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)

A procedure for the purification of cholesterol ester hydrolase from bovine adrenal cortical 105000 x g supernatant is described. Preincubation of a crude enzyme extract with [gamma-32P]ATP followed by purification resulted in the isolation of a phosphorylated preparation of cholesterol ester hydrolase. The phosphorylated cholesterol ester hydrolase appeared to be composed of 4 subunits, each having a molecular weight of 41000 +/- 280, only one of which may be phosphorylated. Preincubation of the crude enzyme preparation with [alpha-32P]ATP followed by purification did not produce a phosphorylated preparation of cholesterol ester hydrolase. Cyclic-AMP-dependent protein kinase, cyclic AMP, ATP and magnesium ions were required for activation of purified cholesterol ester hydrolase in vitro and the time course of activation closely paralleled the time course of phosphorylation of the enzyme. The addition of ATP, cyclic AMP and magnesium ions to the bovine adrenal cortical 105000 x g supernatant produced a 2.5-fold stimulation in cholesterol ester hydrolase activity. This stimulation was abolished if protein kinase inhibitor was added prior to the addition of ATP cyclic AMP and magensium ions. The addition of magnesium ions or calcium ions to a crude preparation of cholesterol ester hydrolase was found to inhibit activity; however the same additions made to a purified preparation of cholesterol ester hydrolase were not inhibitory. The decrease in cholesterol ester hydrolase activity on incubation with magnesium ion was accompanied by a loss of 32P radioactivity from the protein. Preincubation of a crude preparation of cholesterol ester hydrolase with alkaline phosphatase resulted in a deactivation of cholesterol ester hydrolase. It is suggested that bovine adrenal cortex cholesterol ester hydrolase is activated by a phosphorylation catalysed by a cyclic-AMP-dependent protein kinase. Deactivation of cholesterol ester hydrolase is accomplished by dephosphorylation catalysed by a phosphoprotein phosphatase, dependent on magnesium or calcium ions.
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PMID:Purification and control of bovine adrenal cortical cholesterol ester hydrolase and evidence for the activation of the enzyme by a phosphorylation. 18 99

The characteristics of neutral cholesteryl ester hydrolase activities found in the microsomal and cytosolic subcellular fractions of rat lactating mammary tissue were investigated. The enzymes were assayed using cholesteryl oleate dispersed as a mixed micelle with phosphatidylcholine and sodium taurocholate (molar ratio 1:4:2) as substrate. This method gave activities approx. 20-fold higher than those seen when cholesteryl oleate was added in ethanol. Addition of phosphatidylcholine and sodium taurocholate to the assays using the ethanol-dissolved substrate did not increase the activities observed. When the cholesteryl oleate was dispersed with phosphatidylcholine only (molar ratio, 1:4) the activity of the two neutral cholesteryl ester hydrolases was also decreased considerably compared to that found with mixed micelles. In this case, however, approx. 60% of the cytosolic, but only 10% of the microsomal activity, was restored by separate addition of sodium taurocholate. The activities of both the microsomal and the cytosolic neutral cholesteryl ester hydrolases were inhibited by MgCl2, and this inhibition was almost completely reversed by the addition of an equimolar concentration of ATP. At a fixed concentration of MgCl2 increasing concentrations of ATP increased the enzyme activities in a dose-dependent way. The activity of the microsomal, but not the cytosolic enzyme was enhanced by a cyclic AMP-dependent protein kinase and both activities were inhibited by alkaline phosphatase (bovine milk). These results provide evidence for the regulation of neutral cholesteryl ester hydrolases in the rat lactating mammary gland by mechanisms involving phosphorylation-dephosphorylation and therefore suggest that these enzymes may be under hormonal control.
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PMID:Neutral cholesteryl ester hydrolase in the rat lactating mammary gland: regulation by phosphorylation-dephosphorylation. 217 66

