Esterastin: a potent inhibitor of lysosomal acid lipase

The effect of a fungal metabolite, esterastin, on lysosomal acid lipase purified from rabbit liver was studied. Esterastin inhibited the enzyme activity very strongly (IC50, about 80 nM). The inhibition of acid lipase by esterastin was competitive with respect to the substrate and the inhibition constant for esterastin was 90 nM. Esterastin was less inhibitory to other lipolytic enzymes, such as pancreatic lipase and carboxylesterase. Thus esterastin is a potent new inhibitor of lysosomal acid lipase.     

Changes in oleic acid oxidation and incorporation into lipids of differentiating L6 myoblasts cultured in normal or fatty acid-supplemented growth medium.

L6 myoblasts accumulate large stores of neutral lipid (predominantly triacylglycerol) when cultured in fatty acid-supplemented growth medium. No accumulation of neutral lipid was evident in myotubes (differentiated myoblasts) when treated similarly. Triacylglycerol accumulation was rapid and dependent on exogenous fatty acid concentration. Triacylglycerol content in myoblasts cultured in fatty acid-supplemented growth medium was approx. 3-fold higher than that in myotubes treated similarly and 2-3-fold higher than that in myoblasts cultured in normal growth medium. Incorporation studies using [I-14C]oleic acid showed that myoblasts and myotubes take up exogenous fatty acid at similar rates. However, cells cultured in fatty acid-supplemented growth medium remove more exogenous fatty acid than do cells cultured in normal growth medium. Over 90% of the incorporated label was found in phospholipid and triacylglycerol fractions in all situations studied. Myoblasts incorporated a more significant proportion (P less than 0.001) of label into triacylglycerol compared with that of myotubes. No differences in fatty acid oxidation rates were detected when differentiating L6 cells cultured in normal growth medium were compared with those cultured in fatty acid-supplemented growth medium. However, fatty acid oxidation rates were observed to increase 3-5-fold upon myoblast differentiation. We conclude that there is a marked change in the pattern of lipid metabolism when myoblasts (primarily triacylglycerol-synthesizing cells) differentiate into myotubes (primarily phospholipid-synthesizing cells). Understanding these changes, which coincide with normal muscle development, may be important, since a defect in this natural switch could explain the observed accumulation of lipid in muscle characteristic of some of the muscular dystrophies and other lipid-storage myopathies.     

Post-heparin triacylglycerol lipases in ovine plasma

Lipoprotein lipase and hepatic lipase have been shown to be present in the post-heparin plasma of sheep. Intravenous injection of heparin into sheep produced a rapid increase in the free fatty acid concentration and lipolytic enzyme activity of the plasma, both peaking within 5-15 min and then falling to pre-heparin levels within 30-60 min. Lipolytic activity was not detected in plasma before heparin treatment. Two distinct lipolytic activities were separated from the plasma by chromatography on heparin-Sepharose 6B. Lipoprotein lipase was identified on the basis that the lipolytic activity was dependent upon the addition of plasma, inhibited by 1M NaCl, and inhibited by a specific antiserum against lipoprotein lipase. The second lipolytic activity of plasma was identified as hepatic lipase, as it was not dependent upon plasma for activity, nor was it inhibited by 1M NaCl or antiserum against lipoprotein lipase. Its properties were identical to the lipase extracted from the liver of sheep. Lipoprotein-lipase activity, but not hepatic-lipase activity, was dependent upon the nutritional state of the sheep at the time of heparin injection. However, hepatic lipase comprised a significant proportion of the total lipolytic activity.     

Tetracycline Inhibition of a Lipase from Corynebacterium acnes

A lipase which hydrolyzes triglycerides (tricaprylin and trilaurin) and naphthyl laurate was obtained from the broth of Corynebacterium acnes cultures by ammonium sulfate fractionation. Ca(2+) and sodium taurocholate stimulated activity of the enzyme. Ethylenediaminetetraacetic acid (EDTA) did not inhibit activity of the Ca(2+)-activated enzyme, but lipolytic activity was inhibited by EDTA in the absence of Ca(2+). Tetracycline (10(-4)m) produced a slight inhibition of the lipase activity with 5 x 10(-5)m or less showing no effect on the lipase activity. However, complete inhibition by tetracycline at 10(-4)m was observed for Ca(2+)-activated enzyme. Tetracycline inhibition of the C. acnes lipase could be demonstrated at concentrations as low as 10(-6)m.     

