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)

Although the renal medulla is rich in triacylglycerols, the lipolysis of these intracellular triacylglycerols by a renomedullary triacylglycerol lipase has not been directly demonstrated. The present study demonstrates triacylglycerol lipase activity localized in the particulate subcellular fractions of rabbit renal medullae. Renomedullary triacylglycerol lipase activity, as determined by the hydrolysis of [14C]triolein to [14C]oleic acid, was observed to have a pH optimum of 5.8. Addition of cAMP/ATP/magnesium acetate resulted in an 80% activation of crude homogenate triacylglycerol lipase activity; addition of exogenous cAMP-dependent protein kinase resulted in a further activation of lipolysis. 3 mM CaCl2 had no effect on basal triacylglycerol lipase activity. 1 M NaCl did not inhibit lipolysis, suggesting that the lipase activity measured was not due to lipoprotein lipase. Endogenous renomedullary triacylglycerols were hydrolysed by a lipase in the 100,000 X g pellet of renomedullary homogenates, resulting in the release of free fatty acids including arachidonic and adrenic acids. Dispersed renomedullary cells were prepared to monitor hormone-sensitive triacylglycerol lipase activity in intact cells. Addition of 10 microM forskolin and 10 microM epinephrine resulted in 8-fold and 50-fold increases in triacylglycerol lipase activity, respectively, as defined by release of free glycerol from the cells. These studies demonstrate that a cAMP-dependent hormone-sensitive triacylglycerol lipase is present in the renal medulla, and is responsible for the hydrolysis of renomedullary triacylglycerols.
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PMID:Triacylglycerol lipase activity in the rabbit renal medulla. 282 29

Fatty acid metabolism in adipocytes is known to be regulated by the intracellular transducer cAMP. This study was undertaken to determine the temporal and hormonal regulation of cAMP-dependent protein kinase during the differentiation of preadipocyte mesenchymal cells to adipocytes. For this we have used a stable cell line (TA1) in which the undifferentiated preadipocyte acquires adipocyte functions and morphology after growth to confluence. We observed that synthesis of type I and II cAMP-dependent protein kinases was induced during the adipogenic conversion of growth-arrested TA1 cells. In preconfluent cells, neither mRNAs encoding regulatory subunits (RI, RII beta) and catalytic subunit (C alpha) nor the peptides themselves were detectable. Within several days of growth arrest at high cell density, mRNAs for RI, RII beta, and C alpha were detectable in total RNA extracted from cell populations. The subunits themselves were detectable in some, but not all, of the cells by indirect immunofluorescence. Immunoblotting of cytosolic extracts indicated that RI and the beta-isoform of RII (mol wt = 52,000) were expressed in these cells. Analysis of subunit presence or absence in single cells by immunofluorescence also indicated that kinase subunit expression preceded the accumulation of lipid droplets within the cells. Further, the subunits were predominantly associated with a reticular cytoplasmic structure (Golgi apparatus?) abutting the nucleus. Conversion of TA1 cells to adipocytes can be accelerated by indomethacin (125 microM) or dexamethasone (1 microM) treatment, compounds that also enhanced the accumulation of RII beta and C alpha mRNAs. Within 2-3 days of addition of indomethacin to confluent cultures, RII beta message content is increased about 20-fold, and protein content is increased about 5-fold relative to those in untreated cultures. C alpha mRNA content is increased about 5-fold relative to that in untreated cells. The response to dexamethasone requires 6-7 days, and changes in RII beta message levels were the most pronounced. We also observed the induction of mRNAs for the functionally relevant mRNA lipoprotein lipase in indomethacin-treated cells. In addition to this apparent transcriptional regulation of kinase subunit expression, we provide evidence for regulation at the posttranscriptional level. Within a differentiated culture, there exist stem cells that can be selected, will repopulate the dish, and will again differentiate into adipocytes upon growth arrest at high cell density. In preconfluent populations of these stems cells, unlike the preconfluent TA1 cells originally plated, both RII beta and C alpha messages were present.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Induction of adenosine 3',5'-monophosphate-dependent protein kinase subunits during adipogenesis in vitro. 284 11

