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)

Human megakaryocyte colony formation was observed in serum-free agar cultures with a medium containing deionized bovine serum albumin, iron-saturated transferrin, 2-mercaptoethanol, and recombinant human interleukin 3 (IL-3). Megakaryocyte colonies were identified in situ by the alkaline phosphatase anti-alkaline phosphatase technique, using monoclonal antibody against platelet glycoprotein IIb/IIIa. Colony numbers reached a peak at day 14, with a mean of 47 megakaryocyte colonies (range 23-104) per 2 x 10(5) bone marrow mononuclear cells. Recombinant human IL-3 stimulated the growth of megakaryocyte progenitors. Similar results were obtained using nonadherent, T-cell-depleted mononuclear cells. These findings suggest that IL-3 stimulates the growth of megakaryocyte progenitors directly without activation of accessory cells, or the requirement of factors present in the serum or plasma. This serum-free culture system may be useful for studying the effects of purified natural and recombinant biological regulators on human megakaryocyte progenitors.
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PMID:Clonal growth of human megakaryocyte progenitors in serum-free cultures: effect of recombinant human interleukin 3. 326 26

The isolated brush border membrane of the tapeworm, Hymenolepis diminuta, hydrolyzes p-nitrophenyl phosphate over a broad pH range. Acid phosphatase activity (pH optimum at 4.0) is inhibited specifically by sodium dodecyl sulfate (SDS) and NaF, while the alkaline phosphatase activity (pH optimum at 8.8) is inhibited specifically by levamisole, 2-mercaptoethanol, and ethylenediaminetetra-acetate (EDTA). These two phosphatase activities are further differentiated in that (1) there is a rapid decrease in alkaline phosphatase activity when the membrane preparation is incubated at pH 4.0, while there is little loss of acid phosphatase activity, and (2) the alkaline phosphatase activity is solubilized with no loss of activity when the membrane is treated with Triton X-100, while such treatment causes a significant loss of acid phosphatase activity. Both activities are nonspecific and hydrolyze a variety of phosphorylated compounds, but the relative activities of the two phosphatases against these substrates vary significantly.
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PMID:Acid phosphatase activity in the isolated brush border membrane of the tapeworm, Hymenolepis diminuta: partial characterization and differentiation from the alkaline phosphatase activity. 341 89

A 15-kilodalton protein has been identified as a major component of the residual protein fraction of mouse epididymal/vas spermatozoal heads, demembraned by treatment with Triton X-100 and sequentially extracted with 1 M NaCl/2-mercaptoethanol/DNase I. Two-dimensional electrophoresis of that protein before and after treatment with alkaline phosphatase indicated that it is present in epididymal/vas spermatozoa as a series of five differentially phosphorylated molecules with pI 6.0-7.0. Cyto-immunofluorescence with an affinity-purified antibody to the 15-kDa protein localized that protein to a circumscribed region of the demembraned mouse sperm head mediad from the dorsal margin. By radioimmunoassay, the 15-kDa protein was shown to be sperm-unique and species-specific. The antibody was nonreactive with homogenates of meiotic spermatogenic cells and round spermatids (stages 1-11) but was reactive with a non-phosphorylated 15.5-kDa protein of elongating spermatids (stages 12-16) and testicular spermatozoa. Following alkaline phosphatase treatment, the spermatozoal 15-kDa protein migrated to the position of the spermatidal 15.5-kDa protein on a sodium dodecyl sulfate gel. Thus, we conclude that the 15-kDa protein of mouse spermatozoa is synthesized during the elongation phase of spermiogenesis (stages 12-16) and is phosphorylated in the terminal period of that phase and/or after excursion of spermatozoa from the seminiferous tubules.
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PMID:Proteins of demembraned mouse sperm heads. Characterization of a major sperm-unique component. 388 61

