Showing papers on "Phosphatase published in 1976"
TL;DR: Two heat-stable and trypsin-labile inhibitors of phosphorylase phosphatase, designated inhibitor-1 and inhibitor-2, were partially purified from extracts of rabbit skeletal muscle by heating and coloumn chromatography using DEAE-dellulose and Bio-gel P-60.
Abstract: Two heat-stable and trypsin-labile inhibitors of phosphorylase phosphatase, designated inhibitor-1 and inhibitor-2, were partially purified from extracts of rabbit skeletal muscle by heating and coloumn chromatography using DEAE-dellulose and Bio-gel P-60. Inhibitor-1 exists in an active phosphorylated form and an inactive dephosphorylated form. The interconversion of phosphorylated inhibitor-1 and dephosphorylated inhibitor-1 is mediated by protein kinase dependent on adenosine 3':5'-monophosphate (cyclic AMP) and a Mn2+-stimulated phosphoprotein phosphatase. Inhibitory activity of inhibitor-2 is not influenced by treatment with either the kinase or the Mn2+-stimulated phosphatase. The molecular weights of inhibitor-1 and inhibitor-2 estimated by sodium dodecylsulfate-polyacrylamide gel electrophoresis are 26000 and 33000 respectively. Both inhibitor-1 and inhibitor-2 inhibit phosphorylase phosphatase by a mechanism which appears to be non-competitive with respect to the substrate phosphorylase a. Inhibitor fractions at early stages of purification also inhibit cyclic-AMP-dependent histone phosphorylation, but this kinase inhibitory activity resides with a protein moiety which is separable from inhibitor-1 and inhibitor-2.
430 citations
TL;DR: Two metabolically distinct types of bone cell populations were isolated from mouse calvaria by a repetitive digestive procedure with a mixture of collagenase and trypsin, and it is postulate that the CT type of populations is enriched in osteoclasts and, possibly, osteocytes, and the PT type of population is enrichment in osteoblasts.
Abstract: Two metabolically distinct types of bone cell populations were isolated from mouse calvaria by a repetitive digestive procedure with a mixture of collagenase and trypsin. Cells released early in the digestion showed approximately two fold increases in cAMP when treated with either parathormone or calcitonin. These populations were denoted CT type. Later eluting cells showed larger parathormone-induced increases in cAMP but did not respond to calcitonin. These populations were denoted PT type. Six metabolic and enzymatic activities were measured in the two types of populations: acid and alkaline phosphatases, hyaluronate synthesis, citrate decarboxylation, prolyl hydroxylase, and general protein synthesis. Although each of these activities was present in both cell types, the basal levels of acid phosphatase and hyaluronate synthesis were higher in the CT cells, whereas alkaline phosphatase, citrate decarboxylation, and prolyl hydroxylase were higher in the PT cells. Parathormone stimulated acid phosphatase...
398 citations
TL;DR: The results suggest that phosphatidylinositol-specific phospholipase C can specifically act on plasma membrane of rat kidney slices.
240 citations
TL;DR: The ATPase activities of myosin and heavy meromyosin, in the presence and absence of F-actin, were not significantly changed (+/- 10%) by phosphorylation of the P-light chain.
Abstract: 1. A method for the isolation of a new enzyme, myosin light-chain phosphatase, from rabbit white skeletal muscle by using a Sepharose-phosphorylated myosin light-chain affinity column is described. 2. The enzyme migrated as a single component on electrophoresis in sodium dodecyl sulphate/polyacrylamide gel at pH7.0, with apparent mol.wt. 70000. 3. The enzyme was highly specific for the phosphorylated P-light chain of myosin, had pH optima at 6.5 and 8.0 and was not inhibited by NaF. 4. A Ca2+-sensitive 'ATPase' (adenosine triphosphatase) system consisting of myosin light-chain kinase, myosin light-chain phosphatase and the P-light chain is described. 5. Evidence is presented for a phosphoryl exchange between Pi, phosphorylated P-light chain and myosin light-chain phosphatase. 6. Heavy meromyosin prepared by chymotryptic digestion can be phosphorylated by myosin light-chain kinase. 7. The ATPase activities of myosin and heavy meromyosin, in the presence and absence of F-actin, were not significantly changed (+/- 10%) by phosphorylation of the P-light chain.
