Perturbations of sarcolemmal and microsomal enzymes by amphiphilic lipids and drugs.

Sarcolemmal (SL) and microsomal (MC) membranes were prepared from adult canine cardiocytes. SL Na+, K+-ATPase (2.35 mumole/min per mg) was enriched 117-fold over the homogenate and MC rotenone-insensitive NADH cytochrome c reductase (RINCR) was enriched 41-fold. Preincubation of SL with 50 microM arachidonyl-CoA (20:4 CoA) stimulated Na+, K+-ATPase almost 2-fold; 250 microM 20:4 CoA inhibited the enzyme by 85%. However, RINCR was inhibited 80% by only 0.2 microM 20:4 CoA. Thus, each of these myocardial lipid-dependent enzymes showed a different sensitivity to perturbation by lipid amphiphiles. In further experiments, SL preincubated with 50 microM 20:4 CoA + 2.5 mM propranolol (which had no effect alone) exhibited a synergistic inhibition of the Na+, K+-ATPase: The enzymatic activity declined 8.5-fold when compared to sarcolemma treated with 50 microM 20:4 CoA alone. Thus, the presence of lipid amphiphiles may result in greater inhibition of the Na+, K+-ATPase when propranolol is present in the membrane.

Inhibition of myocardial rotenone-insensitive NADH cytochrome c reductase by amphiphilic compounds.

Because myocardial ischemia is correlated with both an elevation of intracellular levels of amphiphilic lipid metabolites and a decrease in the rotenone-insensitive NADH cytochrome c reductase (RINCR), we investigated the effects in vitro of some amphiphilic lipid metabolites and synthetic detergents on the activity of RINCR-enriched subfractions of microsomes from isolated cardiac myocytes. RINCR activity was unaffected in vitro by the addition of lysophosphatidylethanolamine (up to 0.5 mM) but was inhibited (maximum 63%) by lysophosphatidylcholine (8 microM). Palmitoyl carnitine (up to 2 mM) was ineffective, but the coenzyme A thioesters of palmitate, stearate, oleate, and arachidonate were inhibitory at concentrations (less than 3 microM) below their critical micellar concentrations. Arachidonyl CoA was approximately one order of magnitude more inhibitory than the other long-chain acyl CoA thioesters. Kinetic analyses revealed the effect of arachidonyl CoA on RINCR activity to be exclusively an alteration of the Vmax with no change in the Km for cytochrome c. The inhibition of myocytic RINCR activity by long-chain acyl CoA may be unrelated to the bulk-phase detergency of this lipid amphiphile since the effects were observed at concentrations below the critical micellar concentration, and other lipid amphiphiles had no effect on RINCR activity. Inhibition of microsomal RINCR activity may result from localized disruption of the membrane microenvironment of the enzyme complex by penetration or dissolution of long-chain acyl CoA into the membrane. The pronounced sensitivity of myocytic RINCR activity to long-chain acyl CoA suggests a relationship between the decreased RINCR activity and the increased levels of this class of lipid metabolites observed in the ischemic myocardium.

Characteristics of multiple forms of the acidic triacylglycerol lipase(s) of canine cardiac myocytes.

Acidic lipase activity was extracted by digitonin treatment from particulate fractions prepared from isolated adult canine myocytes. Both methylumbelliferyloleate (MUO) and trioleoylglycerol were hydrolyzed with an apparent Km of 13 and 135 microM, respectively. The primary products of trioleoylglycerol lipolysis were oleic acid and 1,2-dioleoylglycerol. Hydrolysis of either MUO or triacylglycerol was stimulated in vitro by the addition of cardiolipin or Triton X-100. Triton X-100 alone was sufficient for maximal stimulation of MUO hydrolysis, but cardiolipin further stimulated triacylglycerol lipolysis in the presence of an optimal concentration of Triton X-100. Cardiolipin increased the Vmax without altering the Km for trioleoylglycerol. Upon gel filtration chromatography the 4-methylumbelliferyloleate and triacylglycerol lipase activities eluted in regions consistent with molecular weights of approx. 47 000 and 55 000, respectively. Chromatofocusing revealed predominantly one form of acidic 4-methylumbelliferyloleate hydrolase (pI approx. 6.3), whereas acidic triacylglycerol lipase activity eluted continuously in the pH gradient from 7.2 to 4.3 with no clearly predominant peak of activity. Two forms of both 4-methylumbelliferyloleate and triacylglycerol lipase were eluted from columns of carboxymethyl Bio-Gel at pH 5.7; one form of each lipase activity was not bound and another form of each lipase was eluted with 50-60 mM KCl. The non-bound forms of each lipase were indistinguishable from their respective carboxymethyl-bound forms on the basis of pH dependency or kinetically (similar Km). The non-bound and carboxymethyl-bound peaks of lipolytic activity differed in the ratios of 4-methylumbelliferyloleate hydrolase to triacylglycerol lipase activity. The results suggest that the cardiac myocyte contains multiple forms of acidic lipase, and that the catalytic units primarily responsible for the hydrolysis of methylumbelliferyl esters and triacylglycerols may not be identical.

