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.     

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.     

Inhibition of the reversible deactivation of chicken adipose tissue hormone-sensitive lipase by heat-stable proteins from adipose tissue and skeletal muscle.

The reversible deactivation of chicken adipose tissue hormone-sensitive lipase is catalyzed by a lipase phosphatase. Heat-stable protein preparations from rat epididymal fat pads, chicken adipose tissue, and rabbit skeletal muscle inhibited lipase phosphatase activity. Phosphatase inhibitor preparations from rat adipose tissue did not inhibit the protein kinase-catalyzed activation of hormone-sensitive lipase, whereas inhibitor preparations from rabbit skeletal muscle were contaminated with protein kinase inhibitor.     

Activation of chicken adipose tissue diglyceride lipase by cyclic AMP-dependent protein kinase and its deactivation by purified protein phosphatase.

Diglyceride lipase of chicken adipose tissue was found to be activated by cyclic AMP-dependent protein kinase to the same extent as hormone-sensitive triglyceride lipase (3-to 10-fold) when lipase assays were carried out in buffers of low ionic strength. Sodium phosphate (50 mM) or sodium chloride (100 mM) preferentially enhanced the basal (nonactivated) form of diglyceride lipase, which minimized the apparent activation by protein kinase. The activated diglyceride lipase was readily deactivated by a pure protein phosphatase from bovine heart (MW 35,000) and the deactivated enzyme was then reactivated by protein kinase.     

Purification and control of bovine adrenal cortical cholesterol ester hydrolase and evidence for the activation of the enzyme by a phosphorylation.

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.     

Lipolysis: pathway under construction

PURPOSE OF REVIEW: The lipolytic catabolism of stored fat in adipose tissue supplies tissues with fatty acids as metabolites and energy substrates during times of food deprivation. This review focuses on the function of recently discovered enzymes in adipose tissue lipolysis and fatty acid mobilization. RECENT FINDINGS: The characterization of hormone-sensitive lipase-deficient mice provided compelling evidence that hormone-sensitive lipase is not uniquely responsible for the hydrolysis of triacylglycerols and diacylglycerols of stored fat. Recently, three different laboratories independently discovered a novel enzyme that also acts in this capacity. We named the enzyme 'adipose triglyceride lipase' in accordance with its predominant expression in adipose tissue, its high substrate specificity for triacylglycerols, and its function in the lipolytic mobilization of fatty acids. Two other research groups showed that adipose triglyceride lipase (named desnutrin and Ca-independent phospholipase A2zeta, respectively) is regulated by the nutritional status and that it might exert acyl-transacylase activity in addition to its activity as triacylglycerol hydrolase. Adipose triglyceride lipase represents a novel type of 'patatin domain-containing' triacylglycerol hydrolase that is more closely related to plant lipases than to other known mammalian metabolic triacylglycerol hydrolases. SUMMARY: Although the regulation of adipose triglyceride lipase and its physiological function remain to be determined in mouse lines that lack or overexpress the enzyme, present data permit the conclusion that adipose triglyceride lipase is involved in the cellular mobilization of fatty acids, and they require a revision of the concept that hormone-sensitive lipase is the only enzyme involved in the lipolysis of adipose tissue triglycerides.     

Acid hydrolases in early and late endosome fractions from rat liver.

We have examined the distribution of the cation-independent mannose 6-phosphate receptor and five acid hydrolases in early and late endosomes and a receptor-recycling fraction isolated from livers of estradiol-treated rats. Enrichment of mannose 6-phosphate receptor mass relative to that of crude liver membranes was comparable in membranes of early and late endosomes but was even greater in membranes of the receptor-recycling fraction. Enrichment of acid hydrolase activities (aryl sulfatase, N-acetyl-beta-glucosaminidase, tartrate-sensitive acid phosphatase, and cholesteryl ester acid hydrolase) and cathepsin D mass was also comparable in early and late endosomes but was considerably lower in the receptor-recycling fraction. The enrichment of two acid hydrolases, acid phosphatase and cholesteryl ester acid hydrolase, in endosomes was severalfold greater than that of the other three examined, about 40% of that found in lysosomes. Acid phosphatase and cholesteryl ester acid hydrolase were partially associated with endosome membranes, whereas cathepsin D was found entirely in the endosome contents. These findings raise the possibility that lysosomal enzymes traverse early endosomes during transport to lysosomes in rat hepatocytes and suggest that the greater enrichment of some acid hydrolases in endosomes is related to their association with endosome membranes. Despite the substantial enrichment of lysosomal enzymes in hepatocytic endosomes, we found that two, cholesteryl ester acid hydrolase and cathepsin D, did not degrade cholesteryl esters and apolipoprotein B-100 of endocytosed low density lipoproteins in vivo, presumably because they are inactive at the pH within endosomes.     

