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 clinical value of alkaline phosphatase isoenzyme analysis is limited by the inability of most electrophoretic methods to resolve the liver and bone isoenzymes. The authors attacked this problem by treating serum samples with neuraminidase, then running treated and untreated samples side-by-side on specially prepared agarose gels. Each isoenzyme showed a characteristic mobility before and after neuraminidase treatment that allowed its identification. The mobility of the bone isoenzyme was most affected, whereas the intestinal isoenzyme was resistant to the action of neuraminidase. In samples with both liver and bone isoenzymes, pretreatment with neuraminidase clearly distinguished the bands, allowing quantitation by densitometry. Using this method, the authors discovered 22 liver isoenzymes in 54 samples that were interpreted as only bone isoenzyme before neuraminidase treatment. They also detected two bone isoenzymes in 35 samples that appeared to contain only liver +/- biliary isoenzymes. In addition, this procedure enabled them to characterize several unusual isoenzymes with respect to mobility, thus avoiding confusion with the other isoenzymes.
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PMID:Agarose gel patterns of alkaline phosphatase isoenzymes before and after treatment with neuraminidase. 339 58

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

This new method for fractionating alkaline phosphatase isoforms in hepatobiliary disorders is based on isoelectric focusing on a mixed-type polyacrylamide support containing an immobilized pH gradient with a superimposed carrier-ampholyte gradient. The high-Mr alkaline phosphatase forms typical of hepatobiliary disease (greater than 1 mega-dalton), which cannot migrate into the Immobiline gel, are disaggregated in zwitterionic detergents (the most effective being sulfobetaine 3-12)--20 g/L in the sample, 5 g/L in the gel--suggesting that they are still complexed with membrane fragments or that they tend to aggregate spontaneously in solution. These isoforms focus in the pI 5-6 range (while alkaline phosphatases in normal serum focus in the pI 4-5 interval) in immobilized pH gradients, but behave as strongly acidic components by agarose isoelectric focusing in the presence of carrier ampholytes, suggesting that they are strongly complexed with the latter. On treatment with neuraminidase, the low-pI isoforms in normal serum focus in the pI 5-6 range typical of the hepatobiliary isoforms, suggesting that the latter are poorly glycosylated. By a second-dimension run, in a porosity gradient, followed by activity staining, all alkaline phosphatase forms that have entered the Immobiline gel in the first dimension (normal forms and high-Mr species) exhibit the same Mr (ca. 140,000 Da), suggesting that no new chains are synthesized in hepatobiliary disorders.
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PMID:Serum alkaline phosphatase isoenzymes in hepatobiliary disorders resolved by use of immobilized pH gradients. 356 49

We attempted to separate bone and liver alkaline phosphatase (EC 3.1.3.1) isoenzymes in human serum by isoelectric focusing on agarose gel. We found that in a pH 3-10 gradient the liver and bone isoenzymes focused into so many bands over a narrow pH range such that the information could not be quantified. However, when the bone isoenzyme in serum was first desialylated at 37 degrees C for a minimum of 6 h, catalyzed by neuraminidase (EC 3.2.1.18) at pH 5.8-6.0, we could detect four distinct bands with pls of 6.7, 6.8, 6.9, and 7.0. Under the same conditions, the liver isoenzyme in human serum focused into one band at pH 7.0. The multiple banding we observed for the desialylated bone isoenzyme has not been previously reported. The method is suited as a qualitative technique for detecting the bone alkaline phosphatase isoenzyme in serum.
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PMID:Isoelectric focusing of neuraminidase-treated alkaline phosphatase isoenzymes on agarose gel. 359 28

HeLa S3 cells produce an alkaline phosphatase indistinguishable from the enzyme from human term placenta. The phosphatase activity in these cells was induced by both prednisolone and sodium butyrate. Both agents stimulated de novo synthesis of the enzyme. The increase in phosphatase activity paralleled the increase in immunoactivity and biosynthesis of placental alkaline phosphatase. The fully processed phosphatase monomer in control, prednisolone-treated or butyrate-treated cells was a 64.5 K polypeptide, measured by both incorporation of L-[35S]methionine into enzyme protein and active-site labeling. The 64.5 K polypeptide was formed by the incorporation of additional N-acetylneuraminic acid moieties to a precursor polypeptide of 61.5 K. However, this biosynthetic pathway was identified only in butyrate-treated cells. In prednisolone-treated cells, the processing of 61.5 K to the 64.5 K monomer was accelerated, and the presence of the 61.5 K precursor could only be detected by either neuraminidase or monensin treatment. Phosphatase mRNA which comigrated with the term placental alkaline phosphatase mRNA of 2.7 kilobases was induced in the presence of either prednisolone or butyrate. Alkaline phosphatase mRNA in untreated HeLa S3 cells migrated slightly faster than the term placental alkaline phosphatase mRNA. Butyrate also induced a second still faster migrating alkaline phosphatase mRNA. Both prednisolone and butyrate increased the steady-state levels of placental alkaline phosphatase mRNA. Our data indicate that the increase in phosphatase mRNA by prednisolone and butyrate resulted in the induction of alkaline phosphatase activity and biosynthesis in HeLa S3 cells. Furthermore, both agents induced the expression of different alkaline phosphatase gene transcripts without altering its protein product.
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PMID:Control of placental alkaline phosphatase gene expression in HeLa cells: induction of synthesis by prednisolone and sodium butyrate. 365 99