Hepatic triacylglycerol lipase activities after induction of diabetes and administration of insulin or glucagon.

Triacylglycerol lipase activities of homogenates and subcellular fractions of rat liver were measured under optimal conditions at pH 7.5 using emulsified tri[1-14C]oleoylglycerol as substrate. Twenty-four hr after administration of streptozotocin, hepatic alkaline lipase activity was 39% of normal, and this lower level of activity was observed at 72 hr and 7 days, after streptozotocin injection. After 24 hr of starvation, lipase activity also was significantly lower (35%) than normal. Insulin (35 U regular/kg body weight) had no acute (90 min) effect on the hepatic lipase activity of either normal or diabetic rats. Chronic insulin administration (4 subcutaneous injections of 10 U protamine zinc insulin/kg at 16-hr intervals) to normal rats provoked a 40% increase in hepatic lipase activity. Diabetic rats given the same insulin treatment showed lipase activity that was significantly higher (155%) than normal. Lipase activity fell to 65% of normal when insulin was withheld (32 hr) from diabetic rats given chronic insulin therapy. Intracardial injection of glucagon (1 mg/kg) into normal rats had no acute (30 min) effect on hepatic alkaline lipase activity. Hepatic alkaline lipase activity varied independently from the concentrations of either glucose or triacylglycerol in the plasma. However, there was an apparent negative correlation between this lipase activity and the concentration of fatty acids in the plasma; lipase activity was highest when fatty acid concentrations were lowest, and lowest when fatty acid concentrations were elevated. From these data we conclude: 1) changes in hepatic alkaline lipase activity ware provoked by chronic, but not acute, alteration of the hormonal and metabolic status of the rat, and 2) changes in hepatic alkaline lipase activity may be mediated through changes in the levels of circulating fatty acids presented to the liver, but the effect is not an immediate one.

Hydrolysis of sarcolemma by lysosomal lipases and inhibition by chlorpromazine.

The susceptibility of the lipids of canine cardiac sarcolemma to attack by soluble lysosomal lipases was studied to simulate in vitro the lipolytic injury that occurs during ischemia. The sarcolemmal fraction was incubated at 37 degrees C with the soluble portion of rat hepatic lysosomes (the lysosol) under conditions (pH 5.0, 5 mM ethylenediaminetetraacetic acid) appropriate for the activity of the major lysosomal lipases. Incubation of sarcolemma with lysosol resulted in a 78% lipolysis of sarcolemmal triacylglycerols, a lesser degradation of glycerophospholipids, and a parallel production of free fatty acids and lysophospholipids. The hydrolysis of sphingomyelin was negligible but was greatly stimulated (75%) by the addition of Triton X-100 (1 mg). Endogenous lipolytic activities of the sarcolemma did not contribute significantly to the observed lipid hydrolysis either in the presence or absence of detergent. The lipolysis of sarcolemmal triacylglycerols, glycerophospholipids, and sphingomyelin (Triton X-100 stimulated) were inhibited by varying concentrations of chlorpromazine. Thus cardiac sarcolemma is susceptible to hydrolysis of lysosomal lipases, and chlorpromazine inhibits this potentially injurious process.

The acute effects of streptozotocin-induced diabetes on rat liver glycerolipid biosynthesis.

