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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hormone-sensitive lipase has been purified from rat adipose tissue to almost 50 per cent protein purity and partially characterized. The isolated enzyme can be phosphorylated by ATP-Mg2+ in the presence of the catalytic subunit of cyclic AMP-dependent protein kinase from the same tissue. Its activity towards emulsified triglyceride is thereby increased two-fold. The enzyme is phosphorylated also in the intact adipocyte, verifying the physiological relevance of the findings with the isolated enzyme. Noradrenaline causes a rapid increase in phosphorylation of the enzyme in intact adipocytes, immediately followed by a marked increase of its activity. Addition of dibutyryl-cyclic AMP to the adipocytes causes the same effects. The extent of phosphorylation of the enzyme after maximal noradrenaline stimulation of the adipocytes is rapidly decreased by insulin addition in close association with inhibition of the lipase activity. The results demonstrate that these hormones regulate the activity of the hormone-sensitive lipase, ie the rate of lipolysis in the adipocytes, by changes of the degree of phosphorylation of the enzyme.
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PMID:Regulation of adipose-tissue lipolysis by phosphorylation of hormone-sensitive lipase. 627 18

The effects of free fatty acids and fatty acyl esters of coenzyme A and carnitine on the activity of a hormone-sensitive lipase preparation made from pigeon adipose tissue were determined. Oleic acid (100 microM) resulted in a 40% inhibition of lipase activity. A more potent inhibition of lipase activity was seen with long-chain fatty acyl CoA compounds. The concentration required for half-maximal inhibition with oleoyl CoA and palmitoyl CoA was 25-40 microM, whereas palmitoyl carnitine stimulated lipase activity. Activated lipase preparations (preincubated with Mg2+, ATP, cyclic AMP and protein kinase) were 4-6 times more sensitive to inhibition by oleoyl CoA than were nonactivated preparations. An increase in cellular levels of fatty acyl coenzyme A could, therefore, contribute to the feedback inhibition of lipolysis in adipose tissue.
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PMID:Inhibition of the hormone-sensitive lipase in adipose tissue by long-chain fatty acyl coenzyme A. 632 7

Incubation of purified hormone-sensitive lipase, 32P-phosphorylated with the catalytic subunit of cyclic AMP-dependent protein kinase and [gamma-32P]ATP-Mg2+, with partially purified protein phosphatase from the same tissue caused a rapid decrease of the 32P content of the enzyme protein. Deactivation of the lipase towards emulsified trioleoylglycerol was temporally related to the dephosphorylation with approx. 80% decrease of both phosphorylation and activity within 30 min. Addition of ATP-Mg and cyclic AMP-dependent protein kinase to the dephosphorylated lipase was shown to rephosphorylate and reactivate the enzyme. These findings are the first direct demonstration of reversible protein phosphatase-catalyzed dephosphorylation/deactivation of hormone-sensitive lipase.
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PMID:Direct evidence for protein phosphatase-catalyzed dephosphorylation/deactivation of hormone-sensitive lipase from adipose tissue. 633 16

In isolated adipocytes, fast-acting lipolytic hormones and insulin have been shown previously to control lipolysis by regulating the activity of hormone-sensitive lipase, the rate-limiting enzyme, through an increase or decrease, respectively, of the extent of phosphorylation of the enzyme. Here, we demonstrate that exposure to lipolytic hormones (corticotropin, noradrenaline) led to phosphorylation at two sites on the Mr 84,000 lipase subunit. One, designated "basal site," was phosphorylated also in the absence of any hormonal stimulation, its phosphorylation apparently not being influenced by hormones. The second, designated "regulatory site," was identical to that phosphorylated by cyclic AMP-dependent protein kinase on the isolated lipase. The regulatory site was not appreciably phosphorylated in the absence of hormones, but exposure of the cells to noradrenaline increased its phosphorylation extent to that of the basal site. Insulin or the beta-adrenergic antagonist propranolol decreased the extent of phosphorylation of the regulatory site to the low level before stimulation, apparently without effect on the basal site. Phosphoserine was the only phosphorylated amino acid residue at both sites. Limited proteolytic digestion indicated that the two sites were separated by less than about 170 amino acid residues. Thus, control of adipose tissue lipolysis by fast-acting lipolytic hormones and by insulin is exerted through the regulation of the phosphorylation state of a single phosphoserine residue in the hormone-sensitive lipase.
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PMID:Hormonal regulation of hormone-sensitive lipase in intact adipocytes: identification of phosphorylated sites and effects on the phosphorylation by lipolytic hormones and insulin. 637 55

