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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)

Isoenzymes of alkaline phosphatase (EC 3.1.3.1) were separated by micro-scale two-dimensional electrophoresis, with isoelectric focusing in capillary gels in the first dimension and polyacrylamide gradient-gel electrophoresis in the second. The isoenzymes detected were identified by several treatments--e.g., incubation with sialidase, papain, Triton X-100, and wheat-germ agglutinin--and by comparison with alkaline phosphatase from liver microsomes. Liver and bone isoforms in normal sera showed overlapping isoelectric points but differed in molecular mass, estimated as 172 and 185 kDa, respectively. Sera of patients with liver disease showed several additional groups of alkaline phosphatase isoforms, two of which were found to consist of multi-molecular complexes. Others probably correspond to incompletely glycated enzyme proteins. A further isoform with a mass of about 250 kDa does not seem to correspond to any known isoform of alkaline phosphatase in serum. With this technique, we demonstrated intra- and interindividual variations of the placental alkaline phosphatase isoenzyme in pregnancy sera.
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PMID:Micro-scale two-dimensional electrophoresis of alkaline phosphatase from serum. 335 9

Membrane and soluble forms of alkaline phosphatase (ALP) were selectively prepared from human placental microsomes by treatment with 1-butanol at pH 8.5 and 5.5, respectively. The purified membrane (mALP) and soluble (sALP) forms were analyzed for chemical compositions. mALP was found to contain 1 mol each of palmitate, stearate, and glycerol/subunit of ALP, which were absent in sALP. Both the forms contained 1 mol of inositol and 2 mol of ethanolamine/subunit. However, none of these compounds was detectable in another soluble form prepared by treatment with papain, which is known to cleave the carboxyl-terminal region. The results suggest that mALP contains diacylglycerol, the removal of which results in its conversion to sALP. We then prepared [3H]ethanolamine-labeled ALP by incubating choriocarcinoma cells (JEG-3) with the isotope. 3H-Labeled sALP was mixed with unlabeled sALP and treated with papain. A 3H-labeled single component was purified from the digests by sequential chromatography through anti-ALP-IgG-Sepharose, concanavalin A-Sepharose, Bio-Gel P-6, and TSK G-2000 columns. Chemical analyses revealed that the purified sample contains the tripeptide Thr-Thr-Asp, ethanolamine, glucosamine, mannose, inositol, and phosphate. Molar ratios of the latter five compounds were calculated to be 2, 1, 3, 1, and 2, respectively, by taking Asp as 1 mol. The tripeptide sequence was identified at positions 482-484 in the primary structure deduced from the cDNA sequence, which predicts a further extension to position 513, containing a hydrophobic amino acid sequence. Taken together, these results suggest that the mature ALP molecule lacks the predicted carboxyl-terminal peptide extension and is attached at Asp484 with a glycosylphospholipid, the components of which are characterized above. The glycosylphospholipid thus attached is considered to function as the membrane anchor of ALP.
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PMID:Chemical characterization of the membrane-anchoring domain of human placental alkaline phosphatase. 339 21

Skeletal growth factor (SGF) activity was extracted from human bone matrix by demineralization and purified under dissociative conditions using hydroxyapatite, HPLC gel-filtration and HPLC reverse-phase chromatography. Human SGF thus purified was characterized chemically and biologically. Purified human SGF stimulated chick embryo bone cell proliferation at picomolar concentrations (half maximum at 2-3 ng/ml) and had little or no activity on other cell types tested (mouse 3T3 and normal rat kidney fibroblasts, embryonic chick intestinal and human placental cells). Human SGF did not displace 125I-labeled epidermal growth factor binding to normal rat kidney cells and did not stimulate normal rat kidney cell colony formation in soft agar. Human SGF activity was sensitive to trypsin, chymotrypsin, papain, dithiothreitol and performic acid but was resistant to heat (upto 70 degrees C), pH (3-10), cyanogen bromide, alkaline phosphatase and neuraminidase and did not bind jack bean concanavalin A or kidney bean lectin. From our chemical and biological studies it appears that human SGF is different from other known polypeptide growth factors: epidermal growth factor, fibroblast growth factor, insulin, insulin-like growth factor-I, platelet-derived growth factor and transforming growth factor.
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PMID:Chemical and biological characterization of low-molecular-weight human skeletal growth factor. 349 Feb 78

