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 placental microsomal 5'-nucleotidase (EC 3.1.3.5) was prepared free of alkaline phosphatase by isoelectric focusing. A total of seven electrophoretic variants were isolated during the preparation of six placentas. Only three to six variants were found in a single placenta. The isoelectric pH's were 6.70, 6.44, 6.23, 6.02, 5.76, 5.63 and 5.44. These were found to be composed of variable quantities of a large, medium and low molecular weight form. The apparent molecular weights of the medium and light form of the enzyme were 86 500 and 43 500, respectively, as estimated from Stokes radius and sedimentation velocity determinations. The electrophoretic variants were not distinguishable with respect to specific activity and Michaelis constants for AMP, GMP or CMP or inhibition by ATP, CTP or adenosine. These electrophoretic variants appeared to be pseudoisozymes based upon different states of aggregation of a common primary sequence. There was a wide range of substrate specificity among nucleoside 5'-monophosphates which included 2-deoxyribose compounds. With AMP as 100, substrate activity was: CMP, 122; NMN, 74; GMP, 68: IMP, 63; XMP, 28 and UDP-glucose, 68. The Michaelis constants for AMP, GMP and CMP ranged from 12-18 muM, from 33-67 muM and from 170-250 muM, respectively. Although 5'-nucleotidase was active in the absence of divalent cation, 5 mM MgCl2 stimulated the enzyme activity to 234% of control and shifted the pH optimum of 9.8 to a plateau from pH 7.4-9.8.
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PMID:Purine catabolism in man: characterization of placental microsomal 5'-nucleotidase. 0 35

A membrane fraction from calf thymocytes was used to investigate molecular and catalytic properties of membrane-bound alkaline phosphatase (ortho-phosphoric-monoester phosphohydrolase EC 3.1.3.1). The principal findings were: 1. Solubilization of membranes with the non-ionic detergent Triton X-100 increases alkaline phosphatase activity by 30-40%. The enzyme activity elutes in a single peak (Stokes' radius = 7.7 nm) after chromatography in Sepharose 6B in the presence of Triton X-100. The activity also sediments as a single component of approx. 6.4 S during centrifugation in sucrose gradients containing Triton X-100. 2. Ion-exchange chromatography and isoelectric focusing in the presence of Triton X-100 indicate substantial charge heterogeneity. Two overlapping bands, a peak at pH 5.92 with a pronounced shoulder at pH 5.29, are apparent by isoelectric focusing. 3. The pH optimum for hydrolysis of p-nitrophenylphosphate (pNPhP) by the undissolved enzyme(s) is 9.57. Half-maximal activity occurs at pH 8.65 and ph 10.45. Triton X-100 has no effect on the pH profile. 4. Catalytic activity is affected by amines, especially analogues of ethanolamine. Diethanolamine exerts a unique stimulatory effect, but does not change the pH dependency. Increasing the concentration of diethanolamine from 0 to 1 M causes a 6-fold increase in Km and a 10-fold increase in the rate of hydrolysis of pNPhP. Glycine is inhibitory. 5. EDTA causes an irreversible loss of activity with t1/2 (1 mM EDTA, pH 8.2, 23 degrees C) = 3.5 h. Optimal activity is achieved in 0.1--1.0 mM Mg2+, although this does not cause the degree of activation reported to occur with the purified enzymes. Other divalent ions are inhibitory. Concentrations required to reduce activity to 50% of control are: Zn2+, 4.0 muM (no added Mg2+) and 30 muM (in the presence of 1 mM Mg2+); Mn2+, 0.25 mM (+/- Mg2+); Ca2+, 20 mM (+/- Mg2+). 6. Monovalent cations have little effect on activity. In the absence of added Mg2+, 50--150 mM Na+ is partially inhibitory, but markedly less so in the presence of 1 mM Mg2+. K+ has no significant effect. 7. Of the substrates tested, pNPhP (Km = 44 muM) was most rapidly hydrolyzed. Other substrates (rate relative to pNPhP) were alpha-naphthylphosphate (0.79), 2'-AMP (0.80), 5'-AMP (0.70), 3'-AMP (0.63), alpha-glycerophosphate (0.47) and glucose 6-phosphate (0.35). Phosphodiesterase activity was less than or equal to 10% of the phosphomonoesterase activity (for pNPhP) as evidenced by the lack of hydrolysis of bis(p-nitrophenyl)-phosphate and cyclic 3',5'-AMP. The ability of these substances to inhibit hydrolysis of pNPhP reflected their capacity as substrates, i.e. the most inhibitory were the most rapidly hydrolyzed.
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PMID:Calf thymus alkaline phosphatase. I. Properties of the membrane-bound enzyme. 1 42