236 citations
TL;DR: N-Ethylaminoethanol proved superior to the now widely used diethanolamine buffer, especially for the enzymes from the intestine and placenta, behaving as an uncompetitive activator.
Abstract: I studied the kinetics and sensitivity toward inhibition by levamisole and R 8231 of the most important human alkaline phosphatase isoenzymes. N-Ethylaminoethanol proved superior to the now widely used diethanolamine buffer, especially for the enzymes from the intestine and placenta, behaving as an uncompetitive activator. The optimum pH largely depends on the substrate concentration. The addition of Mg2+ has no effect on the activities. The meaning of Km-values for alkaline phosphatases is questioned. Isoenzymes from human liver, bone, kidney, and spleen are strongly inhibited by levamisole or R 8231 at concentrations that barely affect the enzymes from intestine or placenta. The inhibition is stereospecific, uncompetitive, and not changed by Mg2+. Inhibition is counteracted by increasing concentrations of N-ethylaminoethanol. The mechanism of inhibition is suggested to be formation of a complex with the phosphoenzyme.
198 citations
TL;DR: The results suggest that phosphoprotein phosphatases I and II are each active toward all of the substrates tested.
173 citations
01 Jan 1976
TL;DR: The main missing link is in the ionic effect whose elucidation might lead to the understanding of the mode of action of insulin, and can presently be explained in molecular terms.
Abstract: At the 1973 meeting on interconvertible enzymes, we summarized the data which establish that the activity of phosphorylase phosphatase in the liver is regulated by the concentration of glucose and that the activity of synthetase phosphatase is controlled by the concentration of phosphorylase a (Hers et al, 1974) The two phosphatases oppose the kinase system which is controlled by the concentration of cyclic AMP, according to the general scheme shown in Figure 1
139 citations
Journal Article•
TL;DR: It is hypothesized that the alkaline phosphatase of human tumor tissues reflects the expression of placental genes corresponding to one or more phases of trophoblastic development.
Abstract: Summary Alkaline phosphatase electrophoretic patterns characteristic of three phases in early human trophoblast development are described in this preliminary communication. Phase 1 (6 to 10 weeks) consists entirely of two heat-sensitive, l-homoarginine-inhibited bands, the slower one of which possesses antigenic determinants of liver-bone-type alkaline phosphatase, whereas the fast band lacks any of the known alkaline phosphatase antigenic determinants. Phase 2 pattern (11 to 13 weeks) is that of a mixture of Phase 1 and Phase 3 isozyme components, the latter exhibiting two isozyme bands with the characteristics of term placental alkaline phosphatase. These three patterns of developmental phase-specific placental alkaline phosphatases correspond in order to non-Regan isoenzyme, a mixture of Regan and non-Regan isozymes and Regan isoenzyme in a variety of human cancer tissues. The biochemical profile characteristic of trophoblast developmental Phase 1 alkaline phosphatase is expressed as 78.5% heat-sensitive inhibition (5 min at 65°), 66.3% l-homoarginine inhibition, and 17.3% l-phenylalanine inhibition where n = 12. It is hypothesized that the alkaline phosphatase of human tumor tissues reflects the expression of placental genes corresponding to one or more phases of trophoblastic development.
102 citations
TL;DR: It is concluded that liver phosphorylase phosph atase and glycogen synthase phosphatase have a common identity and that coordinate regulation of the phosphat enzyme-catalyzed activation of glycogen synthesase and inactivation of phosphoryLase occurs in vivo.
