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)

Guanylate cyclase is dephosphorylated in response to the interaction of egg peptides with a spermatozoan surface receptor (Suzuki, N., Shimomura, H., Radany, E. W., Ramarao, C. S., Ward, G. E., Bentley, J. K., and Garbers, D. L. (1984) J. Biol. Chem. 259, 14874-14879). Here, the phosphorylated form of guanylate cyclase was purified to apparent homogeneity from detergent-solubilized spermatozoan membranes by the use of GTP-agarose, DEAE-Sephacel, and concanavalin A-Sepharose chromatography. To prevent dephosphorylation of the enzyme during purification, glycerol (35%) was required in all buffers. Following purification, a single protein-staining band of Mr 160,000 was obtained on sodium dodecyl sulfate-polyacrylamide gels. The final specific activity of the purified enzyme was 83 mumol of cyclic GMP formed/min/mg of protein at 30 degrees C, an activity 5-fold higher than that observed with the purified, dephosphorylated form of guanylate cyclase. A preparation containing protein phosphatase from spermatozoa, or highly purified alkaline phosphatase (from Escherichia coli), catalyzed the dephosphorylation of the enzyme; this resulted in a subsequent decrease in guanylate cyclase activity and a shift in the Mr from 160,000 to 150,000. The phosphate content of the high Mr form of the enzyme was 14.6 mol/mol protein whereas the phosphate content of the low Mr form was 1.6 mol/mol protein. All phosphate was localized on serine residues. The Mr 160,000 form of guanylate cyclase demonstrated positive cooperative kinetics with respect to MnGTP while the Mr 150,000 form displayed linear, Michaelis-Menten type kinetics. The phosphorylation state of the membrane form of guanylate cyclase, therefore, appears to dictate not only the absolute activity of the enzyme but also the degree of cooperative interaction between catalytic or GTP-binding sites.
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PMID:Purification and properties of the phosphorylated form of guanylate cyclase. 289 12

Acute renal failure was diagnosed by clinical, necropsy and histological criteria in 39 flocks (20 low ground, 13 hill and six marginal upland) in areas served by six veterinary investigation centres. Forty-eight lambs of 12 different breeds or crosses were investigated. The mean age of affected lambs was 38 days (range seven to 84 days); 21 lambs (44 per cent) were aged seven to 28 days, while only eight (17 per cent) were older than two months. Mortality in clinically affected lambs was almost 100 per cent, with no response to various treatments. Histological examination showed that 40 lambs (83 per cent) had nephrosis, while the rest had toxic tubular necrosis, interstitial nephritis or tubular damage associated with oxalate crystal deposits. Only about half of the lambs had any evidence of enteric infections or enteropathy. Acutely ill lambs had azotaemia, haemoconcentration and proteinuria; some lambs had glycosuria or haematuria. Samples of plasma from 22 lambs with nephrosis were compared with similar samples from 82 incontact but asymptomatic lambs. The clinically affected group had significantly elevated plasma urea, creatinine, total protein, globulin, phosphorus and chloride concentrations and significantly reduced plasma calcium concentrations compared with healthy lambs. Affected lambs had a significant reduction also in the calcium:phosphorus ratio. No significant differences between groups was found in plasma concentrations of albumin, glucose, lactate, glycerol, creatine kinase, alkaline phosphatase, sodium, potassium or magnesium.
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PMID:Acute nephropathy in young lambs. 291 11

Organic solvents (ethylene glycol, glycerol, dimethyl sulphoxide, dimethylformamide, dioxane, methanol and propanol-2, as well as sucrose and urea) have been included in aqueous two-phase (liquid-liquid) systems comprised of water, dextran and poly(ethylene glycol). The concentration of the organic solvent was in most cases 20% (w/w). The influence of these solvents on the phase-forming properties, the volume ratio, the freezing point and the partitioning of a polymer-bound ligand, Procion Red HE-3B poly(ethylene glycol), has been studied. The partition coefficients for alkaline phosphatase decrease with ethylene glycol, glycerol, sucrose and urea (factors of 0.25-0.5), but increase with the other substances (factors of 1.2-1.6). The temperature effects on the partitioning of alkaline phosphatase from calf intestine as well as of phosphofructokinase from yeast in systems containing ethylene glycol have been studied and compared with partitioning in standard systems, not containing solvents. The possible uses of the above systems for partitioning studies of enzymes are discussed.
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PMID:Effects of organic solvents on the partitioning of enzymes in aqueous two-phase systems. 295 91

