Reversible protein kinase activation of hormone-sensitive lipase from chicken adipose tissue

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.     

The identification and characterization of protein kinase C activity in fetal membranes

Protein kinase C activity was demonstrated in cytosolic fractions prepared from human amnion and decidua vera tissues. The enzyme has been partially purified and was found to be glycerophospholipid-dependent. Phosphatidylserine was most active in the stimulation of protein kinase C. Ca2+ was also required for the expression of the enzyme activity. In the presence of unsaturated diacylglycerols, maximum activation of protein kinase C was observed at suboptimal concentrations of Ca2+. A possible role of phospholipid-dependent protein kinase C in the regulation of arachidonic acid release in this tissue is discussed.     

Involvement of cyclic AMP-dependent protein kinase on the phosphorylase kinase inhibition by glucose-6-phosphate in adipose tissue extracts

In order to achieve further clarification of the regulation of glycogenolysis in adipose tissue, we studied the effect of glucose-6-phosphate on phosphorylase activation in Sephadex G-25 filtrate of adipose tissue. The activity of phosphorylase kinase was decreased by 50% and by 75% in the presence of 0.5 mM and 2 mM of glucose-6-phosphate, respectively. This inhibition could be partially prevented by 0.5 mM AMP. Furthermore, we investigated the influence of glucose-6-phosphate on the effect of cyclic-AMP-dependent protein kinase on the activation of phosphorylase. The addition of cyclic-AMP and cyclic-AMP-dependent protein kinase caused a decrease in the inhibition of the phosphorylase activation by glucose-6-phosphate. Also, the glucose-6-phosphate at physiological concentration, decreased adipose tissue cyclic-AMP-dependent protein kinase activity.     

Enzymes regulating glycogen metabolism in swine subcutaneous adipose tissue. I. Phosphorylase and phosphorylase phosphatase.

Glycogen phosphorylase from swine adipose tissue was purified nearly 700-fold using ethanol precipitation, DEAE-cellulose adsorption, AMP-agarose affinity chromatography, and agarose gel filtration. The purified enzyme migrated as one major and several minor components during polyacrylamide gel electrophoresis. Activity was associated with the major component and at least one of the minor components. The molecular weight of the disaggregated, reduced, and alkylated enzyme, estimated by polyacrylamide gel electrophoresis performed in the presence of sodium dodecyl sulfate, was 90,000. Stability of the purified enzyme was considerably increased in the presence of AMP. The isoelectric pH of the enzyme in crude homogenates was 6.3. The sedimentation coefficient of the purified enzyme (7.9 S) and that in crude homogenates (7.3 S) was determined by sucrose density gradient sedimentation. Optimal pH for activity was between pH 6.5 and 7.1. Apparent Km values for glycogen and inorganic phosphate were 0.9 mg/ml and 6.6 mM, respectively. The Ka for AMP was 0.21 mM. Enzyme activity was increased by K2SO4, KF, KCl, and MgCl2 and decreased by NaCl, Na2SO4, D-glucose, and ATP. Inhibition by glucose was noncompetitive with the activator AMP; inhibition by ATP was partially competitive with AMP. The purified enzyme was activated by incubation with skeletal muscle phosphorylase kinase. Enzyme in crude homogenates was activated by the addition of MgCl2 and ATP; activation was not blocked by addition of protein kinase inhibitor, suggesting that phosphorylase kinase in homogenates of swine adipose tissue is present largely in an activated form. Deactivation of phosphorylase a by phosphorylase phosphatase was studied using enzyme purified approximately 200-fold from swine adipose tissue by ethanol precipitation, DEAE-cellulose chromatography, and gel filtration. The Km of the adipose tissue phosphatase for skeletal muscle phosphorylase a was 6 muM. The purified swine adipose tissue phosphorylase, labeled with 32-P, was inactivated and dephosphorylated by the adipose tissue phosphatase. Dephosphorylation of both skeletal muscle and adipose tissue substrates was inhibited by AMP and glucose reversed this inhibition. Several lines of evidence suggest that AMP inhibition was due to an action on the substrate rather than on the enzyme. We have previously reported that the system for phosphorylase activation in rat fat cells differs in some important characteristics from that in skeletal muscle. However, both swine fat phosphorylase and phosphorylase phosphatase have major properties very similar to those described for the enzymes from skeletal muscle.     

