Soluble Subcellular Fractions Research Articles

Plasmodium falciparum Rab5B is an N-terminally myristoylated Rab GTPase that is targeted to the parasite's plasma and food vacuole membranes.

Plasmodium falciparum (Pf) has a family of 11 Rab GTPases to regulate its vesicular transport. However, PfRab5B is unique in lacking a C-terminal geranyl-geranylation motif, while having N-terminal palmitoylation and myristoylation motifs. We show that the N-terminal glycine is required for PfRab5B myristoylation in vitro and when an N-terminal PfRab5B fragment possessing both acylation motifs is fused to GFP and expressed in transgenic P. falciparum parasites, the chimeric PfRab5B protein localizes to the plasma membrane. Upon substitution of the modified glycine by alanine the staining becomes diffuse and GFP is found in soluble subcellular fractions. Immuno-electron microscopy shows endogenous PfRab5B decorating the parasite's plasma and food vacuole membranes. Using reverse genetics rab5b couldn't be deleted from the haploid genome of asexual blood stage P. berghei parasites. The failure of PbRab5A or PbRab5C to complement for loss of PbRab5B function indicates non-overlapping roles for the three Plasmodium Rab5s, with PfRab5B involved in trafficking MSP1 to the food vacuole membrane and CK1 to the plasma membrane. We discuss similarities between Plasmodium Rab5B and Arabidopsis thaliana ARA6, a similarly unusual Rab5-like GTPase of plants.

Entamoeba histolytica: Soluble and membrane-associated neutral sphingomyelinase-C and other unidentified esterase activity

Sphingomyelinase (SMase) activity was measured in Entamoeba histolytica particulate and soluble subcellular fractions. The effects on SMase of incubation time, total protein concentration, pH, and several divalent cations were determined. SMase-C and other unidentified esterase activity were detected in soluble and particulate fractions. SMase-C was 94.5–96.0% higher than the unidentified esterase activity. Soluble and insoluble SMase-C specific activities increased with protein dose and incubation time. Soluble and insoluble SMase-C activities were maximum at pH 7.5 and were dependent on Mg 2+, Mn 2+, or Co 2+, and inhibited by Zn 2+, Hg 2+, Ca 2+, and EDTA. SMase-C was active in the pH range of 3–10 and its maximum activity was at pH 7.5. The soluble and insoluble SMases have remarkably similar physicochemical properties, strongly suggesting that E. histolytica has just one isoform of neutral SMase-C that had not been described before and might be essential for E. histolytica metabolism or virulence.

ACTIVITY OF INTRACELLULAR PHOSPHOLIPASE A1AND A2IN GIARDIA LAMBLIA

Neither phospholipase A1 (PLA A1) nor phospholipase A2 (PLA A2), nor their respective genes, have been identified in Giardia lamblia, even though they are essential for lipid metabolism in this parasite. A method to identify, isolate, and characterize these enzymes is needed. The activities of PLA A1 and PLA A2 were analyzed in a total extract (TE) and in vesicular (P30) and soluble (S30) subcellular fractions of G. lamblia trophozoites; the effects of several chemical and physicochemical factors on their activities were investigated. The assays were performed using substrate labeled with 14C, and the mass of the 14C-product was quantified. PLA A1 and PLA A2 activity was present in the TE and the P30 and S30 fractions, and it was dependent on pH and the concentrations of protein and Ca2+. In all trophozoite preparations, PLA A1 and PLA A2 activities were inhibited by ethylenediaminetetraacetic acid and Rosenthal's inhibitor. These results suggest that G. lamblia possesses several PLA A1 and PLA A2 isoforms that may be soluble or associated with membranes. In addition to participating in G. lamblia phospholipid metabolism, PLA A1 and PLA A2 could play important roles in the cytopathogenicity of this parasite.

Trichomonas vaginalis: identification of a phospholipase A-dependent hemolytic activity in a vesicular subcellular fraction.