94 citations
TL;DR: Both enzymes recovered in the same manner temporally after oral glucose administration to adrenalectomized, fasted rats, suggesting an integrated regulatory mechanism for the two phosphatase.
84 citations
TL;DR: Low- and high-molecular-weight acid phosphatases were characterized by their elution volumes, specific inhibition and activity with two substrates and it is suggested that the distribution pattern found om rat tissues could be common to all eukaryotic cells.
TL;DR: 31P NMR spectra of phosphate and phosphonate complexes of Escherichia coli alkaline phosphatase have been obtained by Fourier transform NMR methods and suggest that the enzyme phosphoryl group is highly strained with an O-P-O bond angle of under 100 degrees.
TL;DR: Sephadex gel filtration was used to characterize phosphomonoesterases in two small takes in northern Sweden and found that phosphate served as an effective inhibitor of phosphatase A but had no such effect on phosphat enzyme B.
Abstract: Sephadex gel filtration was used to characterize phosphomonoesterases in two small takes in northern Sweden. Two fractions, here termed phosphatase A and phosphatase B, were found both as free enzymes and associated with seston. The activity of phosphatase A was correlated with the presence of algal biomass. Phosphatase B, on the other hand, was derived from zooplankton. Phosphate served as an effective inhibitor of phosphatase A but had no such effect on phosphatase B. Both fractions had pH optima between 6.5 and 7.0.
TL;DR: The progesterone-induced purple phosphatase isolated from the uterine flushings of pigs is activated by a variety of reagents that cleave disulfide bonds, including 2-mercaptoethanol, dithiothreitol, L-ascorbate, L
TL;DR: A new method for the synthesis of benzoyl phosphate was reported, which has the advantages of more rapid procedure, lower cost, and higher yield.
Abstract: A new method for the synthesis of benzoyl phosphate was reported. The advantages are: 1. more rapid procedure; 2. lower cost; 3. higher yield.
TL;DR: Potato acid phosphatase (EC 3.1.3.2) was used to remove the eight phosphate groups from alphas1-casein, which is homogeneous on gel electrophoresis with a slower mobility than native alphas 1- casein and has an amino acid composition which is identical to native alasts1-casesin.
01 Jan 1976
TL;DR: In this article, two distinct phosphorylase kinase phosphatases specific for the two sites of the primary site and secondary site were identified, indicating that reversal of the hormonal stimulation is controlled by the relative activities of two enzymes with opposing functions.
Abstract: Although the scheme hormone leads to raised cyclic AMP levels leads to activated protein kinase leads to phosphorylated protein leads to physiological response may represent an outline for the action of several hormones, in the best understood example, namely regulation of glucogen metabolism in mammalian muscle, the picture is more complex. Modification of phosphorylase kinase by cyclic AMP-dependent protein kinase, after stimulation by adrenaline, leads to phosphorylation of the enzyme at two sites. Activation is associated exclusively with the phosphorylation of the primary site, but the secondary phosphorylation indirectly antagonizes the primary phosphorylation in that it is necessary to render the primary site susceptible to dephosphorylation. The recent separation of two distinct phosphorylase kinase phosphatases specific for the two sites shows that reversal of the hormonal stimulation is controlled by the relative activities of two enzymes with opposing functions. Glycogen synthetase, which is phosphorylated and inactivated by cyclic AMP-dependent protein kinase, is also under the control of insulin. Although insulin appears to stimulate glycogen synthetase by reversal of the inactivation catalysed by the cyclic AMP-dependent protein kinase, tissue cyclic AMP concentrations do not alter. The recent identification of a second glycogen synthetase kinase, unaffected by cyclic AMP, therefore raises the possibility that insulin action may also be mediated through phosphorylation-dephosphorylation mechanisms, which antagonize those mediated through cyclic AMP-dependent protein kinase.
TL;DR: The unicellular alga Chlamydomonas reinhardi produces two constitutive acid phosphatases and three depressible phosphatase that can utilize napthyl phosphate as a substrate that differ in their heat sensitivity and affinity for glycerophosphate.