The microheterogeneity of human serum alkaline phosphatase (ALP) was investigated by means of isoelectric focusing. Liver and bone isoenzymes focused in a similar pattern, with about 10 bands located between pH 3.7 and 4.9, but differed in the relative intensity of the various bands. Intestinal ALP exhibited 7 to 8 bands at pH 4.9-5.1, and the placental enzyme showed 2 to 3 bands at pH 4.9. Mild digestion with neuraminidase revealed that the banding of liver and bone isoenzymes was at least partly due to differences in the sialic acid content of the various fractions. Extensively desialylated liver and bone isoenzymes showed apparently identical patterns with 6 to 7 bands focused at pH 6.2-6.7. Isoelectric focusing is a useful method for characterizing the microheterogeneity of alkaline phosphatase isoenzymes. The clinical value of this method seems to be limited, however, since it did not distinguish between liver and bone isoenzymes and failed to detect 'specific' isoelectric fractions correlated to various diseases.
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PMID:Microheterogeneity of serum alkaline phosphatase isoenzymes as revealed by isoelectric focusing. 367 27

The major source of rat serum alkaline phosphatase (ALP) is well known to be from the intestinal enzyme, but it is still unclear whether it is from the duodenal or the ileal enzyme. The organic origin was investigated by means of two-dimensional electrophoresis. Major isoelectric points and molecular masses for activities of duodenal enzyme treated with both phosphatidylinositol-specific phospholipase C and neuraminidase were identified apparently with those of the major serum enzyme. In organ culture, the normal duodenal enzyme was released in the highest amounts to the culture medium. These results indicate that the major source of serum ALP in adult rats is basically from the duodenal enzyme. On the other hand, lectin affinity chromatography for ALPs showed that the ALP in the medium from culture duodenum and liver had the same complex-type sugar chain as with the ALP in the duodenal tissue. Although the duodenal ALP induced by glucosamine in vitro had the hybrid-type chain, sugar chains of the induced ALP in the culture medium were of the complex type, indicating that medial ALPs possessing the same sugar chain as the native duodenal enzyme, complex type, are mainly released from their tissues in normal conditions.
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PMID:Blood appearance of rat alkaline phosphatase originating from the duodenum in vitro. 369 1

Isoelectric focusing and gradient polyacrylamide gel electrophoresis were used to define the physical differences between canine liver alkaline phosphatase (LAP) and steroid induced alkaline phosphatase (SIAP). LAP has an isoelectric point (pI) of pH 4.3 and a molecular radius (Mr) of 100,000, while SIAP has a pI of pH 3.5 and an Mr of 110,000. After removal of sialic acid residues by neuraminidase, the two isoenzymes were still distinct. The pIs of both LAP and SIAP were increased with the pI of LAP becoming pH 4.7 to 4.8 and that of SIAP becoming pH 4.5 to 4.6. On gradient polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, SIAP gave a single band of Mr 100 000 after neuraminidase treatment, while LAP increased in molecular size in spite of the denaturing conditions of the electrophoretic separation.
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PMID:The effect of neuraminidase on the molecular weight and the isoelectric point of the steroid induced alkaline phosphatase of dogs. 376 Feb 70

Of 98 patients' specimens examined for alkaline phosphatase (EC 3.1.3.1) isoenzymes by electrophoresis on cellulose acetate membrane after incubation with neuraminidase, 50 showed only a single liver or bone isoenzyme staining band; in 15 of these, the tissue origin of the fraction could not be accurately identified from its electrophoretic location. In the remaining 48 specimens, both liver and bone fractions were identifiable, but in only 25 of these was the electrophoretic resolution sufficient to yield separate peaks on densitometry. In contrast, both liver and bone alkaline phosphatase isoenzymes were identified in 95 of the 98 specimens by affinity electrophoresis involving wheat-germ lectin, the detection of both fractions being in agreement with the results of sequential heat inactivation. The tissue origin of the enzyme bands was readily ascertainable from their consistent electrophoretic location in this medium, and in 89 of the specimens the isoenzyme fractions could be resolved into separate peaks on densitometry. We conclude that resolution of liver and bone alkaline phosphatase by incubation with neuraminidase followed by cellulose acetate electrophoresis is greatly inferior to that obtained by wheat-germ lectin affinity electrophoresis.
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PMID:Incubation with neuraminidase and affinity electrophoresis with wheat-germ lectin compared for separating and quantifying alkaline phosphatase isoenzymes in plasma. 383 70

The major scrapie prion protein, designated PrP 27-30, exhibited both charge and size heterogeneity after purification from infected hamster brains. Eight or more discrete charge isomers of PrP 27-30 with isoelectric points ranging from approximately pH 4.6 to 7.9 were found by using non-equilibrium pH gradient electrophoresis in the first dimension followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. The charge isomers were detected by silver staining as well as by radioiodination. The procedures used to disaggregate PrP 27-30 before electrophoresis in the first dimension do not appear to be responsible for the charge heterogeneity. However, heating PrP 27-30 to 100 degrees C for 15 min in 0.1 N NaOH or 0.1 N HCl resulted in modification of the protein and alteration of its electrophoretic pattern. A PrP 27-30 fragment (molecular weight, 17,100 to 21,900) obtained by cyanogen bromide cleavage also exhibited charge and size heterogeneity. Periodic acid-Schiff staining of PrP 27-30 electrophoresed into sodium dodecyl sulfate-polyacrylamide gels demonstrated that carbohydrate residues are attached to the protein. Digestion of PrP 27-30 with neuraminidase and endo-beta-N-acetylglucosaminidase H resulted in significant changes in the isoelectric pH of PrP 27-30 isomers, whereas digestion with alkaline phosphatase had no effect. Our results demonstrate that PrP 27-30 is a sialoglycoprotein; this is consistent with several properties of this protein and of the scrapie prion.
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PMID:Scrapie PrP 27-30 is a sialoglycoprotein. 391 76


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