Studies of lysosomal sialic acid metabolism: retention of sialic acid by Salla disease lysosomes

Purified rat liver lysosomes were incubated in 0.2 M sialic acid resulting in an increase in lysosomal free sialic acid of 3.8 +/- 1.5 nmol/unit beta hexosaminidase. Sialic acid loss by these lysosomes was stimulated 2-3 fold by 25 mM sodium phosphate. Loss of sialic acid by lysosomes from cultured human diploid fibroblasts was similar to that observed in rat liver lysosomes while loss of sialic acid by lysosomes from cultured fibroblasts from a patient with infantile Salla disease occurred much more slowly. Salla disease appears to be the consequence of defective lysosomal transport of sialic acid and is analogous to cystinosis, a disorder of lysosomal amino acid transport.     

Dibutyryl cyclic AMP decreases the rate of lipoprotein lipase synthesis in cultured adipocytes

The regulation of avian lipoprotein lipase by dibutyryl cyclic AMP in cultured adipocytes was studied with quantitative and specific methods for the measurements of enzyme catalytic activity, enzyme protein mass, and immunoadsorption of labeled enzyme. Incubation of adipocytes in 0.5 mM dibutyryl cyclic AMP plus 0.5 mM theophylline results in a time-dependent decrease in cell lipoprotein lipase catalytic activity. The activity is decreased by 70% in 4 h and over 90% by 12 h. The decrease in cellular catalytic activity is due to a decrease in both enzyme content and enzyme catalytic efficiency. 4 h after exposure of adipocytes to cAMP, enzyme protein was decreased from 3.58 +/- 0.5 to 1.92 +/- 0.1 ng/dish and specific activity from 15.1 +/- 2.1 to 8.4 +/- 1.1 nmol/ng. In the presence of 0.5 mM theophylline, the dibutyryl cyclic AMP-mediated decrease in lipoprotein lipase activity was half-maximal at less than 25 microM dibutyryl cyclic AMP. The rate of lipoprotein lipase synthesis was estimated by measuring the incorporation of L-[35S]methionine into enzyme protein during 30 min. A method for the quantitative immunoadsorption of lipoprotein lipase from cell lysates was developed. Utilizing this immunoadsorption technique, the rate of incorporation of L-[35S]methionine into lipoprotein lipase was 0.0026 +/- 0.002%, when expressed as a percentage of that incorporated into total trichloroacetic acid-precipitable counts. By 2 h after exposure of adipocytes to 0.5 mM dibutyryl cAMP, the relative synthesis rate had already decreased to 64 +/- 4% of the control rate. After 16 h the synthesis rate was 43.2 +/- 13.8% of the control rate. The observed decreased synthesis rate could account for most of the decreased cellular enzyme content and diminished enzyme secretion rate.     

Synthesis of lipoprotein lipase in cultured avian granulosa cells

Avian granulosa cells cultured as a homogeneous parenchymal population contain lipolytic activity. This activity is stimulated 2--5-fold by serum, inhibited 90% by 1 M NaCl and inhibited 80% by specific anti-lipoprotein lipase immunoglobulins. 85% of the activity binds to heparin-Sepharose 4B, and 70% of bound activity is eluted with 1.5 M NaCl. Thus, the lipolytic activity of cultured granulosa cells is lipoprotein lipase. Granulosa cells were shown to synthesize lipoprotein lipase in culture by incorporating [3H]leucine into the enzyme protein, as measured with an immunoadsorption technique. Finally, colchicine was shown to increase intracellular lipolytic activity, suggesting an inhibition of secretion of this enzyme by cultured granulosa cells.     

Cholesteryl ester storage disease and Wolman disease: phenotypic variants of lysosomal acid cholesteryl ester hydrolase deficiency