Abstract: The unicellular alga Chlamydomonas reinhardi produces two constitutive acid phosphatases and three depressible phosphatases (a neutral and two alkaline ones) that can utilize napthyl phosphate as a substrate. Specific mutants depressible phosphatase were used to investigate biochemical properties and the cytochemical localization of these enzymes. The two constitutive phosphatases show similar pH optima (about 5.0) and Km values (2 x 10(-3) to 3.3 x 10(-3) M) but differ in their heat sensitivity and affinity for glycerophosphate.
TL;DR: The control by light of the activities of the Calvin cycle enzymes involves a reduced agent formed by the photosynthetic electron transport chain, which seems appropriate to hypothesize that the light activation of the enzymes is not a single mechanism.
Abstract: The effect of light in activating fructose-1,6 biphosphate phosphatase (E.C. 3.1.3.11), sedoheptulose-1,7, biphosphate phosphatase (E.C. 3.1.3.11), ribulose-5 phosphate kinase (E.C. 2.7.1.19), ribulose-1,5 biphosphate carboxylase (E.C. 4.1.1.39) and (NADPH) glyceraldehyde-3 phosphate dehydrogenase (E.C. 1.2.1.13) in intact spinach chloroplasts in the presence of antimycin A, tetramethylethylenediamine (TMEDA) or chlorophenyl-1,1-dimethylurea (CMU) was examined. Antimycin A and TMEDA were added as stimulating agents for photosynthetic electron transfer in intact chloroplasts while CMU was added for its inhibitory characteristics.
Light exerted its control through the mediation of the photosynthetic electron transfer. Antimycin A and TMEDA promoted the light activation. CMU nullified the light activation as well as the stimulatory effect of antimycin A and TMEDA.
Thus the control by light of the activities of the Calvin cycle enzymes involves a reduced agent formed by the photosynthetic electron transport chain. From the presently available evidence, it seems appropriate to hypothesize that the light activation of the enzymes is not a single mechanism. In fact three types of enzymes can be distinguished: Ru-5 P kinase and (NADPH) G-3 P dehydrogenase, maximal activation of which appears within the first minute of illumination and is promoted by antimycin A and by TMEDA; F-1,6 P2 phosphatase and S-1,7 P2 phosphatase, ferredoxin-dependent enzymes, activation of which is slightly slower but is also promoted by antimycin A and by TMEDA; finally Ru-1,5 P2 carboxylase, activation of which is still slower and characterized by the absence of any response to antimycin A as well as to TMEDA.
TL;DR: Using this CDP-diglyceride Sephrose affinity column, this work was able to resolve the membrane associated 3-sn-phosphatidyl'1- Sn-glycerol (PG) synthesizing system present in Bacillus licheniformis into two activities.
Abstract: Cytidinediphospho-sn-1,2-diaclglycerol (CDP-diglyceride) has been covalently linked to Sephrose 4B via adipic acid dihydrazide spacer arm forming an effective affinity chromatography column. This liponucleo-tide ligand and sn-glycero-3-phosphate are subtracts for the formation of 3-sn-phoshatidyl-1'-sn-glycero-3'-phosphate (PGP) catalyzed in both eukaryotic and prokaryotic organisms by sn-glycero-3-phosphate: CMP phosphatidlytranferase (PGP synthetase). Using this CDP-diglyceride Sephrose affinity column we were able to resolve the membrane associated 3-sn-phosphatidyl'1-sn-glycerol (PG) synthesizing system present in Bacillus licheniformis into two activities. A PGP synthetase activity was adsorbed to the affinity column and was eluted using buffer containg CDP-diglyceride; a PGP phosphatease acactivity had no affinity for the column. Both PGP synthase and PGP phosphatase of B. licheniformis were associated with a membrane component of the cell as evidenced by sucrose gradient centrifugation, differential centrifugation, and solubilization by buffers containing detergent...