Short term regulation of hepatic cholesterol ester hydrolase by reversible phosphorylation is described. Two different kinase systems seem to be involved in this regulation. The addition of ATP, cyclic AMP and Mg2+ to rat liver 104,000 X g supernatant (S104) produced a 100-140% increase in cholesterol ester hydrolase activity. This stimulation was abolished when protein kinase inhibitor was added prior to the addition of ATP, cyclic AMP and Mg2+. Cholesterol ester hydrolase activity was also stimulated when calcium ions, phosphatidylserine, and diolein were added to S104 along with ATP and Mg2+. Diolein in this reaction could be substituted by phorbol 12-myristate 13-acetate. Preincubation of S104 with alkaline phosphatase resulted in a deactivation of cholesterol ester hydrolase. The addition of increasing concentrations of Mg2+ to S104 produced increasing inhibition of cholesterol ester hydrolase activity, and this effect was blocked by NaF. It is suggested that rat liver cholesterol ester hydrolase is activated by cyclic AMP dependent protein kinase and protein kinase C. Deactivation is accomplished by dephosphorylation catalyzed by a phosphoprotein phosphatase, dependent on Mg2+.
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PMID:Activation of rat liver cholesterol ester hydrolase by cAMP-dependent protein kinase and protein kinase C. 255 47

Okadaic acid, a potent and specific inhibitor of protein phosphatases 1 (IC50 10-20 nM) and 2A (IC50 0.05-2 nM) caused early and sustained inhibitions of microsomal cholesterol ester hydrolase activity in hepatocyte suspensions. The changes in the kinetic properties of the esterase and its response to exogenous alkaline phosphatase and cyclic AMP-dependent protein kinase after cell exposure to 1 microM or 1 nM okadaic acid differed markedly among themselves, which suggests the involvement of both protein phosphatases 1 and 2A in the regulation of the microsomal hydrolysis of cholesterol esters. Furthermore, the inhibitory effect of okadaic acid is likely to be independent of the dibutyryl-cyclic AMP promoted cell events leading to stimulation of esterase activity.
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PMID:Inhibition of microsomal cholesterol ester hydrolase by okadaic acid in isolated rat hepatocytes. 754 88

The regulation of neutral cholesterol ester hydrolase activity by changes in its phosphorylation state was studied in rat liver microsomes. Treatment with cAMP-dependent protein kinase resulted in increased enzyme activity, which was further enhanced by the addition of cAMP and MgATP. Consistent activations were also achieved with MgCl2 and MgATP, the magnesium effect being abolished by ethylenediaminetetraacetic acid and adenosine triphosphate. Cholesterol ester hydrolase was activated twofold by free calcium and Ca2+/calmodulin; this latter effect was blocked by the chelator ethylene-glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid and the calmodulin antagonist trifluoperazine. The phosphatase inhibitors pyrophosphate and glycerophosphate led to marked and dose-dependent increases in esterase activity, whereas okadaic acid elicited no effect. Furthermore, pyrophosphate and okadaic acid did not change the increases in enzyme activity promoted by Ca2+, Ca2+/calmodulin, Mg2+ and MgATP. Cholesterol ester hydrolase was inactivated in a concentration-dependent manner by nonspecific alkaline phosphatases. In cAMP-dependent protein kinase/cAMP- or Ca2+/calmodulin-activated microsomes, a time-dependent loss of activation in cholesteryl oleate hydrolysis was caused by alkaline phosphatase. These findings suggest that microsomal cholesterol ester hydrolase is activated through cAMP and Ca2+/calmodulin phosphorylation, whereas enzyme deactivation is dependent on phosphatase action.
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PMID:Regulation of rat liver microsomal cholesterol ester hydrolase by reversible phosphorylation. 813 99