Trichomonad total extracts (TTE), or vesicular (P30) and soluble (530) subcellular fractions from 3 pathogenic Trichomonas vaginalis strains (GT-3. GT-13. and GT-15), lysed both human and Sprague-Dawley rat erythrocytes in a time- and dose-dependent manner. The entire hemolytic activity of TTE was located in P30, showing 2 peaks of maximum activity, one at pH 6.0 and another at pH 8.0. in the presence of 1 mM Ca2+. Hemolytic activity on rat erythrocytes was greater at pH 6.0 16.71 +/- 0.33 hemolytic units IHU]/mg/hr to 11.60 +/- 0.24 HU/mg/hr) than at pH 8.0 (3.81 +/- 0.30 HU/mg/hr to 5.75 +/- 0.65 HU/mg/hr). and it was greater than that on human red blood cells at pH 6.0 (2.67 +/- 0.19 HU/mg/hr to 4.08 +/- 0.15 HU/mg/hr) or pH 8.0 (2.24 +/- 0.0 9 HU/mg/hr to 2.81 +/- 0.06 HU/mg/hr). The alkaline and acidic hemolytic activity diminished (60-93% at pH 6.0 and 78-93% at pH 8.0) by the effect of 80 microM Rosenthal's inhibitor, which also inhibited 27-45% and 29-54% trichomonad alkaline and acidic phospholipase A activities, respectively. Vesicles, vacuoles, and hydrogenosomes were rich in P30. Trichomonas vaginalis has a hemolytic PLA, which could be involved in its cytopathogenic mechanism.

Relative Increase in Alzheimer's Disease of Soluble Forms of Cerebral Aβ Amyloid Protein Precursor Containing the Kunitz Protease Inhibitory Domain

Although a number of studies have examined amyloid precursor protein (APP) mRNA levels in Alzheimer's disease (AD), no clear consensus has emerged as to whether the levels of transcripts for isoforms containing a Kunitz protease inhibitory (KPI)-encoded region are increased or decreased in AD. Here we compare AD and control brain for the relative amounts of APP protein containing KPI to APP protein lacking this domain. APP protein was purified from the soluble subcellular fraction and Triton X-100 membrane pellet extract of one hemisphere of AD (n = 10), normal (n = 7), and neurological control (n = 5) brains. The amount of KPI-containing APP in the purified protein samples was determined using two independent assay methods. The first assay exploited the inhibitory action of KPI-containing APP on trypsin. The second assay employed reflectance analysis of Western blots. The proportion of KPI-containing forms of APP in the soluble subcellular fraction of AD brains is significantly elevated (p < 0.01) compared with controls. Species containing a KPI domain comprise 32-41 and 76-77% of purified soluble APP from control and AD brains, respectively. For purified membrane-associated APP, 72-77 and 65-82% of control and AD samples, respectively, contain a KPI domain. Since KPI-containing species of APP may be more amyloidogenic (Ho, L., Fukuchi, K., and Yonkin, S. G. (1996) J. Biol. Chem. 271, 30929-30934), our findings support an imbalance of isoforms as one possible mechanism for amyloid deposition in sporadic AD.

Partial purification of a diacylglycerol lipase from bovine aorta.

A diacylglycerol (DG) lipase has been purified from a soluble subcellular fraction of bovine aorta by (NH4)2SO4 precipitation in the presence of 5.0% (w/v) Triton X-100, followed by chromatography on DEAE-Sephacel, heparin-Sepharose and octyl-Sepharose in the presence of either CHAPS or Triton X-100 detergents. Under basal conditions, the hydrolysis of a short-chain [3H]dioctanoylglycerol ([3H]diC8) substrate was much greater than that of a long-chain 1-[1-14C]palmitoyl-2-oleoyl-sn-glycerol (1-[14C]POG) substrate. Lipase activity measured with 1-[14C]POG was markedly enhanced by Triton X-100. In the presence of 0.1% Triton X-100, specific enzyme activities in the octyl-Sepharose fraction determined with 1-[14C]POG or 1-stearoyl-2-[1-14C]-arachidonoyl-sn-glycerol as substrates were the same as that measured with [3H]diC8. MgCl2 (5mM) or CaCl2 (2 mM) also selectively stimulated lipase activity (up to 10-13-fold) measured with the long-chain (1-[14C]POG) substrate only. The increase in relative specific activity in the octyl-Sepharose fraction was 60-fold and 155-fold, based on hydrolysis of [3H]diC8 and 1-[14C]POG (+ Triton X-100), respectively. Unlabelled diC8 was a competitive inhibitor of 1-[14C]POG hydrolysis, suggesting that a single lipase hydrolyses both the short-chain and long-chain DG substrates; selective stimulatory effects of non-ionic detergents and bivalent cations on the hydrolysis of 1-[14C]POG may be due to effects on the physical properties of the substrate preparation. Monoacylglycerol lipase, DG kinase and cholesterol esterase activities could not be detected in the partially purified lipase preparation.