The lysosomal enzyme responsible for cholesteryl ester hydrolysis, acid cholesteryl ester hydrolase, or acid lipase (E.C.3.1.1.13) plays an important role in cellular cholesterol metabolism. Loss of the activity of this enzyme in tissues of individuals with both Wolman disease and cholesteryl ester storage disease is believed to play a causal role in these conditions. The objectives of our studies were not only to directly compare and contrast the clinical features of Wolman disease and cholesteryl ester storage disease but also to determine the reasons(s) for the varied phenotype expression of acid cholesteryl ester hydrolase deficiency. Although both diseases manifest a type II hyperlipoproteinemic phenotype and hepatomegaly secondary to lipid accumulation, a more malignant clinical course with more significant hepatic and adrenal manifestations was observed in the patient with Wolman disease. However, the acid cholesteryl ester hydrolase activity in cultured fibroblasts in both diseases was virtually absent. In addition, fibroblasts from both Wolman disease and cholesteryl ester storage disease were able to utilize exogenously supplied enzyme, suggesting that neither disease was due to defective enzyme delivery by the mannose-6-phosphate receptor pathway. Coculture and cell fusion of fibroblasts from Wolman disease and cholesteryl ester storage disease subjects did not lead to correction of the enzyme deficiency, indicating that these disorders are allelic. However, the activities of the hepatic acid and neutral lipase in these two clinical variants were quite different. Hepatic acid lipase activity was only 4% normal in Wolman disease, but the activity was 23% normal in cholesteryl ester storage disease. The hepatic neutral lipase activity was normal in Wolman disease but increased more than twofold in cholesteryl ester storage disease. These combined results indicate that the clinical heterogeneity in acid cholesteryl ester hydrolase deficiency can be explained by a varied hepatic metabolic response to an allelic mutation.     

Triacylglycerol lipase activities of cultured rat L6 myoblasts

The utilization of exogenous triacylglycerol by fusing and non-fusing rat L6 myoblasts grown in culture was investigated. Although small quantities of triacylglycerol were accumulated by both cell lines during an incubation of 2 h, no evidence could be found for the presence of lipoprotein lipase, either in the cells or released into the medium. Cell homogenate studies confirmed the absence of lipoprotein lipase but revealed the presence of an acid lipase having a pH optimum at 4.6. Acid lipase activity was mainly associated with a 15 000 g pellet and was capable of hydrolysing triolein at maximum velocity in the millimolar range. Unlike lipoprotein lipase, acid lipase was strongly inhibited by serum and preliminary investigations suggest that the inhibitory component of serum is located amongst the higher density lipoproteins. It is likely that the acid lipase is of lysosomal origin and is responsible for the hydrolysis of internalized triacylglycerol for subsequent utilization by the cell.     

Studies on the hormone-sensitive lipase of adipose tissue

Sucrose gradient centrifugation has been used to examine the triglyceride lipases present in extracts of rat epididymal adipose tissue. The aqueous infranatant recovered between the pellet and fat cake of tissue homogenates which had been centrifuged at 40,000 g was shown to contain two types of triglyceride lipase activity. One of these appears in the 15s region and has been identified as the active form of the "hormone-sensitive lipase" believed to be responsible for initiating the hydrolysis of tissue triglyceride stores in response to lipolytic stimuli. The activity of this enzyme was selectively increased in extracts prepared from tissue exposed to epinephrine and decreased in extracts of insulin-treated tissue. The increased lipolytic activity of extracts of tissue from fasted or fasted-refed rats was also found largely in this region. When the tissue was incubated with orthophosphate-(32)P, radioactivity was incorporated into a protein migrating at 15s. A second peak of triglyceride lipase activity appeared in the 6s region coincident with the location of the monoglyceride and diglyceride lipase activities. The amount of 6s triglyceride lipase activity did not correlate with changes in the lipolytic activity of the tissue from which the extracts were prepared, and its physiological function remains to be elucidated. The lipoprotein lipase and the short-chain triglyceride lipase ("tributyrinase") each moved more slowly in the gradient than the 6s triglyceride lipase. Both the 6s and 15s enzymes were shown to be present in washed adipocytes isolated from the tissue by collagenase digestion.     

Proton-translocating ATPase and lysosomal cystine transport

A proton-translocating ATPase was identified in highly purified lysosomes from Epstein-Barr virus-transformed human lymphoblasts. Activity of this ATPase caused acidification of highly purified, fluorescein isothiocyanate dextran-loaded lysosomes and correlated with the ATP-dependent efflux of lysosomal cystine. The lysosomal ATPase was distinct from mitochondrial F1-ATPase in its responses to a variety of inhibitors. Although ATP-dependent lysosomal cystine efflux is not demonstrable in cultured lymphoblasts from individuals with nephropathic cystinosis, ATPase activity and acidification in lysosomes from these cells is comparable to that in noncystinotic lysosomes. ATPase activity in lymphoblasts from normal individuals was 543 +/- 79 nmol/mg/min while in lymphoblasts from cystinotic individuals this activity was 541 +/- 25 nmol/mg/min. ATP-dependent acidification of lysosomes from normals was -0.5 +/- 0.1 pH units compared to -0.5 +/- 0.1 pH units in cystinotic lysosomes. Activity of the lysosomal proton-translocating ATPase is a necessary, but not sufficient, condition for lysosomal cystine efflux.     