Lipolysis of intracellular triglycerides in the heart has been shown to be regulated by hormones. However, activation of myocardial triglyceride lipase in a cell-free system has not been directly demonstrated. In the present studies, initial attempts to demonstrate cAMP-dependent activation of triglyceride lipase using the 1,000 X g supernatant fraction (S1) of mouse heart homogenate were unsuccessful, presumably due to the masking effects of high levels of lipoprotein lipase activity even when assayed at pH 7.4 and in the absence of apolipoprotein C-II. Myocardial lipoprotein lipase in the 40,000 X g supernatant fraction was then removed by heparin-Sepharose affinity chromatography. The lipoprotein lipase-free fractions were shown to contain neutral triglyceride lipase and neutral cholesterol esterase of about equal activities. The triglyceride lipase and cholesterol esterase activities fell progressively during preincubation in the presence of 5 mM Mg2+. Additions of cAMP and ATP resulted in 40-70% activation of both triglyceride lipase and cholesterol esterase. The activation was blocked by protein kinase inhibitor and was restored by the addition of exogenous cAMP-dependent protein kinase. Since lipoprotein lipase has no activity toward cholesteryl oleate, activation of cholesterol esterase in untreated S1 was readily demonstrable. Both triglyceride lipase and cholesterol esterase activities were present in homogenates prepared from isolated rat heart myocytes. We conclude that the myocardium contains a hormone-sensitive lipase that is regulated in a fashion similar to that of the adipose tissue enzyme.
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PMID:Activation of myocardial neutral triglyceride lipase and neutral cholesterol esterase by cAMP-dependent protein kinase. 298 7

The effect of CAM [cyclic AMP, Mg-ATP, and 3-isobutyl, 1-methylxanthine (MIX)] on triacylglycerol (TG) lipase activity in extracts from heparin-perfused rat heart was determined. TG lipase activity in homogenate, 10,000g supernatant, 105,000g supernatant, ammonium sulfate supernatant, and the eluate from heparin-Sepharose was increased between 62 and 151% when incubated with a combination of 0.3 mM cyclic AMP, 5 mM MgCl2, and 2 mM ATP. The addition of Mg-ATP + cyclic AMP caused a greater activation of TG lipase in the various fractions than did Mg-ATP + MIX or cyclic AMP + MIX. These results suggest that activation may be mediated by the classical cyclic AMP-protein kinase cascade. Control and CAM-stimulated activities were increased by heparin and inhibited by NaCl and protamine sulfate. In the absence of serum in the assay, the CAM system caused a relatively greater stimulation of lipolytic activity in each fraction compared to when serum was present in the assay. However, the absolute values were 6.1 to 16.3-fold greater with serum in the assay than without serum. In a similar manner, TG lipase activity was stimulated by CAM between 1.75 and 4.26-fold at pH 7.4, and only between 1.62 and 2.51-fold at pH 8.1. However, the absolute values at pH 8.1 were 6.77 to 31.83-fold greater than those seen at pH 7.4. These data demonstrate, for the first time, the cyclic AMP activation of a TG lipase above basal levels in cell-free fractions of rat heart. It is intriguing to speculate that the intracellular fraction of lipoprotein lipase may play a role in the hormonal regulation of cardiac TG lipolysis.
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PMID:Cyclic AMP activation of a triglyceride lipase in broken cell preparations of rat heart. 301 40