Melatonin interactions with cultured murine B16 melanoma cells.

Both in vitro and in vivo observations have suggested that melatonin modulates malignant cell growth. The present studies aimed to characterize the interactions of melatonin with cultured murine B16 melanoma cells. Time- and temperature-dependent specific melatonin accumulation by B16 murine melanoma cells was observed. B16 cells possessed a high affinity binding site (KD = 1.4 nM) which exhibited structural specificity in its affinity for analogues of melatonin (melatonin > 6-hydroxymelatonin = N-acetyl-5-hydroxytryptamine > 5-methoxytryptamine >> 5-hydroxytryptamine). Evidence for a lower affinity uptake system without structural specificity was also observed. Ninety-five per cent of the specific cell-associated melatonin in B16 cells was present in the soluble subcellular fraction of lysed cells; more than 97% of the cell-associated radioactivity was authentic melatonin. When the solubilized cell extracts from the binding assay were analysed by gel filtration immediately, all of the bound counts eluted at the void volume. Continuous exposure to melatonin for 48-120 h did not affect B16 cell proliferation as determined by cell counts, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay or [3H]thymidine incorporation. After 8-h pulse exposures to melatonin daily for 3 days, a 15% stimulation of B16 cell proliferation (p < 0.02) was observed at melatonin concentrations of 0.1 and 1 nM. The anti-oestrogen, tamoxifen, inhibited B16 cell growth and increased specific melatonin accumulation by B16 cells at 1 x 10(-6) M (p < 0.02). Cultured B16 murine melanoma cells possessed a specific, high affinity uptake system for melatonin which appeared to be altered by anti-oestrogen exposure.

Distribution of calcium/calmodulin-dependent protein kinase II in rat ileal enterocytes.

Ca2+/calmodulin (CaM)-dependent protein kinase II is a major effector of the Ca2+ signaling pathway. It has a wide tissue distribution and phosphorylates multiple substrates. Villus enterocytes from rat ileum contain a Ca2+/CaM-dependent kinase activity that phosphorylates the exogenous neural substrate synapsin I. This phosphorylation is blocked by a specific peptide inhibitor. Antibodies made to rat brain Ca2+/CaM-dependent protein kinase II label a single band with a relative molecular mass of approximately 50 kDa in isolated rat enterocytes by immunoblot. Almost one-half of this immunoreactive protein is preferentially found in a particulate compared with a soluble subcellular fraction of the enterocytes. Virtually all of the 50-kDa band in the particulate fraction is insoluble in nonionic detergent, suggesting that the kinase is associated with the enterocyte cytoskeleton. Antibodies to Ca2+/CaM-dependent protein kinase II immunocytochemically detect fibrillar structures concentrated in the terminal web region of intestinal epithelial cells that colocalized with myosin II. This enzyme may have a role in regulating the intestinal epithelial cytoskeleton.