Reversible ATP-dependent inactivation of glycerolphosphate acyltransferase from rat adipose tissue

Microsomal glycerolphosphate acyltransferase from rat adipose tissue is shown to be inactivated with time upon incubation with ATP. The inactivation can be observed in postmitochondrial supernatant as well as in washed microsomes. However, the effect is more pronounced upon addition of the cytosolic fraction. This activity is specific for ATP, is dependent on the nucleotide concentration, and is prevented when ATP is substituted by beta,gamma-methylene-ATP. Some protection is provided by amiloride but not by EGTA or cAMP-protein kinase inhibitor. Also, the level of enzyme inactivation is not modified by addition of cAMP-dependent protein kinase and its substrates. Inactivated glycerol-phosphate acyltransferase from ATP-treated microsomes can be reactivated by incubation with partially purified protein phosphatase from rat liver. These results suggest the existence in adipose tissue of a protein kinase (cAMP independent) that may be involved in the regulation of glycerolphosphate acyltransferase.     

Hormone-sensitive lipase of rat adipose tissue: correlation of activity with a protein of molecular weight 84 000

Hormone-sensitive lipase of rat adipose tissue was partially purified. The enzyme retained its capacity to be activated by cyclic AMP-dependent protein kinase throughout purification. When the partially purified 32P-labeled preparation was subjected to two-dimensional gel electrophoresis, the enzyme activity was found to be associated with a 32P-labeled protein of molecular weight 84 000. The result suggests that this 32P-labeled protein represents hormone-sensitive lipase or the catalytic subunit of the enzyme.     

Effect of cyclic AMP-dependent protein kinase on insulin receptor tyrosine kinase activity.

To explain the insulin resistance induced by catecholamines, we studied the tyrosine kinase activity of insulin receptors in a state characterized by elevated noradrenaline concentrations in vivo, i.e. cold-acclimation. Insulin receptors were partially purified from brown adipose tissue of 3-week- or 48 h-cold-acclimated mice. Insulin-stimulated receptor autophosphorylation and tyrosine kinase activity of insulin receptors prepared from cold-acclimated mice were decreased. Since the effect of noradrenaline is mediated by cyclic AMP and cyclic AMP-dependent protein kinase, we tested the effect of the purified catalytic subunit of this enzyme on insulin receptors purified by wheat-germ agglutinin chromatography. The catalytic subunit had no effect on basal phosphorylation, but completely inhibited the insulin-stimulated receptor phosphorylation. Similarly, receptor kinase activity towards exogenous substrates such as histone or a tyrosine-containing copolymer was abolished. This inhibitory effect was observed with receptors prepared from brown adipose tissue, isolated hepatocytes and skeletal muscle. The same results were obtained on epidermal-growth-factor receptors. Further, the catalytic subunit exerted a comparable effect on the phosphorylation of highly purified insulin receptors. To explain this inhibition, we were able to rule out the following phenomena: a change in insulin binding, a change in the Km of the enzyme for ATP, activation of a phosphatase activity present in the insulin-receptor preparation, depletion of ATP, and phosphorylation of a serine residue of the receptor. These results suggest that the alteration in the insulin-receptor tyrosine kinase activity induced by cyclic AMP-dependent protein kinase could contribute to the insulin resistance produced by catecholamines.     

Insulin action rapidly decreases multifunctional protein kinase activity in rat adipose tissue