Oligomycin-sensitive particulate ATPase (MB ATPase) from L. donovani promastigotes was solubilized by chloroform treatment. Polyacrylamide gel electrophoresis revealed several protein bands, with the major one possessing ATPase activity. The solubilized enzyme had Mg2+-ATPase and Ca2+-ATPase but no K+-dependent alkaline phosphatase activity. The Mg2+-ATPase activity was stimulated by monovalent cations and was not sensitive to oligomycin. Hence it is referred to as F1 ATPase. It had optimum activity at pH 7.6 and 30 degrees C. The Arrhenius plot for MB ATPase was biphasic with activation energies (Ea) of 16.2 and 3.4 kcal mol-1, while F1 ATPase exhibited a linear plot with Ea = 10.1 kcal mol-1. Lineweaver-Burk plots were biphasic with Km values of 0.17 and 1.25 mM for MB ATPase and 0.18 and 1.33 mM for F1 ATPase. The enzyme could be preserved at -15 degrees C in Tris-SO2-(4)-EDTA-ATP-glycerol (t1/2 = 20 days).
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PMID:Solubilization and kinetic characterization of mitochondrial adenosine triphosphatase from Leishmania donovani promastigotes. 297 May 89

Human bone cell cultures were established by maintaining collagenase-treated bone fragments in low Ca++ medium. The resulting cell cultures exhibited a high level of alkaline phosphatase activity and produced a significant increase in intracellular cAMP when exposed to the 1-34 fragment of human parathyroid hormone. With continued culture, the cells formed a thick, extracellular matrix that mineralized when cultures were provided daily with normal levels of calcium, fresh ascorbic acid (50 micrograms/ml) and 10 mM beta-glycerol phosphate. Biosynthetically, these cells produced type I collagen (without any type III collagen), and the bone-specific protein, osteonectin. In addition, the cells produced sulfated macromolecules electrophoretically identical to those positively identified as the bone proteoglycan in parallel cultures of fetal bovine bone cells. This technique provides a useful system for the study of osteoblast metabolism in vitro.
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PMID:Human bone cells in vitro. 299 72

When 1 mM ATP is added to human dermal fibroblasts (DF) in monolayer culture permeabilized by glycerol, they undergo a rapid reduction in length and their intracellular actin filaments aggregate. This process is referred to as cell contraction. Treating glycerol-permeabilized DF with alkaline phosphatase before adding 1 mM ATP should cause dephosphorylation. Dephosphorylated preparations do not undergo cell contraction initiated by ATP. When myosin light-chain kinase (MLCK) isolated from turkey gizzard is added with cofactors to cells dephosphorylated by alkaline phosphatase treatment, contraction is restored. DF incubated for 24 h with db cAMP or cholera toxin show elevated intracellular concentrations of cAMP and little cell contraction. Contraction is reestablished when MLCK with cofactors is incubated with these preparations before ATP is added. Fibroblasts from Epidermolysis Bullosa dystrophica recessive patients produce excess cAMP. Those cells show minimal contraction, however; treating them with MLCK and cofactors renews contraction brought about by ATP. When DF are incubated with trifluoperazine to block calmodulin-dependent enzyme reactions, cell contraction is inhibited. Adding cytochalasin B disrupts microfilaments and also inhibits contraction. This work supports the idea that myosin ATPase is critical to cell contraction. Myosin ATPase is dependent on the phosphorylation of the regulatory peptide, myosin light chain. Elevating intracellular concentrations of cAMP or treatment of permeabilized cell preparations with alkaline phosphatase may inhibit myosin ATPase activity. The restoration of phosphorylation by adding MLCK with cofactors served to reestablish cell contraction.
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PMID:ATP-induced cell contraction in dermal fibroblasts: effects of cAMP and myosin light-chain kinase. 301 87

1-Acyl-sn-glycerol-3-phosphate acyltransferase (also called lysophosphatidic acid acyltransferase) which catalyzes the acylation of 1-acyl-sn-glycerol-3-phosphate to phosphatidic acid is generally assayed by the use of a radioactive substrate followed by a time-consuming chromatographic separation of substrate and product. We report a direct and highly sensitive nonchromatographic assay for this enzyme based on the ability of Escherichia coli alkaline phosphatase to dephosphorylate 1-acyl-sn-glycerol-3-phosphate but not phosphatidic acid. This selective hydrolysis coupled with the use of 32P-labeled 1-acyl-sn-glycerol-3-phosphate as substrate permits measurement of the product, 32P-labeled phosphatidic acid by solvent extraction or precipitation. We also report a series of enzymatic reactions for the efficient conversion of 32Pi to 32P-labeled 1-acyl-sn-glycerol-3-phosphate.
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PMID:A direct nonchromatographic assay for 1-acyl-sn-glycerol-3-phosphate acyltransferase. 305 7