Inhibition of diacylglycerol metabolism in isolated cardiac myocytes by U-57 908 (RHC 80267), a diacylglycerol lipase inhibitor

U-57 908 (RHC 80267) inhibited diacylglycerol (DG) lipase activity in soluble and microsomal subcellular fractions from cardiac myocytes isolated from adult rat hearts; half-maximal inhibition was observed at a concentration of 3.5 microM. Monoacylglycerol lipase activity was much less sensitive to inhibition, but U-57 908 reduced lipoprotein lipase activity in cardiac myocytes with the same sensitivity as observed for DG lipase. DG kinase activity was not inhibited by U-57 908. DG metabolism by intact cardiac myocytes was studied in incubations with a cell-permeable DG analog, [3H]-dioctanoylglycerol (diC8). DiC8 was mainly metabolized by conversion to mono-octanoylglycerol (monoC8) and glycerol (lipase pathway); much less radioactivity was incorporated into the triacylglycerol and total phospholipid fractions. U-57 908 reduced the loss of radioactivity from the exogenous diC8 substrate, with a corresponding decline in the formation of radiolabelled monoC8 and glycerol. The incorporation of radioactivity into phospholipids was slightly reduced, but triacylglycerol synthesis from diC8 was increased in the presence of U-57 908. Therefore, U-57 908 is an effective inhibitor of DG metabolism by the lipase pathway in intact cardiac myocytes.

Altered protein kinase C activity in different subfields of hippocampus following cerebral ischemia

Protein kinase C (PKC) activity was determined in different (membrane, nuclear and soluble) subcellular fractions prepared separately from the CA1 and CA3 subfields of Mongolian gerbils hippocampus at various time intervals following a single 5-min occlusion of the common carotid arteries. Soluble and nuclear PKC activities of the CA1 sector were found to be elevated at 24 hours following the ischemic injury, while PKC activities did not increase in the CA3 region until the 3rd day after ischemia. The ratio of soluble/membrane-associated PKC activities followed a similar pattern, predominantly because the activation/elevation and then down regulation of the cytosolic enzyme pool changing correspondingly to the ongoing pathological processes. PKC activity returned to the normal level in each subfraction of the CA3 subfield by the 7th day. However, PKC activity remained elevated in the soluble fraction of the CA1 sector even after the delayed death of pyramidal neurons, presumably because of the reactive response of astrocytes. Conceivably, the transient activation and rapid down regulation of PKC in the CA1 sector may contribute to the initiation of postischemic neuronal death in the CA1 subfield.

Expression of a functionally active cardiac fatty acid-binding protein in the yeast, Saccharomyces cerevisiae

The unicellular eukaryotic microorganism, Saccharomyces cerevisiae, transformed with a plasmid containing a cDNA fragment encoding bovine heart fatty acid-binding protein (H-FABP) under the control of the inducible yeast GAL10 promoter, expressed FABP during growth on galactose. The maximum level of immunoreactive FABP, identical in size to native protein as judged from SDS-polyacrylamide gel electrophoresis, was reached after approximately 16 hours of induction. Analysis of particulate and soluble subcellular fractions showed that FABP was exclusively associated with the cytosol. FABP expressed in yeast cells was functional as was demonstrated by its capacity to bind 14C-oleic acid in an in vitro assay. Growth of the transformants on galactose as the carbon source was significantly retarded at 37 degrees C. Whereas the fatty acid pattern of total lipids was not altered in transformed cells, desaturation of exogenously added 14C-palmitic acid was significantly reduced both at 30 and 37 degrees C. The lowest percentage of radioactively labeled unsaturated fatty acids was found in the phospholipid fraction.