31P NMR has been used to investigate the nature of the two chemically distinct phosphorylation sites of ATP-citrate lyase from rat liver. The "regulatory" or "structural" phosphorylation site is acid stable and known to be phosphoserine. The "catalytic" site is very acid labile and has been suggested by different workers to contain either phosphohistidine or an acyl phosphate group. We have demonstrated the presence of both endogenous phosphoserine and phosphoserine introduced after treatment of the lyase with the catalytic subunit of cAMP-dependent protein kinase. This structural phosphate group could be titrated and was readily removed by alkaline phosphatase; these facts, together with the narrow line width of the 31P NMR signal, suggest that it is relatively mobile and located near the surface of the protein. 31P NMR spectra of ATP-citrate lyase that had previously been exposed to fairly high concentrations of potassium chloride (1.5 M), or that had been denatured in detergent and 2-mercaptoethanol, clearly identified phosphohistidine as the catalytic phosphate group. That phosphohistidine is indeed a catalytic intermediate was demonstrated by the disappearance of the resonance in the presence of the substrates citrate and coenzyme A. The line width of the phosphohistidine resonance indicated that the catalytic phosphohistidine residue has negligible residual mobility on the protein. These results are consistent with the pattern of earlier observations on the chemical environments of phospho groups that serve a regulatory or structural role as opposed to a catalytic function in proteins.
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PMID:Phosphorus-31 nuclear magnetic resonance study of the active site phosphohistidine and regulatory phosphoserine residues of rat liver ATP-citrate lyase. 393 62

1. The purification of a nuclease from rat-liver mitochondria is described. The mitochondria are rendered soluble by treatment with Triton X-100 and, after fractionation with ammonium sulphate and acetone, the active fraction is further purified by chromatography on DEAE-cellulose and Sephadex G-75 to give a purification of over 700-fold. 2. The purified enzyme was only very slightly contaminated with deoxyribonuclease II, phosphodiesterase and phosphomonoesterase. The individual activities of these enzymes did not exceed 0.1% of the activity of the liver nuclease. 3. The purified enzyme attacked RNA more rapidly than denatured DNA and hydrolysed native DNA more slowly than denatured DNA. 4. There is some evidence to suggest that the nucleolytic activity of the purified preparation towards native DNA, denatured DNA and RNA is associated with a single protein. 5. The enzyme is relatively labile but is stabilized in the presence of 20% (w/v) glycerol or 10mm-2-mercaptoethanol.
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PMID:The purification from rat liver of a nuclease hydrolysing ribonucleic acid and deoxyribonucleic acid. 591 28

Spectrophotometric and isoelectric focusing (IEF) electrophoretic characterization of the alkaline phosphatase (ALKP) of the mosquito, Culex tarsalis, are presented. With p-nitrophenylphosphate (Pnp) as substrate, ALKP was optimally active at 37 degrees C, pH 8.0, 30 mM MgCl2, Vmax was 35.8 mumoles/10 min and the Km was 5.7 mM, with no demonstrable requirement for Zn2+. The spectrophotometric enzyme(s) was stimulated by dithiothreitol, 2-mercaptoethanol, and poly-vinylpyrollidone (PVP); inhibited by NaF, several alternative cations (Ca2+, Ba2+, Fe2+, Cu2+), and EDTA. ALKP activity was cyclic during the 15 day post-adult emergence period of the study. No significant differences were noted between the specific activities of males and females. IEF electrophoresis revealed 6 ALKP isozymes detected with alpha-naphthylphosphate within the pH range 4.0-5.5, with a second group of 3 rather indistinct species in the pH 6.0-7.0 range. IEF ALKP isozymes were stimulated by Mg2+ and PVP and inhibited by EDTA (except ALKP5.0) and cysteine; partial inhibition with phenylalanine. IEF detection of ALKP activity with Pnp indicated that the majority of the activity was localized in the pH 4.0-5.5 range, in close agreement with the alpha-naphthylphosphate results.
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PMID:Alkaline phosphatases of the mosquito, Culex tarsalis Coquillett. 646 96

Studied was the effect of beta-glucuronidase, hydrocortisone, and mercaptoethanol in bull semen on the activity of the alkaline phosphatase and the viability and fertilizing capacity of spermatozoa. It was found that the beta-glucuronidase enzyme in conc. of 270 UI per cu. cm of semen enhanced the activity of one of the forms of AP in agar electrophoresis (AP-1) with the simultaneous enhancement of the thermal resistance of spermatozoa at 46 degrees C and their fertilizing capacity by 9.6 per cent at first insemination. At minimum concentration hydrocortisone (1 X 10(-6) M) lowered the heat resistance of spermatozoa at 39 degrees C. Mercaptoethanol was found to lower by 3 per cent the activity of semen alkaline phosphatase. It is suggested to use the beta-glucuronidase enzyme in the practice of artificial insemination out of all other biologically active agents studied.
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PMID:[Effect of biologically active substances on the alkaline phosphatase activity, viability and fertility of bull spermatozoa]. 688 16