TL;DR: A model is proposed for the relationship between the active and inactive forms of the enzyme, and the possible regulation of this interconversion, which is fundamental to understanding of the molecular mechanisms of the regulation of glycogen metabolism.
TL;DR: Diphosphoglycerate mutase has been purified to homogeneity from outdated human erythrocytes, and the amino acid composition of the homogeneous protein has been determined.
Abstract: Diphosphoglycerate mutase has been purified to homogeneity from outdated human erythrocytes. The native enzyme has a molecular weight of 57 000 as determined by equilibrium centrifugation and exclusion chromatography. Disc gel electrophoresis in the presence of sodium dodecyl sulfate yields a single protein band with a molecular weight of about 26 500, indicating that diphosphoglycerate mutase is comprised of two subunits of similar mass. The enzyme exhibits the following intrinsic activities: diphosphoglyceratemutase, monophosphoglycerate mutase, and 2,3-diphosphoglycerate phosphatase. The latter activity is enhanced in the presence of either organic or inorganic anions. Glycolate-2-P, particularly, has a profound activating effect. Nonspecific phosphatase and enolase activities are absent. The enzyme has an extinction coefficient at 280 nm of 1.65 cm2/mg. The amino acid composition of the homogeneous protein has been determined.
TL;DR: Insulin administration reduced the sensitivity of the phosphatase reaction to glycogen inhibition, which could explain, at least in part, the increased phosphat enzyme activity noted in the insulin-treated, fasted rats since glycogen was routinely added to the homogenizing buffer.
TL;DR: Glycogen synthase b was purified from rabbit liver by a procedure involving isolation of the glycogen-enzyme complex, DEAE-cellulose chromatography, and affinity chromatography and appeared to be homogeneous by the criterion of polyacrylamide disc gel electrophoresis.
Abstract: Glycogen synthase b was purified from rabbit liver by a procedure involving isolation of the glycogen-enzyme complex, DEAE-cellulose chromatography, and affinity chromatography. The purified enzyme had a specific activity of 25 mumol of glucose transferred from UDPglucose into glycogen per min per mg of protein at 30 degrees C in the presence of 10 mM glucose 6-P, and appeared to be homogeneous by the criterion of polyacrylamide disc gel electrophoresis. The b form was convertible into the a form by a rabbit-liver protein phosphatase. A subunit size of 85 000 was determined by electrophoresis in sodium dodecyl sulfate and molecular weights of 183 000 +/- 20 000 and 170 000 +/- 21 000 were determined for the a and b forms of the enzyme, respectively. On conversion of the a into the b form, 1.13 mol of phosphate was incorporated per 85 000 g of protein. The degree of phosphorylation and loss of glycogen synthase a activity paralleled each other.
TL;DR: In this paper, the spectral properties and enzymatic activities of cobalt alkaline phosphatase have been investigated and it has been shown that magnesium increases the intensity of the absorption and magnetic circular dichroism (MCD) signals of the 4-Co(II) enzyme without evidence of changes in coordination geometry.
Abstract: Alkaline phosphatase of Escherichia coli, isolated by procedures which do not alter its intrinsic metal content, contains 1.3 +/- 0.3 g-atom(s) of magnesium and 4.0 +/- 0.2 g-atoms of zinc per mol of molecular weight 89 000 (Bosron et al., 1975). Substitution of Co(II) for Zn(II) and/or Mg(II) results in spectral properties which can be correlated with enzymatic activity. Magnesium does not activate the apoenzyme but augments the activity of 2-Co(II) enzyme almost 3-fold and that of the 4-Co(II) enzyme 1.3-fold. The magnesium-induced increase in activity of the 2-Co(II) enzyme is accompanied by spectral changes which are consistent with an alteration from largely octahedral-like to pentacoordinate-like coordination geometry. Magnesium increases the intensity of the absorption and magnetic circular dichroism (MCD) signals of the 4-Co(II) enzyme but without evidence of changes in coordination geometry. Cobalt when bound to the magnesium sites results in octahedral-like EPR spectra, unperturbed by phosphate which significantly affects cobalt at the pentacoordinate-like sites. In the absence of magnesium, 6 g-atoms of cobalt are required to maximize the spectral properties, but activity does not increase further after the addition of only 4 g-atoms of cobalt, while activity is optimal with only 2 g-atoms of cobalt. Hydrogen-tritium exchange measurements indicate that magnesium also stabilizes the dynamic structural properties of the apo- and 2-Co(II) enzymes but has little effect on the structure of 4-Co(II) phosphatase. The response to magnesium of both the spectral properties and enzymatic activities of cobalt alkaline phosphatase demonstrates that magnesium regulates cobalt (and zinc) binding and modulates the activity of the resultant products.