Metabolism of dioctanoylglycerol by isolated cardiac myocytes

Diacylglycerol (DG) metabolism by intact cardiac myocytes isolated from adult rat hearts and by broken cell preparations from myocytes was investigated in experiments with [ 3H]dioctanoyglycerol (diC8), a cell-permeable diacylglycerol analog. In a low-speed super-natant fraction, the K m and V max for DG kinase activity (formation of phosphatidic acid) was 22 μ m and 110 nmol/h/mg, respectively, whereas the K m and V max for DG lipase activity (formation of monoacylglycerol) was 80 μ m and 1000 nmol/h/mg. At a substrate concentration of 80 μ m diC8, lipase activity was 7-fold greater than kinase activity. The majority of DG kinase activity was recovered in the soluble subcellular fraction; DG lipase activity was localized in a microsomal fraction. When [ 3H]diC8 was incubated with intact cardiac myocytes, 10-fold more radioactivity was incorporated into the products of the lipase pathway (monoacylglycerol and free glycerol) as compared to incorporation into the total phospholipid fraction which contained phosphatidic acid. This predominance of metabolism by hydrolysis through the lipase pathway was observed consistently when the incubation time, content of cardiac myocytes and concentration of exogenous diC8 was varied. Therefore, results from both in vitro determinations of enzyme activities in broken cell preparations and flux studies with intact cells have indicated that the lipase pathway is the principal enzymatic mechanism for the metabolism of diC8 in cardiac myocytes.

Transglutaminase-catalyzed incorporation of [2,5-3H]histamine into a Mr 84000 particulate protein in pancreatic islets

Rat pancreatic islet homogenates catalyze the incorporation of [2,5-3H]histamine into endogenous proteins recovered in both the stacking gel and a Mr 84000 protein separated by polyacrylamide electrophoresis. The labelling of these proteins represents a Ca2+-dependent process inhibited by glycine methylester, but not sarcosine methylester, and enhanced after preincubation of the islets at a high concentration of D-glucose. Although transglutaminase activity is found in both soluble and particulate subcellular fractions, the endogenous transglutaminase substrates were located mainly in particulate, possibly membrane-associated, material.

Monoacylglycerol lipase activity in cardiac myocytes.

Monoacylglycerol lipase activity in homogenates of isolated myocardial cells (myocytes) from rat hearts was recovered in both particulate and soluble subcellular fractions. The activity present in the microsomal (100,000 X g pellet) fraction was solubilized by treatment with Triton X-100 and combined with the 100,000 X g supernatant fraction; the properties of monoacylglycerol lipase were investigated with this soluble enzyme preparation. The Km for the hydrolysis of a 2-monoolein substrate was 16 microM. The rates of hydrolysis of 1-monoolein and 2-monoolein were identical, and 1-monoolein was a competitive inhibitor (Ki = 20 microM) of the hydrolysis of 2-monoolein. Monoacylglycerol lipase activity was regulated by product inhibition according to the following order of potency: fatty acyl CoA greater than free fatty acids greater than fatty acyl carnitine.

Involvement of a low-molecular-weight substance in in vitro activation of the molybdoenzyme respiratory nitrate reductase from a chlB mutant of Escherichia coli.

The soluble subcellular fraction of a chlB mutant contains an inactive precursor form of the molybdoenzyme nitrate reductase, which can be activated by the addition to the soluble fraction of protein FA, which is thought to be the active product of the chlB locus. Dialysis or desalting of the chlB soluble fraction leads to the loss of nitrate reductase activation, indicating that some low-molecular-weight material is required for the activation. The protein FA-dependent activation of nitrate reductase can be restored to the desalted chlB soluble fraction by the addition of a clarified extract obtained after heating the chlB soluble fraction at 100 degrees C for 8 min. The heat-stable substance present in this preparation has a molecular weight of approximately 1,000. This substance is distinct from the active molybdenum cofactor since its activity is unimpaired in heat-treated extracts prepared from the organism grown in the presence of tungstate, which leads to loss of cofactor activity. Mutations at the chlA or chlE locus, which are required for molybdenum cofactor biosynthesis, similarly do not affect the activity of the heat-treated extract in the in vitro activation process. Moreover, the active material can be separated from the molybdenum cofactor activity by gel filtration. None of the other known pleiotropic chlorate resistance loci (chlD, chlG) are required for the expression of its activity. Magnesium ATP appears to have a role in the formation of the active substance. We conclude that a low-molecular-weight substance, distinct from the active molybdenum cofactor, is required to bestow activity on the molybdoenzyme nitrate reductase during its biosynthesis.

Purification and characterization of estrogen-2/4-hydroxylase activity from rabbit hypothalami: peroxidase-mediated catechol estrogen formation.