A repressible alkaline phosphatase has been isolated from the extreme bacterial thermophile. Thermus aquaticus, and has been purified to homogeneity as judged by disc acrylamide electrophoresis and sodium dodecyl sulfate electrophoresis. Upon investigation, the purified enzyme was shown to hydrolyze certain phosphodiesters in addition to a wide variety of phosphomonoesters. The diesters included bis-p-nitro-phenyl phosphate and thymidine 3'-monophospho-p-nitro-phenyl ester. The temperature optimum for the diesterase activity was 80--85 degrees at pH 7.2. Orthophosphate competitively inhibited both activities. Nucleotides such as AMP, ADP, and ATP also inhibited both esterase activities as did alpha-D-glucose 1-phosphate and alpha-sodium glycerol phosphate. The isoelectric point of the enzyme was determined to be 8.4.
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PMID:Repressible alkaline phosphatase from Thermus aquaticus: associated phosphodiesterase activity. 1

1. Plasma membranes were isolated from ascites hepatoma AH-130 and rat livers with or without partial hepatectomy or bile duct ligation. Reciprocal manifestations of two marker enzymes for plasma membranes were observed in these membrane preparations; alkaline phosphatase activity was found much higher in the hepatoma membrane than in any preparations of the liver membranes, whereas 5'-nucleotidase activity was much lower in the former than in the latter. 2. Effects of lectins and anti-plasma membrane antiserum on these two marker enzymes were examined. The results showed that about 50% of apparent activity of 5'-nucleotidase found in the hepatoma membrane was exhibited by alkaline phosphatase. 3. Localizations of alkaline phosphatase and 5'-nucleotidase in polyacrylamide gels after electrophoresis were demonstrated using 5'-AMP and 5-Br, 4-Cl-indoxylphosphate as substrate. There was a difference in electrophoretic mobility between the alkaline phosphatase of the hepatoma and that of the liver.
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PMID:Contrast manifestation of alkaline phosphatase and 5'-nucleotidase in plasma membranes isolated from rat liver and ascites hepatoma. 2 Sep 52

Alkaline phosphatase was purified from plasma membranes of rat ascites hepatoma AH-130, the homogenate of which had 50-fold higher specific activity than that found in the liver homogenate. The presence of Triton X-100, 0.5%, was essential to avoid its aggregation and to stabilize its activity. The purified enzyme, a glycoprotien, was homogeneous in polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated a protein molecular weight of 140,000. The addition of beta-mercaptoethanol caused the dissociation of the alkaline phosphatase into two subunits of identical molecular weight, 72,000. Isoelectric focusing revealed that the pI of this enzyme is 4.7. The pH optimum for the purified enzyme was 10.5 or higher with p-nitrophenylphosphate, and slightly lower pH values (pH 9.5--10.2) were obtained when other substrates were used. Of the substrates tested, p-nitrophenylphosphate (Km-0.3 mM) was most rapidly hydrolyzed. Vmax values of other substrates relative to that of p-nitrophenylphosphate were as follows; beta-glycerophosphate, 76%; 5'-TMP, 82%; 5'-AMP, 62%; 5'-IMP, 43%; glucose-6-phosphate, 39%; ADP, 36% and ATP, 15%. More than 90% of the activity of the purified enzyme was irreversibly lost when it was heated at 55 degrees C for 30 min, or exposed either to 10 mM beta-mercaptoethanol for 10 min to 3 M urea for 30 min, or to an acidic pH below pH 5.0 for 2 h. Of the effects by divalent cations, Mg2+ activated the enzyme by 20% whereas Zn2+ strongly inhibited it by 95% at 0.5 mM. EDTA at higher than 1 mM inactivated the enzyme irreversibly, although the effect of EDTA at lower than 0.1 mM was reversible by the addition of divalent cations, particularly by Mg2+. The enzyme was most strongly inhibited by L-histidine among the amino acids tested, and also strongly inhibited by imidazole. These results suggest that alkaline phosphatase of rat hepatoma AH-130 is very similar to that of rat liver in most of the properties reported so far.
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PMID:Purification and characterization of alkaline phosphatase from plasma membranes of rat ascites hepatoma. 2 78