DNases A1 and A2 have been purified to homogeneity from the hepatopancreas of Achatina fulica by a series of steps: acetate buffer extraction, ammonium sulfate precipitation and column chromatography on hydroxylapatite, phosphocellulose, Blue-Sepharose, and poly(A)-Sepharose. The purified enzymes are free of acidic phosphomonoesterase, phosphodiesterase, and RNase activities. They are slightly acidic glycoproteins with identical isoelectric point (6.90). On 0.1% SDS gel electrophoresis, DNase A2 had a molecular weight of 30,000 when dissolved in 1% SDS, but it had molecular weights of 17,500, 8,000, and 4,800 when dissolved in 1% SDS and 1% 2-mercaptoethanol. This was evidence that the enzyme consists of three different subunits joined by interchain disulfide bonds. DNases A1 and A2 are endonucleases working at acidic pH (3.5--6.0) and do not require divalent cations for their activities. The enzymes degrade poly(dA) 5 times faster and poly(dT) 3 times faster than heat-denatured DNA under optimal conditions but do not appreciably digest poly(dG) and poly(dC). We developed an analytical procedure for oligodeoxynucleotides by high-performance liquid chromatography. The phosphomonoester end group and the mode of degradation were examined by the method. The termini produced by the enzymes have 3'-phosphoryl and 5'-hydroxy end groups. The products of exhaustive hydrolysis contain di-, tri-, tetra-, and pentanucleotides and mononucleotide was barely detected. The hydrolyzing activities of DNases A1 and A2 are stimulated by polyamines such as spermine, spermidine, and putrescine, but are inhibited by synthetic polynucleotides and various drugs. Adenosine deaminase highly active on oligoadenylic acids was found in a crude DNase A fraction. The enzyme preparation has higher activity on 3'-adenylic acid than on 5'-adenylic acid. The first adenosine residue of oligoadenylic acids was deaminated considerably more rapidly than the second or succeeding ones.
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PMID:DNase A, a poly(dA) and poly(dT)-specific deoxyribonuclease from Achatina fulica. Purification and characterization. 733 15

Two forms of alkaline phosphatase exist in the integument of the "white pupae" (wp) and dark pupae (dp) mutant strains of Ceratitis capitata, during transition from larvae to pupae. They were separated by DEAE-cellulose chromatography. Both isoenzymes have a molecular weight of approximately 180,000 and two pH optima, at 9.4 and at 11.0. The isoenzymes of the "dark pupae" mutant catalyze the hydrolysis of phosphotyrosine and beta-glycerophosphate but not phosphoserine, phosphothreonine, ATP, and AMP. In contrast, the isoenzymes of the white pupae mutant hydrolyze all the substrates tested. The ALPase 1 of the dark pupae mutant was inhibited by L-tyrosine, but L-phenylalanine had no effect on either isoenzyme. The effects of divalent cations, EDTA, temperature, urea, and 2-mercaptoethanol were also investigated. Electrophoretic analysis did not reveal any variants of the larval and pupal isoenzymes, but ALPase A, an adult stage-specific isoenzyme, was found to be polymorphic. The electrophoretic variants were shown to be controlled by three codominant alleles located on the third chromosome of Ceratitis capitata. Since we found no hybrid enzyme, we conclude that ALPase A is monomeric.
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PMID:Biochemical and genetic studies on alkaline phosphatase of Ceratitis capitata. 812 97

Venom alkaline phosphatase was detected using a blotting method following electrophoresis. The enzyme gave strong reactions in some venoms, but was absent in other venoms, some within the same species. The mol. wt of the enzyme is close to 100,000 and its pI is between 3.6 and 4.8. The enzyme was inactivated by EDTA and 2-mercaptoethanol, and lost activity by freezing and thawing. Endogenous venom alkaline phosphatase can interfere with alkaline phosphatase-based detection methods. Pre-screening for endogenous venom alkaline phosphatase is recommended prior to using alkaline phosphatase-based detection methods when studying snake venom.
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PMID:Detection of alkaline phosphatase in venom by blotting methods. 815 62


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