ATP-citrate lyase in vivo contains three phosphorylation sites on two tryptic peptides (peptides A and B). These phosphorylation sites are under hormonal control. Multifunctional protein kinase (MFPK) from rat liver phosphorylates peptide B on serine and threonine residues whereas cAMP-dependent protein kinase phosphorylates peptide A on a serine residue (Ramakrishna, S., and Benjamin, W. B. (1985) J. Biol. Chem. 260, 12280-12286). We now report that rat adipose tissue MFPK also phosphorylates serine and threonine residues of peptide B of ATP-citrate lyase. When the activity of MFPK was assayed using partially purified (by chromatography on phosphocellulose) cytosol fractions from insulin-treated adipose tissue, it was found that MFPK activity was decreased by over 55%. This decrease in MFPK activity occurs at physiological concentrations of insulin (EC50 = 1 x 10(-10) M). Its onset is rapid and almost maximal at 5 min after the addition of insulin. Even when new protein synthesis is inhibited by cycloheximide, extracts from insulin-treated fat pads have less MFPK activity compared to the control. The insulin effect is maintained after further chromatography on a gel filtration column suggesting that the decrease in MFPK activity is not due to a low molecular weight inhibitor. The insulin-induced decrease in MFPK activity is due to a decrease in Vmax whereas the affinity of this enzyme toward ATP-citrate lyase or ATP is unchanged.     

Genetic and biochemical evidence that trehalase is a substrate of cAMP-dependent protein kinase in yeast

In Saccharomyces cerevisiae, trehalase activity in crude extracts obtained from wild type cells was activated about 3-fold by preincubation with cAMP and ATP. The inactive trehalase fractionated by DEAE-Sephacel chromatography was activated by the addition of the cAMP-dependent protein kinase fraction from wild type cells in the presence of cAMP and ATP. Using the crude extract obtained from bcy1 mutant cells which were deficient in the regulatory subunit of cAMP-dependent protein kinase, the stimulation of trehalase activity was observed in the absence of cAMP. The cAMP-dependent protein kinase of CYR3 mutant cells which had a high Ka value for cAMP in the phosphorylation reaction required a high cAMP concentration for activation of trehalase. Increased activation of partially purified inactive trehalase (Mr = 320,000) was observed to correlate with increased phosphorylation of a protein (Mr = 80,000) identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The assay results using various mutants altered in cAMP metabolism indicated that the activation and phosphorylation of inactive trehalase fractions depended on the cAMP concentration accumulated in mutant cells. Inactivation and dephosphorylation of active trehalase fractions were observed by treatment with alkaline phosphatase or crude cell extracts. The results indicated that the conversion of inactive form of trehalase to the active form is regulated by cAMP through cAMP-dependent protein kinase.     

Triglyceride, diglyceride, monoglyceride, and cholesterol ester hydrolases in chicken adipose tissue activated by adenosine 3':5'-Monophosphate-dependent protein kinase. Chromatographic resolution and immunochemical differentiation from lipoprotein lipase.

Hormone-sensitive lipase and cholesterol ester hydrolase of chicken adipose tissue were markedly activated by adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase (on the average, 235 to 275%; occasionally as much as 1000%). Diglyceride and monoglyceride hydrolases were also activated, but to a lesser extent (60 to 87%). The activation of all four hydrolases was inhibited by protein kinase inhibitor and reversed by the addition of exogenous protein kinase. Following activation by cAMP-dependent protein kinase, all four hydrolases were deactivated in a Mg2+-dependent reaction and then reactivated to or near initial levels on incubation with cAMP and Mg2+-ATP. The reversible deactivation is assumed to reflect activity of one or more protein phosphatases. The maximum activation obtainable for the four hydrolases decreased when the tissue had been previously exposed to glucagon, indicating that the glucagon-induced activation was probably similar to or identical with the activation demonstrated in cell-free preparations. The pH optima for the four hydrolase activities were similar (7.13 to 7.38). Although the absolute activities and relative degrees of kinase activation differed according to the particular emulsified substrates used, the results do not rule out the possibility that all four hydrolase activities are referable to a single hormone-sensitive hydrolase. Hormone-sensitive acyl hydrolases were separated from lipoprotein lipase by heparin-Sepharose affinity chromatography. Lipoprotein lipase was active against triolein, diolein, and monoolein, but not cholesterol oleate. Incubation of lipoprotein lipase with exogenous protein kinase, cAMP, and Mg2+ATP had no effect on any of the three hydrolase activities. Lipoprotein lipase was further purified to homogeneity and used to prepare antiserum in rabbits. The immunoglobin G fraction from these antisera completely inhibited lipoprotein lipase eluted from heparin-Sepharose columns. However, the hormone-sensitive hydrolase activities (not retained on heparin-Sepharose affinity chromatography) were not inhibited by anti-lipoprotein lipase immunoglobin G, and anti-lopoprotein lipase immunoglobin G did not affect the activation process in crude fractions. Thus, hormone-sensitive lipase and lipoprotein lipase, functionally distinct enzymes, have been physically resolved and immunochemically distinguished. Apparently lipoprotein lipase activity is not regulated, at least directly, by cAMP-dependent protein kinase.     