Protein kinase-mediated activation of temperature-labile and temperature-stable cholesteryl ester hydrolases in the rat testis

Both temperature-stable and temperature-labile testicular cholesteryl ester hydrolases are shown to be regulated by an endogenous cAMP-dependent protein kinase activity. The temperature-stable form (Mr = 28,000) was activated 3-fold by the endogenous kinase. This activation was completely blocked by protein kinase inhibitor. Following purification by high performance gel permeation chromatography, the temperature-stable form could also be activated 2-fold by bovine heart protein kinase, type I. The partially purified endogenous protein kinase, type I, which was completely separated from hydrolase activity by ion exchange chromatography, increased hydrolase activity 2-fold in the presence of optimal concentrations of cAMP, ATP, and Mg2+. Cholesteryl ester hydrolase activity could be stabilized indefinitely at -10 degrees C with the addition of 0.1 mM thioglycolate, but not by other thiol reagents. In contrast, the endogenous protein kinase activity was lost from 104,000 X g supernatants after 14 days. However, the property of activation could be restored by addition of bovine heart protein kinase. The temperature-labile hydrolase (Mr = 72,000) could be totally inactivated by a Mg2+-dependent, fluoride-sensitive cytosolic factor and reactivated by cAMP-dependent protein kinase. These observations strongly suggest that the inactivating factor is a phosphoprotein phosphatase.     

Topological studies on rat liver microsomal cholesterol ester hydrolase

Lateral and transversal distribution of cholesterol ester hydrolase activity in rat liver microsomal membranes has been studied. Total cholesterol ester hydrolase activity was found predominantly (75%) in rough microsomes though specific esterase activities were similar in rough and smooth microsomal fractions. The transversal asymmetry of the enzyme was examined using the criteria of protease sensitivity and latency of mannose-6-phosphate phosphatase. Cholesterol ester hydrolase resulted drastically inhibited by proteolysis with trypsin when microsomal integrity had been previously disrupted with sodium deoxycholate or sodium taurocholate. Under these conditions, most lumenal mannose-6-phosphate phosphatase activity was destroyed. However, cholesterol esterase was unaffected by preincubating microsomes with the detergent alone, which led to the complete expression of latent mannose-6-phosphate phosphatase or by preincubating them with trypsin, where less than a 15% of the lumenal mannose-6-phosphate phosphatase was lost. These findings suggest that cholesterol ester hydrolase activity is located on the lumenal surface of the hepatic microsomal vesicles.     

Purification and subunit structure of rat-liver phosphoprotein phosphatase, whose molecular weight is 260000 by gel filtration (phosphatase IB)

1. Phosphoprotein phosphatase IB is a form of rat liver phosphoprotein phosphatase, distinguished from the previously studied phosphoprotein phosphatase II [Tamura et al. (1980) Eur. J. Biochem. 104, 347-355] by earlier elution from DEAE-cellulose, by higher molecular weight on gel filtration (260000) and by lower activity toward phosphorylase alpha. This enzyme was purified to apparent homogeneity by chromatography on DEAE-cellulose, aminohexyl--Sepharose-4B, histone--Sepharose-4B, protamine--Sepharose-4B and Sephadex G-200. 2. The molecular weight of purified phosphatase IB was 260000 by gel filtration and 185000 from S20,W and Stokes' radius. Using histone phosphatase activity as the reference for comparison, the phosphorylase phosphatase activity of purified phosphatase IB was only one-fifth that of phosphatase II. 3. Sodium dodecyl sulfate gel electrophoresis revealed that phosphatase IB contains three types of subunit, namely alpha, beta and gamma, whose molecular weights are 35000, 69000 and 58000, respectively. The alpha subunit is identical to the alpha subunit of phosphatase II. While the beta subunit is also identical or similar to the beta subunit of phoshatase II, the gamma subunit appears to be unique to phosphatase IB. 4. When purified phosphatase IB was treated with 2-mercaptoethanol at -20 degrees C, the enzyme was dissociated to release the catalytically active alpha subunit. Along with this dissociation, there was a 7.4-fold increase in phosphorylase phosphatase activity; but histone phosphatase activity increased only 1.6-fold. The possible functions of the gamma subunit are discussed in relation to this activation of enzyme.     