Purified chondrocytic alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) from bovine fetal epiphyseal cartilage hydrolyzes a variety of phosphate esters as well as ATP and inorganic pyrophosphate. Optimal activities for p-nitrophenyl phosphate, ATP and inorganic pyrophosphate are found at pH 10.5, 10.0 and 8.5, respectively. The latter two substrates exhibit substrate inhibition at high concentrations. p-Nitrophenyl phosphate demonstrates decreasing pH optima with decreasng substrate concentration. Heat inactivation studies indicate that both phosphorolytic and pyrophosphorolytic cleavage occur at the same site on the enzyme. Mg2+ (0.1-10.0 mM) and Mn2+ (0.01-0.1 mM) show a small stimulation of p-nitrophenyl phosphate-splitting activity at pH 10.5. Levamisole, Pi, CN-, Zn2+ and L-phenylalanine are all reversible inhibitors of the phosphomonoesterase activity. Pi is a competitive inhibitor with a Ki of 10.0 mM. Levamisole and Zn2+ are potent non-competitive inhibitors with inhibition constants of 0.05 and 0.04 mM, respectively. The chondrocytic alkaline phosphatase is inhibited irreversibly by Be2+, EDTA, EGTA, ethane-1-hydroxydiphosphonate, dichloromethane diphosphonate, L-cysteine, phenyl-methylsulfonyl fluoride, N-ethylmaleimide and iodoacetamide. NaCL, KCL and Na2SO4 at 0.5-1.0 M inhibit the enzyme. At pH 8.5, the cleavage of inorganic pyrophosphate (pyrophosphate phosphohydrolase, EC 3.6.1.1) by the chondrocytic enzyme is slightly enhanced by low levels of Mg2+ and depressed by concentrations higher than 1mM. Ca2+ show only inhibition. Similar effects of Mg2+ and Ca2+ on the associated ATPase (ATP phosphohydrolase, EC 3.1.6.3) activity were observed. Arrhenius studies using p-nitrophenyl phosphate and AMP as substrates have accounted for the ten-fold difference in V in terms of small differences in both the enthalpies and entropies of activation which are 700 cal/mol and 2.3 cal/degree per mol, respectively.
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PMID:Enzymatic characterization of the chondrocytic alkaline phosphatase isolated from bovine fetal epiphyseal cartilage. 4 Jun 3

The sum of the amounts of NAD + NADH was determined from the same acid tissue extract with the aid of a highly specific radioimmunoassay for 5'-AMP. NAD was converted to 5'-AMP via ADP-ribose by alkaline treatment while NADH was converted first to ADP-ribose by incubation of the acid extract at 25 degrees C followed by alkaline conversion to 5'-AMP. Removal of phosphate groups in NADP and NADPH by treatment of the extracts with alkaline phosphatase extended the procedure to the quantification of NADP(H). When combined with enzymic analyses of the oxidized coenzyme forms, NAD/NADH and NADP/NADPH ratios could also be obtained from the same extracts. The sensitivity of the test allows quantification of pyridine nucleotides in the range of 0.1--10 pmol.
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PMID:Determination by radioimmunoassay of the sum of oxidized and reduced forms of NAD and NADP in picomole quantities from the same acid extract. 4 Jul 93