Modulation of acetyl-CoA:1-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF) acetyltransferase in human polymorphonuclears. The role of cyclic AMP-dependent and phospholipid sensitive, calcium-dependent protein kinases

In order to characterize the mechanism of activation of the enzyme 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase (EC 2.3.1.67) which is the limiting step in the regulation of the synthesis of the potent inflammatory mediator 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; homogenates from human polymorphonuclear leukocytes were incubated in the presence of the catalytic subunit of cyclic AMP-dependent protein kinase and in the presence of a partially purified phospholipid sensitive, calcium-dependent protein kinase (PrKC). The first kinase was found to enhance up to 3-fold acetyltransferase activity in a dose- and time-dependent manner. In homogenates from PMN previously stimulated with complement-coated zymosan particles, the decay of acetyltransferase activity was partially prevented by the addition of soybean trypsin inhibitor and almost completely inhibited when the homogenates were supplemented with inhibitors of alkaline phosphatase such as 50 mM KF and 100 microM paranitrophenylphosphate. Under these conditions it was possible to initiate the decay of acetyltransferase activity by adding an excess of alkaline phosphatase. Preincubation of PMN with 12-O-tetradecanoylphorbol-13-acetate previous or simultaneously to the addition of ionophore A23187 reduced the increase in acetyltransferase produced by ionophore A23187, whereas the generation of superoxide anions was enhanced. Addition of partially purified PrKC to homogenates from ionophore A23187-stimulated PMN, reduced acetyltransferase activity by 63%, whereas only a 16% inhibition was observed on homogenates from resting PMN. These data indicate the modulation of acetyltransferase activity in human polymorphonuclear leukocytes by a phosphorylation-dephosphorylation mechanism linked to cyclic AMP-dependent protein kinase. Phospholipid sensitive, calcium-dependent protein kinase seems not to be involved in the mechanism of activation, but, most probably, in the generation of negative activation signals.     

Studies on insulin-stimulated phosphorylation of acetyl-CoA carboxylase, ATP citrate lyase and other proteins in rat epididymal adipose tissue. Evidence for activation of a cyclic AMP-independent protein kinase.

Protein kinase activity in high-speed supernatant fractions prepared from rat epididymal adipose tissue previously incubated in the absence or presence of insulin was investigated by following the incorporation of 32P from [gamma-32P]ATP into phosphoproteins separated by sodium dodecyl sulphate/polyacrylamide-gel electro-phoresis. Incorporation of 32P into several endogenous proteins in the supernatant fractions from insulin-treated tissue was significantly increased. These included acetyl-CoA carboxylase and ATP citrate lyase (which exhibit increased phosphorylation within fat-cells exposed to insulin), together with two unknown proteins of subunit Mr 78000 and 43000. The protein kinase activity increased by insulin was distinct from cyclic AMP-dependent protein kinase, was not dependent on Ca2+ and was not appreciably affected by dialysis or gel filtration. The rate of phosphorylation of added purified fat-cell acetyl-CoA carboxylase and ATP citrate lyase was also increased by 60-90% in high-speed-supernatant fractions prepared from insulin-treated tissue. No evidence for any persistent changes in phosphoprotein phosphatase activity was found. It is concluded that insulin action on acetyl-CoA carboxylase, ATP citrate lyase and other intracellular proteins exhibiting increased phosphorylation involves an increase in cyclic AMP-independent protein kinase activity in the cytoplasm. The possibility that the increase reflects translocation from the plasma membrane, perhaps after phosphorylation by the protein tyrosine kinase associated with insulin receptors, is discussed.     

