors may derive from increasing cell shedding of the tumor cells with elevated levels of membrane fragments in serum, which is a well known phenomenon in liver tumors. H-Mr AP was not more detectable in the serum after successful tumor treatment. These data suggest that H-Mr AP was produced by the tumors and that this parameter may be a serological marker for some solid tumors even in the presence of normal total AP serum activity.
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PMID:High-molecular-mass or macromolecular alkaline phosphatase in sera of children with solid tumors. 815 5

Before fertilization, equine spermatozoa adhere to oviduct epithelial cells (OEC) of the mare. The biochemical basis for this adhesion has not been determined. Our objective was to produce an antiserum to block this interaction. Ejaculated spermatozoa were subjected to nitrogen cavitation and spermatozoal plasma membranes enriched by sucrose density gradient centrifugation; membrane enrichment was confirmed by comparative alkaline phosphatase analysis, electron microscopy, and one- and two-dimensional PAGE. Periacrosomal plasma membrane was used as an immunogen for the production of an antiserum, which recognized several components of spermatozoal plasma membrane on Western blots. Antigen-binding fragments (Fab) were isolated by papain digestion from a specific antiserum and from nonimmunized rabbit IgG (control). The periacrosomal regions of epididymal and ejaculated spermatozoa were immunolabeled with antiserum Fab but not control Fab. The immunoneutralizing activity of antiserum Fab was tested in fluorescent cell-binding assays by competitive inhibition of the binding of spermatozoa to OEC monolayers or explants. In both assays, binding of spermatozoa to OEC was reduced as the concentration of specific Fab increased. These results suggest that one or more protein or glycoprotein components of the rostral spermatozoal plasma membrane mediate adhesion between spermatozoa and oviduct epithelium in vitro.
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PMID:Antibody directed against plasma membrane components of equine spermatozoa inhibits adhesion of spermatozoa to oviduct epithelial cells in vitro. 904 18


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