TL;DR: A repressible alkaline phosphatase has been isolated from the extreme bacterial thermophile, Thermus aquaticus and Amino acid analysis of the protein revealed little in the amino acid composition to separate it from other mesophilic enzymes which have been previously studied.
TL;DR: Screening for the phosphatases in placentae was carried out to establish the frequency of the L-leucine-sensitive placental phenotypes and to provide enough enzyme to enable purification and characterisation of cancer tissue.
Abstract: FISHMAN et al. reported that human placental alkaline phosphatase (Regan isoenzyme) was found in the serum and tumour tissue of a patient with lung cancer1. Two electrophoretically different variant forms of Regan alkaline phosphatase with a greater sensitivity to inhibition by L-leucine have since been described: the ‘Nagao isoenzyme’2, and a hepatoma-specific alkaline phosphatase3–5. Inglis et al.6 reported that the L-leucine-sensitive Nagao isoenzyme found in some cancer patients closely resembled the ‘D variant’ of human placental alkaline phosphatase, a rare phenotype defined electrophoretically on starch gel which is found in less than 1% of the general population of pregnancies7. An example was reported of a pregnancy serum D-phenotype enzyme presumably of placental origin, confirmed by electrophoresis established as L-leucine-sensitive6. Most interesting was the occurrence of the variant at high frequency in the ovarian cancer patients surveyed6,8. To extend the observations of Inglis et aL, screening for the phosphatases in placentae was carried out, both to establish the frequency of the L-leucine-sensitive placental phenotypes (which should match the frequency of the electrophoretically defined D variant phenotypes) and to provide enough enzyme to enable purification and characterisation. Supply of cancer tissue is generally restrictive for this purpose.
TL;DR: Three major cytoplasmic and two major wall-bound acid phosphatases were separated by gel filtration on Sephadex G-75 and ion-exchange chromatography on carboxymethyl cellulose, and some of their characteristics were compared.
Abstract: In wheat (Triticum aestivum) roots, about 67% of the total activity of acid phosphatase was associated with the cell wall debris, and 35% of it was released from the wall by incubation with 0.8 M N...
TL;DR: Chick intestinal brush border proteins were examined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate and a large molecular weight protein present in the vitamin D-deficient brush borders diminishes and a larger protein appears.
TL;DR: Alkaline phosphatase purified from a Burkitt lymphoma cell line and Moloney-virus-induced murine leukemia (YAC) showed unique catalytic properties in substrate specificity and inhibition by cysteamine-S-phosphate.
Abstract: Alkaline phosphatase [orthophosphoricmonoester phosphohydrolase (alkaline pH optimum), EC 3.1.3.1] purified from a Burkitt lymphoma cell line (Daudi) and Moloney-virus-induced murine leukemia (YAC) showed unique catalytic properties in substrate specificity and inhibition by cysteamine-S-phosphate. It migrated on polyacrylamide gel electrophoresis in a single activity band. Alkaline phosphatase with similar properties was found in several human lymphoblastoid cell lines, in chronic lymphatic leukemic cells, in organs of leukemic mice, and in sera of patients with certain lymphoproliferative disorders.