Activation of lipoprotein lipase by blood platelets

When an homogenate of human blood platelets is added to preparations of partially purified adipose tissue lipoprotein lipase, the lipase activity is markedly enhanced. The degree of activation is related to the amount of platelet homogenate in the reaction mixture. The lipoprotein lipase activator in platelets is heat labile, nondialyzable, partially resistant to papain, and present mostly in the particulate fraction of platelet homogenates.     

Human adipose tissue hormone-sensitive lipase: identification and comparison with other species

The mRNA for human hormone-sensitive lipase (HSL) was identified using Northern blot analysis and a cDNA-probe for rat HSL. As in the rat, human adipose tissue expresses a single mRNA species of 3.3 kb. Using Western blotting with a polyclonal rabbit antibody towards rat adipose tissue HSL, the corresponding enzyme in human adipose tissue was identified with an apparent 88 kDa polypeptide, thus slightly larger than the rat and bovine 84 kDa, and the mouse and guinea-pig 82 kDa species. Additional evidence for the identification was provided by the inhibition of HSL diacylglycerol lipase activity by the anti-rat HSL antibody, and by NaF, DFP and Hg2+, known inhibitors of HSL. The concentration of the enzyme, as reflected by its activity per g tissue and the specific activity was about two thirds of that in the rat adipose tissue (200 g rats). The identification of the human enzyme protein made it possible to directly demonstrate its phosphorylation by cAMP-dependent protein kinase, thus extending the previous report regarding activation of the lipase with this kinase and ATP-Mg2+ in human adipose tissue extracts (Khoo, J.C., Aquino, A.A. and Steinberg, D. (1974) J. Clin. Invest. 53, 1124-1131).     

Cyclic 3',5'-adenosine monophosphate-dependent kinase and phosphoproteins in human amnion

3',5'-Cyclic adenosine monophosphate (cAMP) modulates prostaglandin production in human amnion membranes. The major effects of cAMP are presumably mediated through the phosphorylation of specific regulatory phosphoproteins following cAMP activation of cAMP-dependent protein kinase. Cyclic AMP-dependent protein kinase and phosphoproteins have not previously been characterized in human amnion. Total homogenates, cytosol, and membrane fractions from human amnion were examined for [3H]cAMP binding activity and cAMP-dependent kinase activity. cAMP-dependent kinase activity was barely detectable in crude amnion fractions. Cytosol was therefore partially purified by DEAE column chromatography for further examination. Two peaks of coincident [3H]cAMP binding and cAMP-dependent kinase activity were demonstrated at 70 and 140 mM NaCl, characteristic of the Type I and Type II cAMP-dependent protein kinase isozymes. [3H]cAMP binding to the material from both peak fractions was saturable and reversible. Scatchard analysis of [3H]cAMP binding to the peak fractions was linear for peak I and curvilinear for peak II. Assuming a one-site model, [3H]cAMP binding to the Type I isozyme showed a KD = 4.17 x 10(-8) M and Bmax = 73 pmole/mg protein; using a two-site model, [3H]cAMP binding to the high-affinity site for the Type II isozyme had a KD = 3.94 x 10(-8) M and Bmax = 6.3 pmole/mg protein. Other cyclic nucleotides competed for these [3H]cAMP binding sites with a potency order of cAMP much greater than cGMP greater than (BU)2cAMP.cAMP caused a dose-dependent increase in cAMP-dependent kinase activity in the peak fractions; half-maximal activation was observed with 5.0 x 10(-8) M cAMP. The ability of cAMP to increase phosphorylation of endogenous proteins in both crude amnion cytosol and cytosol from cultures of amnion epithelial cells was assessed using [32P]ATP, SDS-polyacrylamide gel electrophoresis and autoradiography. cAMP stimulated 32P incorporation into three proteins having Mr = 80,000, 54,000, and 43,000 (P less than .01). Half-maximal 32P incorporation into these proteins occurred at 1.0 x 10(-7) M cAMP. cAMP-dependent kinase is present in human amnion; specific cAMP-enhanced phosphoproteins are also present. Hormones elevating cAMP levels in amnion may exert their effects by activating cAMP-dependent kinase and phosphorylating these phosphoproteins.     