We have studied the effect of choline on the activity and temperature dependency of the brush-border alkaline phosphatase isoenzymes from rat intestine (tissue-specific type), and from kidney and placenta (tissue-nonspecific type). The removal of choline with phospholipase D resulted in the loss of enzyme activity in all the membranes, whereas in situ loss in the discontinuity of Arrhenius plots occurred in the kidney and the placental membranes, but not in the intestinal membranes. The lost activity was restored either by addition of free choline or phosphatidylcholine or by the removal of the enzyme from the membrane surface. Intestinal enzyme was removed by papain, while the tissue-nonspecific enzyme was released by subtilisin and by phosphatidylinositol-specific phospholipase C. The enzyme from kidney and placental membranes aggregated (rho = 1.13) upon removal of choline, and addition of choline resulted in disaggregation (rho = 1.03). Conversion of discontinuous to continuous linear plots of alkaline phosphatase in the kidney and placental membranes paralleled the increase in membrane phosphatidic acid content, and the decrease in total phosphatidylcholines. The intestinal enzyme produced plots with break points at all phosphatidic acid/phosphatidylcholine ratios. The change brought about by treatment with phospholipidase D was not due to changes in the half-saturation kinetics (Km) for the substrate. Based on these studies we conclude that the active site of the tissue-nonspecific phosphatase is approximated to exterior membrane cholines, as in the case of the intestinal isoenzyme; that despite similar effects on the membrane content of phospholipids, phospholipase D treatment caused much greater effects on the tissue-nonspecific enzyme, as assessed by Arrhenius plots and density centrifugation; that these effects are due to different protein structures rather than to a lipid milieu unique to each brush-border membrane.
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PMID:The role of choline on the activity-temperature relationship of brush-border alkaline phosphatase. 355 47

Horse kidney brush border membrane proteins were incorporated into phosphatidylcholine vesicles. Structural analysis of proteoliposomes prepared with various lipid:protein ratios showed that: (a) only a few of the proteins present in the crude brush border extract are integrated, (b) all known membrane hydrolases are integrated, and (c) these proteoliposomes are homogeneous vesicles. Papain solubilization of brush border membrane hydrolases, i.e. aminopeptidase M, neutral alpha-glucosidase, gamma-glutamyltransferase and alkaline phosphatase, performed in parallel on native membrane vesicles and proteoliposomes, revealed similar kinetics. Analysis of membrane vesicles and proteoliposomes on sucrose density gradients either without any treatment, or after papain treatment showed that: (a) in proteoliposomes, neutral alpha-glucosidase is associated with radiolabelled phosphatidylcholine, and (b) papain-treated vesicles and proteoliposomes released enzyme activity in the same way. These results suggest that the integration mechanism of brush border membrane proteins may be similar in proteoliposomes and native membrane vesicles. Transport experiments under equilibrium exchange conditions showed that the uptake properties of proteoliposomes are similar to those of brush border membrane vesicles.
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PMID:Reconstitution of brush border membrane proteins in phosphatidylcholine vesicles. Biochemical and functional characterization. 374 73

Benzoyl- and isopentenoyl phosphoric triamides (BPA and IPA) strongly inhibited urease activities from jack bean, soybean, watermelon seed, Proteus mirabilis, P. rettgeri, P. vulgaris, Mycobacterium smegmatis, and Ureaplasma urealyticum. Their I50 values (the final concentration causing 50% inhibition), independent of enzyme source, were 2-21 nM, which are about 1,000-fold lower than that of caprylohydroxamic acid, one of the most potent urease inhibitors. ATP-urea amidolyase activity was inhibited 50% by BPA at a higher concentration of 0.28 mM, but was not affected by IPA even at 1.3 mM. Thirteen kinds of hydrolases (trypsin, chymotrypsin, thermolysin, leucine aminopeptidase, papain, lipase, alpha-amylase, glucuronidase, asparaginase, arylsulfatase, alkaline phosphatase, acid phosphatase, and true cholinesterase), two oxidoreductases (catalase and alcohol dehydrogenase), three transferases (glutamic-oxaloacetic aminotransferase, gamma-glutamyl transpeptidase, and arylsulfotransferase) and two kinases (pyruvate kinase and creatine kinase) were not affected at all even at 1 mM BPA and IPA. Exceptionally, pseudo-cholinesterase from human serum was inhibited by BPA and IPA, whose I50 values were 70 nM and 10 muM, respectively, using acetylthiocholine as a substrate. These values increased to 0.55 muM and 54 muM, respectively, when acetylcholine was used as a substrate. These results show that N-acylphosphoric triamides potently and specifically inhibit urease activity at concentrations of nM order.
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PMID:Specific inhibition of urease by N-acylphosphoric triamides. 384 42

The quantitative release of enterokinase from isolated rat enterocytes following treatment with taurocholate-taurodeoxycholate, papain, chymotrypsin, elastase, carbamylcholine, and cholecystokinin-octapeptide was examined. Alkaline phosphatase and lactate dehydrogenase activities were evaluated simultaneously to check for specificity. Bile salts promoted a concentration-dependent release of all enzymes. Concomitantly, bile salts also led to cell destruction in proportion to the amount of enzymes released. Proteases caused the release of enterokinase and alkaline phosphatase with no concomitant increase of lactate dehydrogenase or cell lysis. At equal concentrations, papain released more enzymes than chymotrypsin and elastase. Chymotrypsin and elastase, however, led to higher ratios of enterokinase to alkaline phosphatase found in the media and suggested a selective release of enterokinase (EK) over that of alkaline phosphatase. Bile salts and pancreatic proteases together seem to have an additive effect of the release of EK. Carbamylcholine and cholecystokinin-octapeptide had no effect on enzyme release. These results suggested that pancreatic proteases are involved in the release of enterokinase by a selective action. Bile salts may also play a role through a nonselective detergent effect.
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PMID:Physiological factors controlling release of enterokinase from rat enterocytes. 390 6