Lipoprotein lipase activity is stimulated by insulin and dexamethasone in cardiomyocytes from diabetic rats.

Type 1 diabetes mellitus reduces lipoprotein lipase (LPL) activity in the heart. The diabetic phenotype of decreased LPL activity in freshly isolated cardiomyocytes persisted after overnight culture (16 h). Total cellular LPL activity was 311+/-56 nmol oleate released x h(-1) x mg(-1) cell protein in diabetic cultured cardiomyocytes compared with 661+/-81 nmol oleate released x h(-1) x mg(-1) cell protein for control cultured cells. Diabetes also resulted in lower heparin-releasable (HR) LPL activity compared with control cells (111+/-25 vs. 432+/-63 nmol x h(-1) x mg(-1) cell protein). In kinetic experiments, the reduction in total cellular LPL and HR-LPL activities in cultured cells from diabetic hearts was due to a decrease in maximal velocity, with no change in apparent Km for substrate (triolein). LPL activity in primary cultures of cardiomyocytes from control rats is stimulated by the combination of insulin (Ins) and dexamethasone (Dex). Overnight treatment of cultured cardiomyocytes from diabetic rats with Ins+Dex elicited an 84% increase in cellular LPL activity (to 572+/-65 nmol x h(-1) x mg(-1) cell protein) and a 194% increase in HR-LPL activity (to 326+/-46 nmol x h(-1) x mg(-1) cell protein). This stimulation occurred at subnanomolar concentrations of the hormones, but neither hormone was effective alone. The amount of immunoreactive LPL protein mass in cultured cardiomyocytes from diabetic hearts was unchanged by Ins+Dex treatment. Addition of oleic acid (60 microM) to the overnight culture medium inhibited the already reduced HR-LPL activity in diabetic cultured cells by 73% (to 30+/-4 nmol x h(-1) x mg(-1) cell protein). The presence of oleic acid also reduced hormone-stimulated HR-LPL activity. Increasing the glucose concentration in the culture medium to 26 mM had no effect on total cellular LPL or HR-LPL activities.     

Diacylglycerol hydrolysis in rat liver lysosomes

The matrix of rat liver lysosomes exhibits high hydrolytic activity towards 1,2-diacylglycerol with an optimum at pH 4.0. The lipolytic reaction follows Michaelis-Menten kinetics (apparent Vmax 470 nmol hydrolysed/min per mg protein; apparent Km 71 microM 1,2-dioleoylglycerol). Formation of 1- and 2-monooleoylglycerols indicates an initial attack at both the primary and secondary ester bonds. The lysosomal matrix also catalyses (re)acylation reactions, i.e. the formation of 1,2-diacylglycerol from 2-monoacylglycerol and free fatty acid. However, (re)acylation proceeds at a far lower rate than deacylation of diacylglycerol. Lysosomal diacylglycerol hydrolysis is sensitive towards non-ionic detergents, cationic amphiphilic drugs and the lipase inhibitor RHC 80267.     

Involvement of glyoxysomal lipase in the hydrolysis of storage triacylglycerols in the cotyledons of soybean seedlings

The total cotyledon extract of soybean (Glycine max [L.] Merr. var. Coker 136) seedlings underwent lipolysis as measured by the release of fatty acids. The highest lipolytic activity occurred at pH 9. This lipolytic activity was absent in the dry seeds and increased after germination concomitant with the decrease in total lipids. Using spherosomes (lipid bodies) isolated from the cotyledons during the peak stage of lipolysis (5-7 days) as substrates, about 40% of the lipase activity was found in the glyoxysomes after organelle breakage had been accounted for; the remaining activity was distributed among other subcellular fractions but none was found in the spherosomal fraction. The glyoxysomal lipase had maximal activity at pH 9, and catalyzed the hydrolysis of tri-, di-, and monoacylglycerols of linoleic acid, the most abundant fatty acid in soybean. The spherosomes contained a neutral lipase that could hydrolyze monolinolein and N-methylindoxylmyristate, but not trilinolein. This spherosomal lipase activity dropped off rapidly during early seedling growth, preceding lipolysis. Spherosomes isolated from either dry or germinated seeds did not possess lipolytic activity, and spherosomes from germinated seeds but not from dry seeds could serve as substrates for the glyoxysomal lipase. It is concluded that the glyoxysomal lipase is the enzyme catalyzing the initial hydrolysis of storage triacylglycerols.     