Studies on protein phosphorylation using subcellular fractions from insulin-treated white adipose tissue of rats

In these studies the incorporation of 32P into proteins within subcellular fractions, obtained from rat white adipose tissue upon incubation in the presence of [gamma-32P]ATP, was investigated. A stable increase in the activity of protein serine(threonine) kinase in high-speed supernatant fractions was observed following treatment of intact tissue with insulin. Protein kinase activity associated with the plasma membrane fraction of cells was diminished in response to insulin, but the decrease was apparently insufficient to account for increases observed in corresponding supernatant fractions. A range of assay conditions was employed to characterize the insulin-stimulated protein serine(threonine) kinase in in supernatant fractions. The insulin-stimulated protein serine(threonine) kinase displays properties that indicate it is distinct from a number of well-characterized protein kinases, including those regulated by cAMP, calcium ions (in the presence or absence of calmodulin or mixtures of phosphatidylserine-diacylglycerol), polyamines, or heparin. There were no apparent effects of insulin on incorporation of 32P into added casein or histones II-S or III-S. The protein serine(threonine) kinase activity (or activities) described here displays properties that also appear to differ from the properties of previously described insulin-stimulated activities able to catalyze the phosphorylation of the ribosomal protein S6. The differences in properties may, in part, be explained by the use of different cell types, but may also indicate that treatment of cells with insulin leads to activation of more than one protein serine(threonine) kinase.     

Regulation of a rat lung protein tyrosine kinase activity by reversible phosphorylation/dephosphorylation

Incubation of a partially purified protein tyrosine kinase from rat lung with Mg2+ and ATP resulted in about 10-15-fold activation of the enzyme activity as judged by the phosphorylation of poly(Glu:Tyr,4:1), an exogenous substrate. The activation was time dependent and was associated with the phosphorylation of a single protein band of 50 kDa. Phosphoamino acid analysis of the phosphorylated protein indicated that tyrosine was the amino acid being phosphorylated. Upon gel filtration on a Sephacryl S-200 column, the phosphorylated protein co-eluted with protein tyrosine kinase and ATP-binding activities, suggesting that all three activities are part of the same protein. In addition, pretreatment of the partially purified protein tyrosine kinase with alkaline phosphatase inhibited its enzyme activity which could be restored by reincubation with Mg2+ and ATP. These data suggest that a temporal relationship exists between the phosphorylation and the activation states of rat lung protein tyrosine kinase, and that the phospho- and dephospho- forms represent the active and inactive (or less active) forms, respectively, of the enzyme.     

Identification of an activator required for elevation of maturation- promoting factor (MPF) activity by gamma-S-ATP

Maturation-promoting factor (MPF) is a cell cycle control element able to cause cells to enter M-phase upon microinjection and will induce metaphase in nuclei incubated in cell extracts. Previous work has shown that MPF is composed of a complex between p34cdc 2 protein kinase and a B-type cyclin. In the present work gamma-S-ATP was found to cause activation of MPF activity in partially purified preparations, but this activation was lost upon chromatography on Matrex Green gel A. Readdition of other Matrex Green fractions to purified MPF restored the ability of gamma-S-ATP to activate MPF for nuclear breakdown as well as phosphorylation of histone H1. Use of the system described here will facilitate study of p34cdc 2 kinase activation and identification of elements involved in MPF regulation.     

Hormone-sensitive lipase of adipose tissue.

Some physiologic aspects of the mobilization and fate of free fatty acids are reviewed. The molecular mechanism of the activation of hormone-sensitive lipase in adipose tissue is then discussed. Recent evidence established that hormone-sensitive lipase, concerned with fat mobilization, is both functionally and immunochemically distinct from lipoprotein lipase, concerned with uptake of plasma triglycerides. Lipoprotein lipase activity is not altered by cyclic AMP-dependent protein kinase. The latter enzyme enhances not only triglyceride hydrolase but also monoglyceride, diglyceride and cholesterol ester hydrolase activities in chicken adipose tissue. Finally, it is shown that the activation of all four acyl hydrolases is reversible, the deactivation being magnesium-dependent. Protein phosphatase fractions from heart and liver active against phosphorylase a can reversibly deactivate adipose tissue hormone-sensitive lipase, implying a low degree of substrate specificity for lipase phosphatase.