1. A method for the preparation of brush border from rabbit kidneys is described. Contamination by other organelles was checked by electron microscopy and by the assay of marker enzymes and was low. 2. Seven enzymes, all hydrolases, were substantially enriched in the brush-border preparation and are considered to be primarily located in this structure. They are: alkaline phosphatase, maltase, trehalase, aminopeptidase A, aminopeptidase M, gamma-glutamyl transpeptidase and a neutral peptidase assayed by its ability to hydrolyse [(125)I]iodoinsulin B chain. 3. Adenosine triphosphatases were also present in the preparation, but showed lower enrichments. 4. Alkaline phosphatase was the most active phosphatase present in the preparation. The weak hydrolysis of AMP may well have been due to this enzyme rather than a specific 5'-nucleotidase. 5. The two disaccharidases in brush border were distinguished by the relative heat-stability of trehalase compared with that of maltase. 6. The individuality of the four peptidases was established by several means. The neutral peptidase and aminopeptidase M, both of which can attack insulin B chain, differed not only in response to inhibitors and activators but also in the inhibitory effect of a guinea-pig antiserum raised to rabbit aminopeptidase M. This antiserum inhibited both the purified and the brush-border activities of aminopeptidase M. The neutral peptidase and gamma-glutamyl transpeptidase were unaffected but aminopeptidase A was weakly inhibited. The characteristic responses to Ca(2+) and serine with borate served to distinguish aminopeptidase A and gamma-glutamyl transpeptidase from other peptidases. 7. No dipeptidases, tripeptidases or carboxypeptidases were identified as brush-border enzymes. 8. Incubation of brush border with papain released almost all the aminopeptidase M activity but only about half the activities of maltase, gamma-glutamyl transpeptidase and aminopeptidase A. No release of alkaline phosphatase, trehalase or the neutral peptidase was observed.
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PMID:Studies on the enzymology of purified preparations of brush border from rabbit kidney. 414 72

Gas vesicles, isolated from lysed Halobacterium halobium cells, gave an amino acid analysis which accounted for 78% of the weight, and the balance was mainly salt and water. One percent of tightly bound d-galactose was found, as well as 2% of phosphate that was not released by treatment which promotes beta-elimination, by hydrolytic release of the galactose, by carboxymethylation of lysine, or by alkaline phosphatase digestion. Only a trace of lipid was detected, and it appeared to have a polyisoprenoid structure. The vesicles were not solubilized by extremes of pH, by agents such as urea, guanidine hydrochloride, formic acid, and detergents, or by organic solvents. Succinylation and carboxymethylation gave partial dispersion, but the products were heterogeneous and of high molecular weight. The amino acid composition of vesicles was independent of fragment size. No band was obtained by polyacrylamide gel electrophoresis, with neutral, acidic, and alkaline systems, with or without sodium dodecyl sulfate and urea, before or after chemical modification. No amino terminus was detected. Electrofocusing of a vesicle dispersion showed a major component with a pI of 4.0 and an amino acid composition of the whole vesicles, and a minor band with pI 3.4 which had an amino acid composition different from whole vesicles. Vesicle protein was resistant to digestion by Pronase, trypsin, thermolysin, and papain. The precipitin reaction with rabbit antivesicle serum was not inhibited by galactose or inorganic phosphate. Succinylated and carboxymethylated vesicles cross-reacted with antivesicle serum. Cell lysates contained material which reacted with antiserum, but it was heterogeneous and mainly larger than 5 x 10(6) daltons. Material from nonvacuolated mutants reacted weakly with antiserum, but the amino acid composition of the precipitated antigen was different from that of vesicles and of soluble cross-reacting material from vacuolated cells.
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PMID:Analysis of Halobacterium halobium gas vesicles. 473 10

Proteins associated with intestinal brush borders and their various fractions were solubilized with sodium dodecyl sulfate and beta-mercaptoethanol, and separated by electrophoresis on acrylamide gels containing sodium dodecyl sulfate. Brush borders contain at least 15 proteins or subunits, ranging in molecular weight from 19,000 to 270,000. The largest proteins (170-270,000 mol wt), including the disaccharidases, are removed from the brush borders by papain. Proteins belonging to the remaining membrane, including alkaline phosphatase, have an intermediate size (53-140,000 mol wt). The proteins corresponding to the filamentous "core" of the microvilli are the smallest (19-45,000). The relative rates of degradation of these proteins were studied by following the rate of decline of (14)C-labeled leucine activity in specific proteins, and by the double isotope technique of Schimke in which leucine-(14)C was given to intact rats intraluminally 10 hr before an intraluminal dose of leucine-(3)H. Heterogeneity of (3)H/(14)C ratios and thus of rates of turnover of brush border proteins was noted. In general, the largest proteins (including the disaccharidases), were turning over the fastest. Other membrane proteins (i.e. alkaline phosphatase) had an intermediate rate of degradation, and "core" proteins turned over slowly. Thus, there was a general correlation between relative degradation rate and size.
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PMID:The relation of size to the relative rates of degradation of intestinal brush border proteins. 505 58


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