Ros 17/2 clonal rat osteosarcoma cells calcify when cultured in the presence of 10 micrograms/ml beta-glycerol phosphate in an agarose gel. Culture in 1% agarose inhibited cell division while allowing cells to remain metabolically active and viable for over 21 days. Serial photography of the same microscopic field shows a progressive deposition of calcium phosphate during the course of the experiment. The deposition of calcium around cells was confirmed by calcium-specific stains, and by energy dispersive X-ray analysis (EDX) during scanning electron microscopy. Cells with high calcium content analyzed by EDX had Ca:P ratios similar to hydroxyapatite. Total calcium progressively increased in beta-glycerol phosphate-treated cultures whereas the control plates maintained a constant calcium content over 16 days. Alkaline phosphatase activity increased with time in culture whereas cells with beta-glycerol phosphate maintained the alkaline phosphatase values achieved at the time of initial calcification. Alkaline phosphatase staining revealed no correlation between the presence of the enzyme activity and calcification. Radioimmunoassay for the bone-specific vitamin K-dependent protein bone Gla protein showed that beta-glycerol phosphate-treated cells accumulate over sixfold greater amounts of this protein. Our studies show that ROS cells can calcify and accumulate bone-specific matrix components when cultured in a 3-dimensional agarose matrix.
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PMID:Calcification of osteoblastlike rat osteosarcoma cells in agarose suspension cultures. 312 Nov 51

The effect of heat shock on Myxococcus xanthus was investigated during both glycerol- and starvation-induced development. Cells heat shocked at 40 degrees C for 1 h prior to a development-inducing signal displayed an accelerated rate of myxospore formation at 30 degrees C. Additionally, M. xanthus cells heat shocked prior to glycerol induction formed a greater total number of myxospores when sporulation was complete than did control cells maintained at 30 degrees C. However, in starvation-induced fruiting cells the total number of myxospores in control and heat-shocked populations was about equal when fruiting body and myxospore formation was complete. When extended heat shock (3 h) was applied to cells prior to development, no acceleration of myxospore formation was observed. Heat shock elicited the premature expression of many developmentally regulated proteins. Cell fractionation and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography revealed the subcellular location and molecular weights of the 18 glycerol-induced and 9 starvation-induced developmental proteins. Comparison with previously identified M. xanthus heat shock proteins showed that nine of the developmental proteins found in glycerol-induced cells and three of the developmental proteins found in starvation-induced cells were heat shock proteins. Furthermore, heat shock increased the activity of alkaline phosphatase, a developmentally regulated enzyme, in vegetative cells, glycerol-induced cells, and starvation-induced cells.
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PMID:Acceleration of starvation- and glycerol-induced myxospore formation by prior heat shock in Myxococcus xanthus. 314 80

The Escherichia coli glpT gene encodes a transport protein that mediates uptake of sn-glycerol-3-phosphate. This permease is a member of a class of bacterial organophosphate permeases which transport substrates by antiport with inorganic phosphate. The glpT gene product, probably an oligomer of a single polypeptide chain, is thought to span the cytoplasmic membrane several times, as predicted by the hydropathic profile. Protein fusions, in which varying lengths of the amino-terminal end of the permease is attached to alkaline phosphatase (phoA) and to beta-galactosidase (lacZ) were constructed. On the assumption that phoA fusions only exhibit high enzymatic activity when fused to extra-cytoplasmic regions of the target protein, whereas lacZ fusions will only be active when the beta-galactosidase portion is attached to cytoplasmic domains of the target protein, the activities of the fusions were used to test a two-dimensional model for the permease. The model proposes that GlpT contains 12 transmembrane segments divided by a larger cytoplasmic region. Despite some limitation caused by hot-spot sites of transpositions, the TnphoA approach was consistent with the model. In contrast, we feel that the enzymatic activity of lacZ fusions is only a limited parameter for studying the topology of a complex membrane protein.
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PMID:The transmembrane topology of the sn-glycerol-3-phosphate permease of Escherichia coli analysed by phoA and lacZ protein fusions. 314 44


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