Three TG lipases have been identified in muscle (i.e., acid, neutral, and alkaline), but as yet we do not know which enzyme is responsible for tissue TG hydrolysis. Over the past 8 yr, work in our laboratory has focused on intracellular lipoprotein lipase (LPL). The results show that this lipase is regulated by the classical cAMP cascade and that the activity of this enzyme is inversely related to endogenous TG concentration. Using these results as a foundation we plan to examine molecular mechanisms involved in the synthesis, compartmentalization, and transport of the alkaline TG lipase. Further, the evidence suggests that this enzyme may be regulated by protein phosphorylation mediated by cyclic AMP-dependent protein kinase. We plan to test this possibility.
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PMID:Role of the alkaline TG lipase in regulating intramuscular TG content. 307 Feb 56

In the mammalian myocardium, an active triglyceride synthesis pathway is operating, (re)esterifying activated fatty acids from endogenous or exogenous sources, with the glycolytically derived three-carbon intermediates dihydroxyacetone-phosphate and glycerol-3-phosphate by the so-called Kennedy pathway. The seven enzymes of triglyceride synthesis are membrane bound and located at the sarcoplasmic reticulum. The first enzyme in the glycerol-3-phosphate pathway, glycerol-3-phosphate acyltransferase, is proposed to be rate limiting for triglyceride formation. This microsomal enzyme is regulated by phosphorylation (inactiycation)-dephosphorylation (activation) coupled to the beta-receptor--adenyl cyclase--protein kinase system. Additional regulatory steps in triglyceride formation are the reactions catalyzed by the microsomal phosphatidic acid phosphatase and diglyceride acyltransferase. Intracellular triglycerides occur as free floating cytosolic droplets, membrane-bound particles and lipid-filled lysosomes. No consensus exists about the metabolically active portion of myocardial triglycerides. Various lipases have been proposed to be involved in endogenous lipolysis: the lysosomal acid, microsomal and soluble neutral triglyceride, intracellular lipoprotein lipases and the microsomal di- and monoglyceridase. It has been acknowledged that the bulk of the intracellular neutral lipase represents the precursor of vascular lipoprotein lipase. The presence of a neutral lipase, as distinct from lipoprotein lipase, in the rat heart was recently advocated. Endogenous lipolysis is a hormone-sensitive process. Hormone-sensitivity may involve direct alteration of enzyme activity by protein phosphorylation-dephosphorylation but is also dependent on the removal rate of product fatty acids, since feedback inhibition is a common property of all lipases in the heart.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Synthesis, storage and degradation of myocardial triglycerides. 331 Oct 5

An attempt was made to activate the capillary-bound fraction of lipoprotein lipase (LPL) with cAMP-dependent protein kinase catalytic subunit (PKC). Following a 30s washout period, hearts were perfused for 1 min with buffer containing heparin. Medium was collected during the second 30s of heparin perfusion. Addition of PKC+Mg-ATP to this capillary bed perfusate increased LPL activity from 6.84 +/- 0.72 nmol/ml/min to 13.76 +/- 1.12 nmol/ml/min (P less than 0.001). A similar 2-fold increase in activity was observed when results were expressed on a mg protein basis. Removal of serum from, or addition of 1.0M NaCl to, the assay system inhibited PKC-stimulated LPL activity approximately 85%. These results indicate that capillary alkaline LPL can be activated by PKC assayed under experimental conditions free of other TG lipases. Moreover, these findings suggest that the intracellular fraction of LPL can be activated by cAMP and that this activation is mediated through protein phosphorylation by cAMP-dependent protein kinase.
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PMID:Protein kinase activation of heparin-releasable lipoprotein lipase in rat heart. 395 70