Streptozotocin-induced diabetes produced a significant rise in rat serum and liver triacylglycerol content and hepatic triacylglycerol biosynthesis measured in vivo. Microsomes, isolated from the livers of streptozotocin-exposed animals (2-72h), exhibited an increased capacity to incorporate sn-[1,3-(14)C]glycerol 3-phosphate into neutral lipid (diacylglycerol and triacylglycerol) in the presence of ATP, CoA and palmitate. The streptozotocin-induced elevation of microsomal neutral lipid production was accompanied by a corresponding rise in the activity of microsomal phosphatidate phosphohydrolase (4-fold after 72 h of streptozotocin exposure). Diabetic-dependent increases in acylglycerol formation, phosphatidate phosphohydrolase activity and serum triacylglycerol and fatty acid levels were reversed by administering insulin (10 units protamine zinc/kg) at 16-h intervals (three separate doses( beginning 24 h after streptozotocin exposure. However, the diabetic-related rise in hepatic triacylglycerol content was only partially corrected by insulin administration. Streptozotocin-relate increases in liver triacylglycerol biosynthesis and phosphatidate phosphohydrolase activity we associated with alterations in plasma factors, since homogenates of hepatocyte monolayers exposed (18h) to plasma isolated from diabetic (72 h exposure to streptozotocin) animals exhibit an increased capacity to incorporate sn-[1,3-(14)C]glycerol 3-phosphate into triacylglycerol compared to homogenates of cells exposed to plasma from control (non-fasted) animals. The importance of these plasma factors in altering hepatic acylglycerol formation was also supported by the observation that hepatocyte monolayers exposed to a mixture of plasma isolated from normal (non-fasted) animals and plasma components elevated in diabetes (glucagon, glucose, oleate and ketones) showed increases in triacylglycerol formation which were similar to those produced by exposure to diabetic plasma. Additional studies demonstrated that fatty acids (oleate) appeared to be the agent primarily responsible for the diabetic plasma-induced rise in monolayer triacylglycerol biosynthesis and phosphatidate phosphohydrolase activity.

Evidence for the existence of only one triacylglycerol lipase of rat liver active at alkaline pH.

There have been numerous reports suggesting the existence of two or more lipases in liver capable of hydrolyzing triacylglycerols at neutral to alkaline pH. We set out to determine if rat liver contains an alkaline triacylglycerol lipase, in addition to heparin-releasable lipase, which has an intracellular localization. We report here the results of studies concerning the pH dependence, subcellular localization and kinetic analysis of the alkaline lipase(s) of rat liver. Homogenates and cytosolic, microsomal and plasma membrane-enriched subfractions all exhibited an optimum of lipase activity at approx. pH 8.0. In no case was there evidence of multiple pH optima in the alkaline ranges of conformity to Michaelis-Menten kinetics were calculated for the microsomal (0.91 +/- 0.12 mM), cytosolic (1.55 +/- 0.38 mM) and plasma membrane-enriched (1.02 +/- 0.04 mM) subfractions. To determine if the com- and subfractions prepared from control livers with those prepared from livers perfused with collagenase. The loss (93%) of lipase activity from both the cytosolic and microsomal subfractions after collagenase perfusion was identical to the loss (93%) of activity from the homogenates, suggesting a common origin with the collagenase-sensitive alkaline lipase on plasma membrane. The characteristics of hydrolysis in vitro of triacylglycerol contained in artificial and natural substrate preparations by the alkaline lipase of rat liver were examined. The artificial substrate preparation was emulsified tri[1-14C]oleoylglycerol prepared by sonication and the natural substrate preparation was a triacylglycerol-rich lipid fraction ('liver fat') prepared from rat liver homogenates. Although the curves were complex, apparent Km values (mean +/- S.W., n = 3-6) over the limited concentration ranges of conformity to Michaelis-Menten kinetics were calculated for the microsomal (0.91 +/- 0.12 mM), cytosolic (1.55 +/- 0.38 mM) and plasma membrane-enriched (1.02 +/- 0.04 mM) subfractions. To determine if the complexity of these kinetics was related to changes in the products of lipolysis, we examined the products after incubations of plasma membrane-enriched fractions with lower and higher concentrations of triacylglycerol. In either case, the products of lipolysis were diacylglycerol, fatty acids and glycerol; no monoacylglycerol accumulated under any circumstances. At the lower concentrations of either tri[1-14C]oleoylglycerol or liver fat, most triacylglycerol hydrolyzed was degraded fully to fatty acids and glycerol. At the higher triacylglycerol concentrations, while complete degradation continued, virtually all of the increased lipolysis of triacylglycerol (over the lipolysis at the lower substrate concentrations) yielded diacylglycerol. The data indicated that the hydrolysis of diacylglycerol by the alkaline lipase of rat liver occurred at a rate slower than that of triacylglycerol. If the same enzyme catalyzes the lipolysis of both tri- and diacylglycerols, triacylglycerols would appear to be preferred...