The regulatory and basal phosphorylation sites of hormone-sensitive lipase are dephosphorylated by protein phosphatase-1, 2A and 2C but not by protein phosphatase-2B

The activity of hormone-sensitive lipase, the rate-limiting enzyme in adipose tissue lipolysis, is controlled by cAMP-mediated phosphorylation at a specific regulatory phosphorylation site. The lipase is also phosphorylated at a site, termed basal, without any effects on its activity [Str?lfors et al. (1984) Proc. Natl Acad. Sci. USA 81, 3317-3321]. The capacity of protein phosphatase-1, 2A, 2B and 2C to dephosphorylate the lipase, selectively phosphorylated by glycogen synthase kinase-4 and cAMP-dependent protein kinase at the basal and regulatory phosphorylation sites, was compared with that towards glycogen phosphorylase and phosphorylase kinase (alpha subunit). Protein phosphatase-1, 2A and 2C were found to dephosphorylate both phosphorylation sites of hormone-sensitive lipase, while protein phosphatase-2B had no measureable activity towards any of the sites. When the activities of protein phosphatase-1, 2A and 2C were normalized with respect to the reference substrates, they were found to dephosphorylate the lipase regulatory site in the approximate relations of 1:4:3 and the basal site in the approximate relations of 1:6:4. Protein phosphatase-1 showed 20% higher and protein phosphatase-2A and 2C 80% higher activity towards the basal site compared to the regulatory site. The two phosphorylation sites of the lipase were comparable to good substrates for protein phosphatase-2A and 2C, but relatively poor substrates for protein phosphatase-1. Protein phosphatase-2C activity towards the lipase was completely dependent on Mg2+ with a half-maximal effect at 3 mM. Protamine increased the lipase dephosphorylation by protein phosphatase-1 3-5-fold with half-maximal effect at 0.6 microgram/ml, and by protein phosphatase-2A about 2-fold with half-maximal effect at 3-5 micrograms/ml, thus illustrating the potential for control of these lipase phosphatase activities.     

Purification, properties, and substrate specificities of phosphoprotein phosphatase(s) from rabbit liver.

The phosphoprotein phosphatase(s) acting on muscle phosphorylase a was purified from rabbit liver by acid precipitation, high speed centrifugation, chromatography on DEAE-Sephadex A-50, Sephadex G-75, and Sepharose-histone. Enzyme activity was recovered in the final step as two distinct peaks tentatively referred to as phosphoprotein phosphatases I and II. Each phosphatase showed a single broad band when examined by sodium dodecyl sulfate gel electrophoresis; the molecular weights derived by this method were approximately 30,500 for phosphoprotein phosphatase I and 34,000 for phosphoprotein phosphatase II. The s20, w value for each enzyme was 3.40. Using this value and values for the Stokes radii, the molecular weight for each enzyme was calculated to be 34,500. Both phosphatases, in addition to catalyzing the conversion of phosphorylase a to b, also catalyzed the dephosphorylation of glycogen synthase D, activated phosphorylase kinase, phosphorylated histone, phosphorylated casein, and the phosphorylated inhibitory component of troponin (TN-I). The relative activities of the phosphatases with respect to phosphorylase a, glycogen synthase D, histone, and casein remained essentially constant throughout the purification. The activities of both phosphatases with different substrates decreased in parallel when they were denatured by incubation at 55 degrees and 65 degrees. The Km values of phosphoprotein phosphatase I for phosphorylase a, histone, and casein were lower than the values obtained for phosphoprotein phosphatase II. With glycogen synthase D as substrate, each enzyme gave essentially the same Km value. Utilizing either enzyme, it was found that activity toward a given substrate was inhibited competitively by each of the alternative substrates. The results suggest that phosphoprotein phosphatases I and II are each active toward all of the substrates tested.     