Hormone-sensitive lipase partially purified from adipose tissue of laying hens was markedly activated by cyclic AMP-dependent protein kinase. Activation was approximately 4-fold (ranging up to as great as 10-fold) compared with the much lower degree of activation obtained with analogous preparations from rat and human adipose tissues (59 and 86%, respectively). The partially purified preparations contained adequate endogenous protein kinase activity to effect complete activation with addition of cyclic AMP, ATP, and Mg(2+). Activation was blocked by protein kinase inhibitor (from rabbit skeletal muscle) but could be restored fully by addition of excess exogenous protein kinase (from bovine skeletal muscle). The fully activated lipase was slowly deactivated by dialysis at 4 degrees C and then rapidly and almost fully reactivated by addition of cyclic AMP and ATP-Mg(2+). Reactivation was blocked by protein kinase inhibitor. This deactivation-reactivation cycle was rapid at 23 degrees C with dialysis against charcoal and could be demonstrated repeatedly using a single preparation. The reversible deactivation of protein kinase-activated enzyme is presumed to reflect the action of a lipase phosphatase. Lipase prepared from tissue previously exposed to glucagon yielded a much smaller degree of activation than lipase prepared from tissue not exposed to the lipolytic hormone, indicating that the physiological hormone-induced activation is probably similar to or identical with the protein kinase activation demonstrated in the cell-free preparations. Under the conditions of assay used, the partially purified lipase fraction contained diglyceride, monoglyceride, and lipoprotein lipase activities. However, treatment with cyclic AMP-dependent protein kinase had virtually no effect on these lipase activities.
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PMID:Reversible protein kinase activation of hormone-sensitive lipase from chicken adipose tissue. 437 88

1. Adrenaline has a biphasic effect on intracellular lipoprotein lipase activity and on endogenous triacylglycerol content in heparin-perfused heart. 2. A high concentration of adrenaline (1 microM in the perfusion buffer) activated endogenous lipoprotein lipase activity and, at the same time, decreased intracellular triacylglycerol stores. 3. In contrast, a low concentration (0.005 microM-adrenaline) inhibited intracellular lipoprotein lipase activity. Under these conditions, cardiac triacylglycerol content was elevated above control values. 4. Perfusing the heart with high and low concentrations of 3-isobutyl-1-methylxanthine elicited a biphasic effect on endogenous lipoprotein lipase activity and triacylglycerol content similar to that seen with adrenaline treatment. 5. The effect of adrenaline on intracellular lipoprotein lipase activity appears to be mediated by cyclic AMP through protein kinase. 6. A possible role for intracellular lipoprotein lipase in the regulation of endogenous triacylglycerol in rat heart is proposed.
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PMID:Possible role of lipoprotein lipase in the regulation of endogenous triacylglycerols in the rat heart. 617 39

Three macrophage cell lines, J774(2), CT2 and J7H1 were compared with respect to synthesis and secretion of lipoprotein lipase. The enzyme activity measured was characterized as lipoprotein lipase on the basis of serum dependence and inhibition by 1 M NaCl. Enzyme activity in all three lines increased with time in culture and the highest activity was found in the medium of the CT2 line which is adenylate cyclase deficient while that in the J7H1 line, cyclic AMP-dependent protein kinase deficient, was intermediate. The half life of the enzyme activity in conditioned medium from all three lines was 30-40 min, suggesting that the different levels of activity observed do represent different levels of enzyme production by the cells. About 80% of the lipoprotein lipase activity from all three lines was present in the medium and 50-70% of cellular activity could be released into the medium by a 3-min exposure to heparin. In addition, 24 h incubation with heparin enhanced enzyme secretion in all three lines. To determine the role of cyclic AMP in the regulation of lipoprotein lipase activity use was made of dibutyryl cAMP, methyl isobutylxanthine (IBMX) and cholera toxin. These agents strikingly depressed lipoprotein lipase activity in the J774(2) line but only dibutyryl cAMP was active in the CT2 line (adenylate cyclase deficient). In the J7H1 (protein kinase deficient) line there was no response to dibutyryl cAMP or IBMX over the first 4 h of incubation. Addition of these agents did not affect total cell protein synthesis. The present findings indicate that in the intact cells changes in cyclic AMP levels are associated with a change in the activity of lipoprotein lipase.
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PMID:Lipoprotein lipase activity in cultured macrophage cell line J774(2) and its increase in variants deficient in adenylate cyclase and cyclic AMP-dependent protein kinase. 618 77


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