The lipolytic action of theophylline was examined using both intact fat cells and a fat globule system. Theophylline had similar lipolytic actions in both systems. However theophylline did not activate hormone-sensitive lipase in the fat globule system as measured with added Ediol. Pretreatment of the fat globules with phospholipase C suppressed theophylline-induced lipolysis, but phospholipase D had no effect. A theophylline-sensitive system was reconstituted from endogenous fat and a lipase fraction. Inhibitors of theophylline-induced lipolysis such as quinine and propranolol inhibited theophylline binding to artificial lipid micelles. Purine nucleosides such as adenosine, inosine and guanosine inhibited theophylline-induced lipolysis in the fat globule system. These results suggest that theophylline has a lipolytic action similar to that of adrenaline. Both share a lipolytic mechanism additional to that involving the activation of hormone sensitive lipase through the cyclic-AMP dependent protein kinase. Phospholipids play an important role in this additional mechanism.
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PMID:The mechanism of the lipolytic action of theophylline in fat cells. 724 46

A triacylglycerol lipase, presumably the first enzyme involved in the mobilization of lipid from the insect fat body, has been purified to homogeneity from the fat body of Manduca sexta. The purification procedure involved polyethyleneglycol precipitation, and chromatography on DEAE-cellulose, phenyl-Sepharose, Q-Sepharose and hydroxylapatite. The final product, a protein with an M(r) = 76,000 by SDS-PAGE, was purified nearly 8000-fold from the original homogenate in a yield of about 11%. The enzyme catalyzed the hydrolysis of tri-, di-, and mono-oleoylglycerols, but showed highest affinity for tri- or dioleoylglycerol. Thus, under initial reaction conditions, the end products of trioleoylglycerol hydrolysis were: free fatty acids (66%), sn-2-monooleoylglycerol (24%), sn-1,2(2,3)-dioleoylglycerol (7%), and glycerol (3%). The fat body lipase exhibited a preference for hydrolyzing the primary ester bonds of acylglycerols, and did not show stereoselectivity toward either the sn-1 or sn-3 position of trioleoylglycerol. The enzyme had a pH optimum of 7.9, and was inhibited by diisopropylfluorophosphate, ATP, ADP, Mg2+, and NaF. The enzyme showed a strong tendency to aggregate, but was stable in detergent solutions at high concentration of glycerol. The polypeptide was phosphorylated by the cAMP-dependent protein kinase from bovine heart; however, phosphorylation did not cause activation of the enzyme. It is suggested that this fat body lipase could be analogous to the "hormone-sensitive lipase" of vertebrate adipose tissue.
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PMID:Purification and properties of a phosphorylatable triacylglycerol lipase from the fat body of an insect, Manduca sexta. 780 79

The release of hepatic triacylglyceride lipase [EC 3.1.1.3] has been examined in isolated hepatocytes in primary culture. The stimulatory release of activity from the hepatocytes into the medium by sodium orthovanadate (vanadate) was observed in a time- and dose-dependent manner. However, insulin failed to have this stimulatory action. Moreover, vanadate rapidly increased the cyclic adenosine monophosphate (cyclic AMP) content in hepatocytes in a time- and dose-dependent manner. The treatment of hepatocytes with H-89, which is a potent cyclic AMP-dependent protein kinase inhibitor, decreased the stimulatory release of hepatic lipase activity by vanadate. The vanadate-stimulated release of the enzyme activity was suppressed by uncouplers. In addition, the incorporation of [3H]leucine into protein was increased in the presence of vanadate. Under the marked inhibition of protein synthesis by cycloheximide, vanadate still showed a full effect on the release of the enzyme activity. These results suggest that the vanadate-stimulated release of hepatic lipase activity from the cultured hepatocytes is associated with a rapid increase in intracellular cyclic AMP content, probably due to an activation of cyclic AMP-dependent protein kinase which requires a metabolic energy process rather than an elevation in enzyme molecule synthesis.
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PMID:Stimulatory release of hepatic lipase activity from cultured rat hepatocytes by sodium orthovanadate: rapid increase in cyclic adenosine monophosphate content. 792 Apr 12

Hormone sensitive lipase (HSL) is an enzyme of relatively broad specificity, having the ability to hydrolyze tri-, di- and mono-acylglycerols as well as cholesterol esters and small water-soluble substrates. This broad specificity allows HSL to perform a variety of functions in several tissues. A key feature of HSL is its ability to be activated via phosphorylation by cyclic AMP-dependent protein kinase. In addition it is phosphorylated at a second site by several kinases, notably AMP-activated protein kinase. Phosphorylation of this site apparently plays a role in rendering the enzyme hormone-insensitive, in that prior phosphorylation at site 2 prevents phosphorylation and activation at site 1 by cyclic AMP-dependent protein kinase. Investigation of the protein phosphatases responsible for dephosphorylation of these sites has indicated that phosphatase 2A plays a predominant role but also that protein phosphatase 2C is a significant phosphatase targeted against both phosphorylation sites. Evidence indicates that HSL has at least three functional domains which contain (a) the phosphorylation sites which control activity, (b) the active site responsible for the catalytic activity and (c) a lipid binding site responsible for anchoring the lipase at the water-lipid interface. Using limited proteolytic studies we have found that it is possible to cleave HSL into several fragments including a stable domain of M(r) approximately 17.6 kDa which contains the active site serine residue. Digestion under similar conditions also generates a stable domain of M(r) approximately 11.5 kDa containing both phosphorylation sites. Furthermore, under appropriate conditions it is possible to digest HSL and retain activity against water-soluble substrates but with the concomitant loss of activity against triacylglycerol, implying that a lipid binding domain is lost during this procedure. HSL is responsible for the neutral cholesterol esterase activity in macrophages and it may play a role in the accumulation of cholesterol esters which occur during the development of foam cells. HSL activity is reduced in macrophage foam cells, at least partly due to increased activity of a cytosolic HSL inhibitor protein. A finding unexplained for many years has been that, although lipolysis can be stimulated 50-100-fold in adipocytes by lipolytic hormones, HSL can apparently only be activated 2-3-fold via phosphorylation in vitro by cyclic AMP-dependent protein kinase. One possibility to explain this discrepancy is that an additional anchoring protein is missing from the in vitro system and indirect evidence is now accumulating for such a protein.
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PMID:The multifunctional role of hormone-sensitive lipase in lipid metabolism. 794 81

