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)

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

Rainbow trout were used to investigate the hormonal regulation by glucagon and insulin of hepatic triacylglycerol (TG) lipase activation. Two purified preparations of the trout hepatic TG lipase enzyme, the 110,000-g preparation and the resuspended ammonium sulfate fraction (ASF), were activated up to 58% with (in mM) 0.5 ATP, 0.01 cAMP, 5 MgCl2, and exogenous protein kinase over control levels. ATP or cAMP alone had no effect on activation. Activation of the trout hepatic lipase was reversible; complete inactivation of the ASF was obtained within 3 h in the presence of exogenous phosphorylase phosphatase. Adenosine 3',5'-cyclic monophosphate (cAMP)/ATP-dependent 32P-phosphorylation of trout hepatic lipase was observed within 5 min of incubation with the cAMP/ATP-Mg2+ activation system and 25 microCi [32P]ATP. Hormonal modulation of trout hepatic lipase phosphorylation was studied in isolated hepatocytes. Hepatocytes were incubated with [32P]-monopotassium phosphate for 3 h, then exposed to mammalian glucagon (GLU). Within 5 min, increased lipolysis was accompanied by a 95% increase in phosphorylation of the enzyme. Mammalian insulin (INS) depressed GLU-stimulated phosphorylation by 56% and inhibited GLU-stimulated lipolysis. These results indicate that GLU and INS modulate lipolysis in trout liver by altering phosphorylation of the TG lipase enzyme.
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PMID:Glucagon and insulin regulate lipolysis in trout liver by altering phosphorylation of triacylglycerol lipase. 834 95

Although G-protein- and protein kinase-mediated pathways have been reported to activate phospholipase D (PLD) following cell stimulation, the relation between these activation pathways and the mechanistic details of lipase stimulation remain unknown. We have studied activation of PLD by GTP gamma S (guanosine 5'-O-(thiotriphosphate)), and its potentiation by ATP, in a cell-free system derived from U937 human promonocytic leukocytes. ATP, in the micromolar to millimolar range, significantly augmented GTP gamma S-stimulated PLD activity (2.6-fold) and the combination resulted in a 15-fold increase in PLD activity compared to control. ATP alone did not stimulate PLD activity. Measurement of endogenous cytosolic ATP levels and nucleotide depletion with activated charcoal demonstrated that stimulation of PLD by GTP gamma S proceeds by both ATP-dependent and -independent pathways. Nucleotide specificity data suggested that the ATP-dependent pathway involves kinase activity. The tyrosine phosphatase inhibitor vanadate augmented PLD activity stimulated by GTP gamma S/ATP by 41% (p < 0.01). Conversely, the tyrosine kinase inhibitors genistein and herbimycin A decreased PLD activity stimulated by GTP gamma S/ATP by 58 and 35%, respectively (p < 0.001 for each). Mixing experiments utilizing subcellular fractions from herbimycin A-treated cells suggested that the relevant tyrosine kinase activity is membrane-associated. Despite its role in ATP-induced potentiation, tyrosine kinase activity is neither necessary nor sufficient for activation of PLD in this system. Protein kinase C (PKC) is unlikely to play a role in potentiation by ATP as PKC activity is not stimulated under conditions of maximal PLD activation.
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PMID:ATP-induced potentiation of G-protein-dependent phospholipase D activity in a cell-free system from U937 promonocytic leukocytes. 837 59

The mechanisms responsible for the diminished lipolytic response of adipocytes to catecholamines after litter removal from lactating rats and their modulation by growth hormone have been investigated. Lactation, litter removal and growth-hormone treatment did not alter the ability of noradrenaline to activate protein kinase A (A-kinase), showing that the defect in signal transduction in rats after litter removal is after A-kinase. Litter removal had no effect on hormone-sensitive lipase activity itself, but the proportion of the lipase associated with the fat droplet was decreased; growth-hormone treatment increased hormone-sensitive lipase activity and the proportion associated with the fat droplet. In addition, a number of other adaptations in the beta-adrenergic signal-transduction system occur during the lactation cycle and in response to growth hormone treatment, including changes in receptor number, adenylate cyclase activity and cyclic AMP phosphodiesterase activity, but a defect in the ability of hormone-sensitive lipase to associate with the lipid droplet appears to be the major reason for the diminished response to catecholamines on litter removal.
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PMID:Mechanisms involved in the adaptations of the adipocyte adrenergic signal-transduction system and their modulation by growth hormone during the lactation cycle in the rat. 838 54

In the previous studies we have shown that testosterone increases lipolytic responsiveness to catecholamines in rat white adipocytes, and that is associated with an up-regulation of beta-adrenergic receptor density. However, the postreceptor events involved in the testosterone induced enhancement of beta-adrenergic receptor activated lipolysis in these cells have not been adequately studied, and were therefore investigated in the present study. Male Sprague Dawley rats were divided into three groups: control, castrated, and castrated treated with testosterone. The beta-adrenergic receptor-mediated cAMP accumulation, measured with RIA after isoproterenol (a beta-adrenergic agonist) stimulation was decreased in castrated rats, and reversed by testosterone treatment, suggesting a testosterone effect at or proximal to adenylate cyclase. However, no differences between the groups were found in abundance of G alpha protein messenger RNAs (G alpha s, G alpha i-1, and G alpha i-2) as analyzed by Northern blot and a solution hybridization RNase protection assay, or in G protein mass measured with a quantitative enzyme-linked immunosorbent assay in fat cell membrane preparation. Lipolysis stimulated by N6-monobutyryl-cAMP was reduced in castrated rats and recovered by testosterone treatment, suggesting that components distal to the adenylate cyclase, i.e. protein kinase A (PKA) and/or hormone sensitive lipase (HSL) also are involved in testosterone regulation of lipolysis. In conclusion, these and previous results suggest that the testosterone-induced increase in lipolytic response to catecholamines in rat white adipocytes is mediated through several events including an increased beta-adrenergic receptor density, probably an increased adenylate cyclase activity and an increased protein kinase A/hormone sensitive lipase activity at the postreceptor level with apparent absence of effect on the expression of G-proteins.
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PMID:Postreceptor events involved in the up-regulation of beta-adrenergic receptor mediated lipolysis by testosterone in rat white adipocytes. 838 92

Mobilization of lipids from adipose tissue during prolonged exercise is of key importance for the supply of energy to the working muscle. During exercise lipid mobilization is mainly stimulated by increased catecholamine production leading to acceleration of the beta-adrenoceptor mediated lipolysis rate in fat cells. This causes breakdown of triglycerides in fat cells to glycerol and free fatty acids, which then are delivered to the blood stream. Decreased insulin production, enhanced adipose tissue blood flow and decreased reesterification of free fatty acids in fat cells contribute to the enhancement of lipid mobilization during strenuous and long-term light exercise. Several additional factors modulate the lipolytic response to exercise as well. Endurance training increases the lipolytic action of catecholamine whereas the opposite occurs during ageing. These alterations are at least in part mediated by changes in the function of the final step in lipolysis activation, the protein kinase-hormone sensitive lipase complex. There are also gender and regional differences in the lipolytic response to exercise. Women mobilize more lipids from the subcutaneous abdominal area than men, whereas a low rate of lipid mobilization from the peripheral subcutaneous areas is observed in either sex. In pathophysiological states, which are associated with catabolism such as fasting and insulin dependent diabetes, there is an enhanced lipolytic response to exercise, because of increased beta-adrenoceptor function.
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PMID:Impact of exercise on adipose tissue metabolism in humans. 858 Oct 90


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