Estrogen-2/4-hydroxylase (E-2/4-H) activity of rabbit hypothalamic tissue was previously found to be localized in the soluble subcellular fraction. In the present study, the enzymatic activity responsible for catechol estrogen formation in this subcellular fraction of the rabbit hypothalamus was purified by ammonium sulfate fractionation, ion exchange chromatography, and chromatofocusing. E-2/4-H activity was found to be associated with a group of hemoproteins with peroxidase activity. The characteristics of this hypothalamic E-2/4-H activity were reestablished in light of a peroxidatic mechanism for catechol estrogen formation. Organic hydroperoxides stimulated E-2/4-H activity, presumably by serving as oxidizing cosubstrate required for peroxidase-mediated reactions. E-2/4-H activity in a 17,500 X g supernatant of hypothalamic tissue was linear with time for at least 10 min and with protein concentration to at least 100 micrograms/150 microliter. It displayed a pH optimum of 6 and an apparent Michaelis-Menten constant (Km) of 32 microM with respect to estradiol. The amounts of 4-hydroxyestradiol formed were comparable to those of 2-hydroxyestradiol. The characteristics established in this study for the peroxidase-type E-2/4-H were distinct from those of the particulate, NADPH-dependent 2-hydroxylases found in rat liver and in porcine blastocyst and ovary. These differences provide a basis for differentiating between the two types of enzymatic activity that can lead to catechol estrogen formation in vitro.

Activation in vitro of respiratory nitrate reductase of Escherichia coli K12 grown in the presence of tungstate. Involvement of molybdenum cofactor.

The chlorate-resistant (chlR) mutants are pleiotropically defective in molybdoenzyme activity. The inactive derivative of the molybdoenzyme, respiratory nitrate reductase, present in the cell-free extract of a chlB mutant, can be activated by the addition of protein FA, the probable active product of the chlB locus. Protein FA addition, however, cannot bring about the activation if 10 mM sodium tungstate is included in the culture medium for the chlB strain. The inclusion of a heat-treated preparation of a wild-type or chlB strain prepared after growth in the absence of tungstate, restores the protein-FA-dependent activation of nitrate reductase. All attempts to activate nitrate reductase in extracts prepared from tungstate-grown wild-type Escherichia coli strains failed. It appears that during growth with tungstate, the possession of the active chlB gene product leads to the synthesis of a nitrate reductase derivative which is distinct from that present in the tungstate-grown chlB mutant. Heat-treated preparations from chlA and chlE mutants which do not possess molybdenum cofactor activity fail to restore the activation. Fractionation by gel filtration of the heat-treated preparation from a wild-type strain produced two active peaks in the eluate of approximate Mr 12000 and less than or equal to 1500. The active material in the heat-treated extract was resistant to exposure to proteinases, but after such treatment the active component, previously of approximate Mr 12000, eluted from the gel filtration column with the material of Mr less than or equal to 1500. The active material is therefore of low molecular mass and can exist either in a protein-bound form or in an apparently free state. Molybdenum cofactor activity, assayed by the complementation of the apoprotein of NADPH:nitrate oxidoreductase in an extract of the nit-1 mutant of Neurospora crassa, gave a profile following gel filtration similar to that of the ability to restore respiratory nitrate reductase activity to the tungstate-grown chlB mutant soluble fraction. This was the case even after proteinase treatment of the heat-stable fraction. Analysis of the chlC (narC) mutant, defective in the structural gene for nitrate reductase, revealed that heat treatment is not necessary for the expression of the active component. Furthermore both the active component and molybdenum cofactor activity are present in corresponding bound and free fractions in the non-heat-treated soluble subcellular fraction.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of cyclic-nucleotide phosphodiesterase activities in resting and [formula omitted] human neutrophils