Adipokinetic hormone-induced mobilization of fat body triglyceride stores in Manduca sexta: role of TG-lipase and lipid droplets

Triglycerides (TG) stores build up in the insect fat body as lipid droplets at times of excess of food. The mobilization of fat body triglyceride (TG) is stimulated by adipokinetic hormones (AKH). The action of AKH involves a rapid activation of cAMP-dependent protein kinase (PKA). Recent in vitro studies have shown that PKA phosphorylates and activates the TG-lipase substrate, the lipid droplets. Conversely, purified TG-lipase from Manduca sexta fat body is phosphorylated by PKA in vitro but is not activated. This study was directed to learn whether or not AKH promotes a change in the state of phosphorylation of the lipase in vivo, and what are the relative contributions of cytosol and lipid droplets to the overall increase of lipolysis triggered by AKH. TG-lipase activity of fat body cytosols isolated from control and AKH-treated insects was determined against the native substrate, in vivo [3H]-TG radiolabeled lipid droplets, obtained from control and AKH-treated insects. The lipase activity of the system composed of AKH-cytosol and AKH-lipid droplets (11.1 +/- 2.1 nmol TG/min-mg) was 3.1-fold higher than that determined with control cytosol and lipid droplets (3.6 +/- 0.5 nmol TG/min-mg). Evaluation of the role of AKH-induced changes in the lipid droplets on lipolysis showed that changes in the lipid droplets are responsible for 70% of the lipolytic response to AKH. The remaining 30% appears to be due to AKH-dependent changes in the cytosol. However, the phosphorylation level of the TG-lipase was unchanged by AKH, indicating that phosphorylation of the TG-lipase plays no role in the activation of lipolysis induced by AKH.     

Heparin decreases the degradation rate of lipoprotein lipase in adipocytes

The mechanism responsible for the stimulation of secretion of lipoprotein lipase by heparin in cultured cells was studied with avian adipocytes in culture. Immunoprecipitation followed by electrophoresis and fluorography were used to isolate and quantitate the radiolabeled enzyme, whereas total lipoprotein lipase was quantitated by radioimmunoassay. Rates of synthesis of lipoprotein lipase were not different for control or heparin treatments as judged by incorporation of L-[35S]methionine counts into lipoprotein lipase during a 20-min pulse. This observation was corroborated in pulse-chase experiments where the calculation of total lipoprotein lipase synthesis, based on the rate of change in enzyme-specific activity during the chase, showed no difference between control (8.13 +/- 3.1) and heparin treatments (9.1 +/- 5.3 ng/h/60-mm dish). Secretion rates of enzyme were calculated from measurements of the radioactivity of the secreted enzyme and the cellular enzyme-specific activity. Degradation rates were calculated by difference between synthesis and secretion rates of enzyme. In control cells 76% of the synthesized enzyme was degraded. Addition of heparin to the culture medium reduced the degradation rate to 21% of the synthetic rate. The presence of heparin in cell media resulted in a decrease in apparent intracellular retention half-time for secreted enzyme from 160 +/- 44 min to 25 +/- 1 min. The above data demonstrate that the increase in lipoprotein lipase protein secretion, observed upon addition of heparin to cultured adipocytes, is due to a decreased degradation rate with no change in synthetic rate. Finally, newly synthesized lipoprotein lipase in cultured adipocytes is secreted constitutively and there is no evidence that it is stored in an intracellular pool.     