Purification of pancreatic carboxylic-ester hydrolase by immunoaffinity and its application to the human bile-salt-stimulated lipase

A column of immobilized antibodies directed against pure human pancreatic carboxylic (cholesterol) ester hydrolase was used to purify in a single step the enzyme from human pancreatic juice as well as carboxylic-ester hydrolases from other species (rat, dog). This immunoaffinity method was also used for the purification of the related bile-salt-stimulated lipase from the human skim milk. The enzymes were homogeneous on SDS-PAGE. The yields obtained were always higher than those previously observed using either conventional or affinity columns. The human and dog carboxylic-ester hydrolases as well as the bile-salt-stimulated lipase, in contrast to the rat enzyme, are glycoproteins. From our results, it can be speculated that these enzymes, which differ in their molecular weight but not in their N-terminal sequences or amino-acid compositions, might have a similar proteic core with a molecular mass between 65 and 75 kDa. The difference in their respective molecular masses might result from a different level of glycosylation of pancreatic carboxylic-ester hydrolases (and milk bile-salt-stimulated lipase).     

Characterization of phosphoprotein phosphatases and phosphorylase phosphatase from yeast

Three peaks of protein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) activity (fractions a, b and c) acting on muscle phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1) were separated by DEAE-cellulose chromatography of yeast extracts. In contrast to fractions a and b, only fraction c was able to liberate phosphate from 32P-labelled inactivated yeast phosphorylase. The activity of fraction c on both substrates was totally dependent on the presence of bivalent metal ions (Mg2+, Mn2+), and was activated by Mg . ATP. Following freezing in the presence of mercaptoethanol, fractions a and b were also able to dephosphorylate yeast phosphorylase. Rabbit muscle phosphoprotein phosphatase inhibitors 1 and 2 showed that yeast phosphatases acting on muscle phosphorylase were inhibited by inhibitor 2 but not by inhibitor 1. The action of fraction c on yeast phosphorylase was not inhibited by either inhibitor. The native yeast phosphorylase phosphatase (EC 3.1.3.17) was purified 8000-fold by ion-exchange chromatography, casein-Sepharose chromatography and Sephadex G-200 gel filtration. The purified enzyme was unable to dephosphorylate rabbit muscle phosphorylase a, but acted on casein phosphate (Km 3.3 mg/ml). Molecular weight was estimated to be 78 000 and pH optimum 6.5-7.5. Activity of the enzyme was dependent on bivalent metal ions (Mg2+, Mn2+) and was inhibited by fluoride (Ki 20 mM) and succinate (Ki 10 mM).     

Direct evidence for protein phosphatase-catalyzed dephosphorylation/deactivation of hormone-sensitive lipase from adipose tissue

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.     

Purification and characterization of a high molecular weight phosphoprotein phosphatase from rabbit liver

A high molecular weight phosphoprotein phosphatase was purified from rabbit liver using high speed centrifugation, acid precipitation, ammonium sulfate fractionation, chromatography on DEAE-cellulose, Sepharose-histone, and Bio-Gel A-0.5m. The purified enzyme showed a single band on a nondenaturing polyacrylamide anionic disc gel which was associated with the enzyme activity. The enzyme was made up of equimolar concentrations of two subunits whose molecular weights were 58,000 (range 58,000-62,000) and 35,000 (range 35,000-38,000). Two other polypeptides (Mr 76,000 and 27,000) were also closely associated with our enzyme preparation, but their roles, if any, in phosphatase activity are not known. The optimum pH for the reaction was 7.5-8.0. Km value of phosphoprotein phosphatase for phosphorylase a was 0.10-0.12 mg/ml. Freezing and thawing of the enzyme in the presence of 0.2 M beta-mercaptoethanol caused an activation (100-140%) of phosphatase activity with a concomitant partial dissociation of the enzyme into a Mr 35,000 catalytic subunit. Divalent cations (Mg2+, Mn2+, and Co2+) and EDTA were inhibitory at concentrations higher than 1 mM. Spermine and spermidine were also found to be inhibitory at 1 mM concentrations. The enzyme was inhibited by nucleotides (ATP, ADP, AMP), PPi, Pi, and NaF; the degree of inhibition was different with each compound and was dependent on their concentrations employed in the assay. Among various types of histones examined, maximum activation of phosphoprotein phosphatase activity was observed with type III and type V histone (Sigma). Further studies with type III histone indicated that it increased both the Km for phosphorylase a and the Vmax of the dephosphorylation reaction. Purified liver phosphatase, in addition to the dephosphorylation of phosphorylase a, also catalyzed the dephosphorylation of 32P-labeled phosphorylase kinase, myosin light chain, myosin, histone III-S, and myelin basic protein. The effects of Mn2+, KCl, and histone III-S on phosphatase activity were variable depending on the substrate used.     