Cyclic nucleotide phosphodiesterase activities in human neutrophils were characterized. Neutrophil sonicates exhibited high-affinity and low-affinity cAMP phosphodiesterase activities, with apparent K m values of 1.9 μM and 112 μM, respectively. No cGMP phosphodiesterase activity was detected. Approx. 70% of cAMP phosphodiesterase activity measured at 1 μM cAMP was present in the soluble subcellular fraction, and the remainder was localized in the particulate fraction. Chromatography of the soluble subcellular fraction on DE-52 ion-exchange resin yielded a low-affinity cAMP phosphodiesterase activity and a high-affinity cAMP phosphodiesterase activity. The soluble high-affinity cAMP phosphodiesterase activity exhibited moderate calmodulin sensitivity. After incubation of intact neutrophils with N- formylmethionylleucylphenylalanine (fMet-Leu-Phe), a 25–30% increase in the activity of the high-affinity cAMP phosphodiesterase activity was observed in the sonicate and in the soluble fraction. Maximal increases were achieved after 2 min of incubation and the increases persisted for at least 10 min. The increase in activity was independent of calmodulin and guanine nucleotide regulatory proteins. These results indicate that a soluble high-affinity cAMP phosphodiesterase comprises the majority of phosphodiesterase activity in neutrophils and that increases in this activity may contribute to the regulation of cAMP levels in neutrophils during activation.

Stimulation of a neutral triacylglycerol hydrolase from rat heart by phosphatidylethanolamine and lysophosphatidylethanolamine.

Triacylglycerol hydrolase activity measured at pH 7.5 in a pH 5.2 precipitate fraction from rat heart was increased two- to three-fold by the presence of phosphatidylethanolamine (PE) or lysophosphatidylethanolamine (LPE). This stimulatory effect also could be obtained in assays with particulate and soluble subcellular fractions and was observed with two different methods of preparing triolein substrate emulsions. Ethanolamine and glycerophosphorylethanolamine had no effect on hydrolase activity, whereas phosphatidylcholine (PC) and acidic phospholipids such as cardiolipin were inhibitory. Palmitic acid, palmityl CoA and palmityl carnitine inhibited PE-stimulated hydrolase activity, but ethyl esters of palmitate had no effect. The preparation of acetone-ether powders resulted in a marked reduction of triacylglycerol hydrolase activity, but PE and LPE now stimulated hydrolase activity by ten-fold or greater, suggesting that these phospholipids may have an obligatory role in modulating triacylglycerol hydrolase activity. Triton X-100 also stimulated hydrolase activity in acetone-ether powders.

Effect of chloroquine on rates of lipolysis in the isolated perfused rat heart and in rat epididymal fat pads

Triglyceride lipase (hydrolase) activity was determined in particulate (17 000 g and 100 000 g pellets) and soluble (100 000 g supernatant) subcellular fractions from rat epididymal fat pad homogenates. Particulate fractions exhibited both an acid (pH 4.5 to 5) and an alkaline (pH 8) pH optimum; in contrast, the soluble fraction was characterized as having a single pH optimum of 7.5. The addition of chloroquine to the triglyceride lipase assay resulted in an inhibition of acid lipase activity (half maximal inhibition at 20 to 25 m m chloroquine) whereas lipase activity measured at pH 7.5 was first stimulated by low concentrations of chloroquine (2 to 10 m m) and then inhibited with higher concentrations. The addition of chloroquine (10 μ m or 50 μ m) to Krebs-Henseleit bicarbonate buffer resulted in negative inotropic and chronotropic effects in isolated perfused rat hearts. Perfusion of rat hearts or preincubation of epididymal fat pads with Krebs-Henseleit bicarbonate buffer containing 10 μ m or 50 μ m chloroquine for 30 min did not decrease basal or epinephrine-stimulated rates of lipolysis as determined by measurements of glycerol release. Similarly, pretreatment of rats with chloroquine 2 h prior to killing had no effect on basal or epinephrine-stimulated rates of lipolysis in perfused hearts or in epididymal fat pads. Since chloroquine is a lysosomotropic agent, it is concluded that the lysosomal acid triglyceride lipase is unlikely to be involved in determining basal or hormone-stimulated rates of lipolysis.