Isolation of a low-temperature adapted lipolytic enzyme from uncultivated micro-organism

Aims: The aim of the study was to isolate a novel lipolytic enzyme from the activated sludge of uncultured micro‐organisms. Methods and Results: The metagenomic DNA was directly extracted from the activated sludge, and a metagenomic library was constructed by using the pUC vector. The library was screened for lipolytic enzyme activity on 1% tributyrin agar plate. A clone among c. 100 000 recombinant libraries showed the lipolytic activity. The putative lipolytic gene encoding lipo1 from the metagenomic library was subcloned and expressed in Escherichia coli BL21 using the pET expression system. The expressed recombinant enzyme was purified by Ni‐nitrilotriacetic acid affinity chromatography and characterized using general substrates of lipolytic property. The gene consisted of 972 bp encoding a polypeptide of 324 amino acids with a molecular mass of 35·6 kDa. Typical residues essential for lipolytic activity such as penta‐peptide (GXSXG) and catalytic triad sequences (Ser166, Asp221 and His258) were detected. The deduced amino acid sequence of lipo1 showed low identity with amino acid sequences of esterase/lipase (32%, ZP_01528487 ) from Pseudomonas mendocina ymp and esterase (31%, AAY45707 ) from uncultured bacterium. This lipolytic enzyme exhibited the highest activity at pH 7·5 and 10°C. At thermal stability analysis, lipo1 was more unstable at 40°C than 10°C. Conclusions: An activity based strategy has been an effective method for fishing out a low‐temperature adapted lipolytic enzyme from the metagenomic library. This lipo1 enzyme can be considered to belong to the hormone‐sensitive lipase family due to the enzyme’s oxyanion hole by the sequence HGGG. Significance and Impact of the Study: Lipo1 is a novel psychrophilic esterase obtained directly from the metagenomic library. Owing its support of significant activity at low temperature, this enzyme is expected to be useful for potential application as a biocatalyst in organic chemistry.     

Acid lipase in rat intestinal mucosa: physiological parameters

Previous studies have shown that up to a half of infused triacylglycerol does not exit the intestine via lymphatics. This suggests the presence of a mucosal lipase which could provide fatty acids for potential transport via the portal vein. The present study describes an acid-active lipase in rat intestinal mucosa. Acid lipase was assayed using a glyceryl tri[14C]oleate emulsion (pH 5.8). Mucosal homogenates were differentially centrifuged to yield cellular organelles and cytosol. Cells were sequentially released from villi using citrate and EDTA. The enzyme was found to be most active in the proximal quarter intestine and in the upper third of villi. Its greatest activity was in the lysosomal fraction. Esophageal diversion demonstrated that lingual lipase was not the precursor of the mucosal acid lipase. Bile salts stimulated activity 3- to 5-fold, but other neutral or anionic detergents were inhibitory. Of the detergents tested, taurocholate at super critical micellar concentrations could restore activity only with SDS. Sepharose 6B chromatography suggested that the enzyme partitioned into an SDS and taurocholate mixed micelle. We conclude that mucosal acid lipase is a distinct, intrinsic enzyme of the intestinal mucosa. It is predominantly lysosomal in origin. The location of its greatest activity in the villus tips of the proximal intestine suggests that it is potentially involved in mucosal triacylglycerol disposal.     

Lipolytic stimulation modulates the subcellular distribution of hormone-sensitive lipase in 3T3-L1 cells

3T3-L1 cells have been a useful model system for studying adipocyte differentiation and metabolism. They acquire a hormone-sensitive lipase during differentiation (Kawamura, M., et al. 1981. Proc. Natl. Acad. Sci. USA. 78: 732-735). In the present study the control of lipolysis in these cells was investigated. Basal glycerol release from cell monolayers was 437 nmol/mg protein per hr, and could be stimulated approximately 6-fold by exposure to 1 microM isoproterenol. Subcellular fractionation of stimulated cells revealed a redistribution of triglyceride lipase activity: loss from the infranatant fraction and increase in the pellet fraction. The redistribution was dosage-dependent and reversible. Treatment of intact cells with 8-bromoadenosine 3':5' cyclic monophosphate elicited similar redistribution of the lipase activity; however, disruption and incubation of untreated cells in the presence of ATP and either cyclic AMP or the catalytic subunit from cAMP-dependent protein kinase did not. The lipase activity in the pellet fraction was increased 3- to 4-fold after maximal lipolytic stimulation of intact cells, whereas phosphorylation of the enzyme in vitro yielded 1.4- to 1.6-fold stimulation in all subcellular fractions from untreated cells. The lipase found in the particulate fraction has the same properties as the previously characterized infranatant enzyme. It is suggested that interaction of the lipase with substrate and associated intracellular membranes may be a novel feature of the regulation of lipolysis.