The three xanthine derivatives, caffeine, theophylline and 3-isobutyl-1-methyl-xanthine (IBMX) produced dose-dependent increases in cyclic AMP concentrations in HeLa cells after long term treatment. Only IBMX produced increases over the first 60 minutes, with a peak of approximately 5-fold control values five to 10 minutes after the addition of the drug. About four hours after the addition of either 0.67 or 1.0 mM IBMX there was a second peak in the concentration of cyclic AMP which was at least as large and usually larger than the peak observed at five to ten minutes. Neither caffeine nor theophylline increased cyclic AMP concentrations above control values until one hour after addition of the compounds, and there was no indication of a peak in the concentration at four hours. Between 24 and 72 hours, all three compounds produced elevations in cyclic AMP levels that were steadily maintained. At any given concentration, the order of potency was IBMX greater than theophylline greater than caffeine. If the xanthine derivatives were removed from the medium after 24 hours of treatment, the cyclic AMP concentrations fell to control levels within one hour. Treatment with 5-iodo-2'-deoxyuridine (IdUrd) or hydrocortisone alone did not change the levels of cyclic AMP, nor did the presence of these inducers of alkaline phosphatase activity alter the effects of the xanthine derivations on cyclic AMP concentrations. The data showed a significant correlation between the magnitude of the increase in cycli AMP concentrations over the period from 24 to 72 hours and the degree of inhibition by the xanthine derivatives of the induction of alkaline phosphatase activity.
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PMID:The inhibition by xanthine phosphodiesterase inhibitors of the induction of alkaline phosphatase activity in HeLa cells: relationship of enzyme activity to cyclic AMP concentrations. 9 Jun 81

Group A streptococcal pyrogenic exotoxin (SPE) type C, produced by strain T18P grown in the presence of 32P, was separated from culture supernatant fluids by using alcohol precipitation. The resulting toxin (EtOH-1) contained 3 X 10(6) to 5 X 10(6) cpm of 32P per milligram of protein. The radiolabel migrated with SPE C during isoelectric focusing in polyacrylamide gels (pI 6.7) and double immunodiffusion, in which the toxin formed a line of identity with highly purified SPE C when reacted with hyperimmune antisera raised against SPE C. The EtOH-1 radiolabeled toxin was pyrogenic and had the capacity to enhance host susceptibility to lethal endotoxin shock. EtOH-1 toxin lost both radiolabel and biological activity after being treated with alkaline phosphatase. The nonspecific lymphocyte mitogenicity of purified unlabeled SPE C was stimulated by adenosine monophosphate but not adenosine, adenosine diphosphate, or adenosine triphosphate. Adenosine monophosphate may function as a cofactor of SPE C and contribute the phosphate group required for biological activity.
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PMID:Natural phosphorylation of group A streptococcal pyrogenic exotoxin type C. 12 Nov 10

Human adult lung fragments removed from macroscopically undamaged and anthracosis exempted zones of lungs of 20 pneumonectomies made for cancer, were tested for 25 enzymic activities. The location and intensities of these enzymic activities were different in the lung tissue components; The bronchial epithelia contained highly active LDH, MDH, SDH, NADH-TR and NADPH-TR, glucose-6-phosphate dehydrogenase, active hydroxyproline-2-epimerase, alkaline phosphatase. Ca2+-activated ATP-ase, and beta-galactosidase. Bronchial and vascular muscles presented intense activities of LDH, MDH and SDH of alkalinephosphatase, AMP-ase and Ca2+-activated ATP-ase, as well as of beta-galactosidase. The alveolar walls presented high activities of SDH, MDH and LDH, of alkaline and acid phosphatases, of beta-galactosidase and of Tween-40 and 60-esterases, of HEP, cytochrome-oxidase and peroxidase. The free alveolar macrophages were active for LDH, MDH, SDH, NADH-TR and NADPH-TR, G1-6-ph-DH, acid and alkaline phosphatase, cytochrome-oxidase and peroxidase, HEP, AMP-ase and Mg2+-activated ATP-ase, Tween-esterases, naphthol-ASD-acetate esterase, and beta-galactosidase. The endothelia contained high activities of alkaline phosphatase, of AMP-ase and Mg2+-activated ATPase, of LDH, MDH and SDH, and of beta-galactosidase. In bronchial lymphoid nodules it was the LDH, MDH, SDH, cytochrome-oxidase and peroxidase, HEP, alkaline phosphatase and AMP-ase, Tween-60-esterase and beta-galactosidase that were active. The interlobular areas of the lung presented intense activities of SDH, MDH, LDH, HEP and cytochrome-oxidase. The activities of the other tested enzymes were weaker or absent in the adult human lung components, the same as those of aminopeptidases which were present only in some free alveolar macrophages. The discussion of some relationships between these enzymic actitivies and the morphology of the human adult lung tissue asserted that the latter could not be considered as a "normal" tissue but as one overstrained by the components of blood and polluted air.
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PMID:Histoenzymology of the lung. I. Enzyme activities of the lung tissue of acult humans; relationships between structure and functions. 14 Mar 14


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