Purification and properties of a heat-stable protein inhibitor of phosphoprotein phosphatase from rabbit liver.

A heat-stable protein inhibitor of phosphoprotein phosphatase has been purified to homogeneity from rabbit liver extract by heating to 95 degrees followed by ion exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. The purified inhibitor showed a single band when examined by gel electrophoresis S20, w and Stokes radius values were 1.45 and 25.5, respectively. Using these two values, the molecular weight and frictional ratio was calculated to be 15,500 and 3.40, respectively. The molecular weight determined by sodium dodecyl sulfate-gel electrophoresis was found to be 14,200. The inhibition of phosphoprotein phosphatase was linear up to 40% inhibition with respect to inhibitor was constant with time of incubation for at least 30 min. The optimum pH for the inhibition was between 6.8 and 7.6. A kinetic analysis of the effect of the inhibitor on the dephosphorylation of [32P]phosphorylase a by rabbit liver phosphoprotein phosphatase indicated a noncompetitive inhibition with respect to phosphorylase a. Purified liver inhibitor inhibited the phosphoprotein phosphatase activity in all rat tissues examined. Utilizing purified rabbit liver phosphoprotein phosphatase, the presence of inhibitor activity was also demonstrated in all rat tissues tested.     

Purification from human plasma of a heparin-released lipase with activity against triglyceride and phospholipids.

A triglyceride lipase different from lipoprotein lipase, but measurable only after intravenous heparin injection, has been isolated from human plasma by sequential use of heparin-Sepharose and concanavalin A-Sepharose affinity chromatography. Using these procedures, phospholipase A1 activity was found to chromatograph identically with the triglyceride lipase. The constancy of the ratio of activities after isoelectric focusing (pI 4.1) and during thermal deactivation indicates that this enzyme has hydrolase activity against both triglycerides and phospholipids. This conclusion was supported further by the homogeneity of the protein as indicated by sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

Hydrolysis of primary and secondary esters of glycerol by pancreatic juice

The relative rates of hydrolysis of the secondary ester in glycerol 1,3-benzylidene 2-oleate and in glycerol 1,3-dihexadecyl ether 2-oleate, and of the primary and secondary esters in triolein were determined. Both unaltered and selectively inactivated rat pancreatic juice were used as sources of enzyme. It was found that rat pancreatic juice contains an enzyme that can hydrolyze fatty acids esterified at the 2-position of a glyceride. This enzyme is not pancreatic lipase. It may be sterol ester hydrolase. Partial glycerides, as well as complete glycerides, can serve as substrates. Pancreatic lipase, if it can hydrolyze the 2-positioned fatty acids of a triglyceride, does so at a very slow rate.     

Phosphoprotein phosphatase associated with rat liver plasma membrane. Properties of phosphorylase phosphatase and phosphohistone phosphatase

Plasma membrane isolated from rat liver contained activities of phosphoprotein phosphatase dephosphorylating [32P]phosphorylase a or [32P]phosphohistone. The properties of the membrane-bound phosphatase were examined using these exogenous substrates. The optimal reaction rate was at pH near neutrality. At concentrations as low as 0.1-1.0 mM, Mg2+ or Mn2+ slightly stimulated the activity for phosphorylase a or phosphohistone, respectively; at higher concentrations, they were inhibitory with both substrates. Co2+ was inhibitory with both substrates, while Ca2+ had no significant effect. The phosphatase activities were inhibited by ATP, ADP, or AMP; the extents of inhibition were in opposite order with the two substrates. Phosphorylase phosphatase activity was strongly inhibited by KF or Pi. Phosphorylase phosphatase activity could be completely solubilized by incubating the membrane with 0.5 M NaCl or trypsin, and this was associated with several-fold activation. While Vmax values were increased, Km values for phosphorylase a were not much affected by these treatments. Unlike the soluble phosphatase, freezing in the presence of mercaptoethanol or by precipitation with ethanol failed to activate or to solubilize the membrane-bound phosphatase. The molecular weights of the NaCl-and the trypsin-solubilized phosphatase were estimated on gel filtration to be about 42,000 and 32,000, respectively. The present results indicate that the phosphoprotein phosphatase associated with liver plasma membrane shares several properties in common with phosphatases from other sources reported, and that, like those in the soluble fraction, it may be bound to some inhibitory proteins.