Temperature-labile cholesterol ester hydrolase (TLCEH) was purified 2,000-fold from rat testis cytosol using sequential ammonium sulfate precipitation, cation exchange chromatography, and isoelectric focusing chromatography. the purified enzyme, which exhibited a single silver-stained band (66 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was inhibited 89% by the elevation of the temperature from 32 to 37 degrees C and 65% by treatment with alkaline phosphatase. Its amino acid composition and amino-terminal sequence differed markedly from those of isoenzymes from other tissues, although 6 of 20 residues were conserved. Polyclonal antibodies raised to TLCEH exhibited no cross-immunoreactivity with cytosolic proteins from other rat tissues and inhibited 70% of testis cytosolic CEH. Western blot analysis demonstrated a high correlation between immunoreactive protein and catalytic activity in the testis during maturation of the rat, with a marked increase at the onset of spermatogenesis. TLCEH was inhibited by physiological levels of Cu2+ (I50 = 0.60 microM) and Zn2+ (I50 = 0.75 microM) and by Cd2+ (I50 = 0.15 microM) but not by 0.5-5 mM Mn2+.
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PMID:Testicular temperature-labile cholesteryl ester hydrolase. Relationship to isoenzymes from other tissues, correlation with spermatogenesis, and inhibition by physiological concentrations of divalent cations. 846 27

Messenger RNA, protein mass and catalytic activity of hepatic neutral cholesteryl ester hydrolase (CEH) were measured in male Sprague-Dawley rats, aged 6, 8, 9.5, 12 and 24 weeks (wks). CEH mRNA increased 101% from 6 to 9.5 wks, corresponding to onset of puberty, and declined by 52% from 12 to 24 wks. CEH mass was highly correlated with mRNA levels at all ages, increasing 170% from 6 to 9.5 wks and declining 61% from 12 to 24 wks. CEH activity was highly correlated with mass and mRNA from 8-24 wks, but was greater at 6 wks than the activity predicted by the measured mass. In all age groups, activity was consistently increased by activation of endogenous protein kinase A and consistently inhibited by alkaline phosphatase, suggesting that age-related differences in catalytic activity were not due to differences in the level of enzyme phosphorylation. These data suggest transcriptional regulation and indicate an important role for CEH in cholesterol homeostasis in the developing rat.
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PMID:Age-related changes in mRNA, protein and catalytic activity of hepatic neutral cholesterol ester hydrolase in male rats: evidence for transcriptional regulation. 869 65

Activity and protein mass of hepatic neutral cholesteryl ester hydrolase (CEH) were measured in liver cytosol and washed microsomes of female Sprague-Dawley rats aged 3, 4, 7, 9, 13, and 16 wk. CEH mRNA was also measured. The microsomal component varied with age and contributed a greater fraction of total activity in females than previously reported in males. Nevertheless, the cytosolic component accounted for 62-80% of activity and 77-94% of immunoreactive protein in postmitochondrial fractions. Cytosolic and microsomal CEH specific activities, relative to total protein, decreased 94 and 83%, respectively, from 3 to 4 wk, prior to onset of puberty at 5 wk, and increased 360 and 137%, respectively, from 12 to 16 wk. These results contrast with an earlier study, in which cytosolic CEH activity of males increased with puberty and declined after 12 wk. Although cytosolic CEH was activated by protein kinase A and inhibited by alkaline phosphatase treatment at all ages, protein kinase activation peaked at 4 wk, coinciding with the initial decrease in specific activity. Specific activity in cytosol and microsomes correlated with CEH mass at all ages, suggesting that this CEH accounts for most variation in cellular activity. In contrast, CEH mRNA varied little from 3-16 wk, indicating that transcriptional regulation does not make a major contribution to the variation in CEH activity and mass in females, although it may make an important contribution to male-female differences in CEH expression. Specific activities of cytosolic and microsomal CEH, relative to immunoreactive CEH protein mass, exhibited changes consistent with posttranslational regulation. These results indicate gender-specific multivalent regulation of hepatic CEH by posttranslational mechanisms during development of female rats.
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PMID:Age-related changes in catalytic activity, enzyme mass, mRNA, and subcellular distribution of hepatic neutral cholesterol ester hydrolase in female rats. 916 52