Bearing in mind the importance of upper-body obesity for the insulin resistance (or metabolic) syndrome and the abnormalities in free fatty acid metabolism associated with this disorder, the regulation of lipolysis in isolated subcutaneous adipocytes was investigated in 13 72-yr old upper-body obese men with insulin resistance and glucose intolerance and in 10 healthy 72-yr-old men. There was a marked resistance to the lipolytic effect of noradrenaline in the metabolic syndrome due to defects at two different levels in the lipolytic cascade. First, an 80-fold decrease in sensitivity to the beta 2-selective agonist terbutaline (P < 0.001) which could be ascribed to a 50% reduced number of beta 2-receptors (P < 0.005) as determined with radioligand binding. The groups did not differ as regards dobutamine (beta 1) or clonidine (alpha-2) sensitivity, nor beta 1-receptor number. The mRNA levels for beta 1- and beta 2-receptors were similar in the two groups. Second, the maximum stimulated lipolytic rate was markedly reduced in the metabolic syndrome. This was true for isoprenaline (nonselective beta-agonist), forskolin (activating adenylyl cyclase), and dibutyryl cAMP (activating protein kinase). In regression analysis, the observed abnormalities in lipolysis regulation correlated in an independent way with the degree of glucose intolerance (r = -0.67) and beta 2-receptor number with insulin resistance (r = 0.67). In conclusion, the results of this study indicate the existence of lipolytic resistance to catecholamines in the adipose tissue of elderly men with the metabolic syndrome, which may be of importance for impaired insulin action and glucose intolerance. The resistance is located at a posttranscriptional level of beta 2-receptor expression and at the protein kinase-hormone sensitive lipase level.
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PMID:Multiple lipolysis defects in the insulin resistance (metabolic) syndrome. 820 Sep 97

Treatment of macrophages with zymosan, 4 beta-phorbol 12-myristate 13-acetate (PMA) and fluoride but not with A 23187 or arachidonic acid (delta Ach) leads to a generation of diacylglycerol (acyl2Gro). Formation of inositol phosphates is achieved with zymosan, only. An elevation of intracellular calcium is obtained with zymosan and A 23187 but not with PMA, fluoride or delta Ach. Prior treatment of the cells with phorbol ester for 3 h which has been shown recently to result in a down-regulation of protein kinase (PK) C-beta but not PKC-delta [Duyster, J., Schwende, H., Fitzke, E., Hidaka H. & Dieter P. (1993) Biochem. J. 292, 203-207] has no effect on the zymosan-induced formation of acyl2Gro or inositol phosphates but inhibits the PMA-induced generation of acyl2Gro. Down-regulation of PKC-delta by prior phorbol ester treatment for 24 h augments the zymosan-induced generation of acyl2Gro and inositol phosphates. The acyl2Gro lipase inhibitor RG 80267 inhibits the PMA-induced and fluoride-induced generation of prostaglandin (PG) E2, reduces the zymosan-induced release of PGE2 by 50% but has no effect on PGE2 formation of unstimulated, A 23187-treated or delta Ach-treated cells. Furthermore, RG 80267 enhances accumulation of delta Ach-labeled acyl2Gro in response to zymosan, PMA and fluoride. These data indicate that zymosan activates a phosphatidylinositol 4,5-bisphosphate-specific phospholipase (PL) C, that generation of acyl2Gro by PMA and fluoride occurs via hydrolysis of other phospholipids, that PKC-beta is involved in the PMA-induced generation of acyl2Gro and PKC-delta negatively modulates the zymosan-induced activation of PLC and PMA and fluoride induce a liberation of delta Ach from acyl2Gro, A 23187 activates the PLA2 pathway and zymosan stimulates both, the acyl2Gro- and PLA2-pathway.
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PMID:Formation of diacylglycerol, inositol phosphates, arachidonic acid and its metabolites in macrophages. 826 66


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