Substrate stabilization of the palmitoyl-coenzyme A hydrolase activity of rat submaxillary gland.

The long-chain acyl-CoA hydrolase (EC 3.1.2.2) activity of rat submaxillary salivary gland, found in the postmicrosomal supernatant fraction, has a pH optimum of 7.4. This hydrolase activity was found to be extremely labile, but inclusion of glycerol or the substrate palmitoyl-CoA in the preparations markedly stabilized the activity. Gel-filtration studies revealed multiple forms of the hydrolase, a lower-molecular-weight species of approx. 45 000 and a higher-molecular-weight species of approx. 130 000 observed when glycerol (20%, v/v) or palmitoyl-CoA (10 micro M) were included in the eluting buffer. This phenomenon is similar to that observed with the palmitoyl-CoA hydrolase of rat brain, except that there is no evidence that the higher-molecular-weight species of the hydrolase of submaxillary gland is generated by substrate-induced dimerization of the lower-molecular-weight species.

Factors affecting the activity and stability of the palmitoyl-coenzyme A hydrolase of rat brain.

Palmitoyl-CoA hydrolase (EC 3.1.2.2) catalyses the irreversible hydrolysis of long-chain acyl-CoA thioesters. This enzyme is found primarily in the postmicrosomal supernatant fraction prepared from homogenates of rat brain. Either of two forms of the hydrolase, a lower-molecular-weight species of approx. 70000 or a higher-molecular-weight species of approx. 130000 can be isolated by gel filtration. The higher-molecular-weight form is obtained from columns of Sephadex G-200 eluted with buffer containing 10mum-palmitoyl-CoA or 20% (v/v) glycerol, whereas the lower-molecular-weight form is obtained when the eluting buffer does not contain palmitoyl-CoA or glycerol. The two forms of the hydrolase have the same pH optimum of 7.5, are equally sensitive to the thiol-blocking reagents p-hydroxymercuribenzoate, HgCl(2), and 5,5'-dithiobis-(2-nitrobenzoic acid), and exhibit the same K(m) (1.8mum) with palmitoyl-CoA as substrate. The two forms differ in the availability or reactivity of certain external thiol groups, as determined by covalent chromatography with activated thiol Sepharose. Dilute solutions of the lower-molecular-weight form of the hydrolase rapidly lose activity (50% in 60min at 0 degrees C), but there is no change in the K(m) with palmitoyl-CoA as substrate during this progressive inactivation. Dilutions of the hydrolase in buffer containing 10mum-palmitoyl-CoA retain full activity. However, addition of palmitoyl-CoA to solutions of the lower-molecular-weight form will not restore previously lost hydrolase activity. The evidence supports the conclusion that the substrate palmitoyl-CoA promotes the formation of a relatively stable dimer from two unstable subunits. This process may not be reversible, since the removal of palmitoyl-CoA or glycerol from solutions of the higher-molecular-weight form does not result in the appearance of the lower-molecular-weight form of the hydrolase.

Effects of small amounts of pentadecan-2-one on the growth of Clostridium butyricum.

Primary alcohols occur as trace lipids and are the only long-chain alcohol species present in Clostridium butyricum. Secondary alcohols do not occur physiologically in this microorganism. Exposure of these cells to the methyl ketone, pentadecan-2-one, results in a marked decrease in the primary alcohol content with the secondary alcohol, pentadadecan-2-ol, becoming the major alcohol present. This change in lipid composition is associated with a significant decrease in growth rate that is proportional to the log of the pentadecan 2-one concentration of the incubation medium. When these cells are incubated with pentadecan-2-ol alone, growth is unaffected. Simultaneous exposure of the bacteria to pentadecan-2-one and a mixture of primary alcohols results in a partial relief of the growth inhibition observed with the ketone alone. These observations indicate that pentadecan-2-one inhibits the formation of primary alcohols that are important for normal growth of this bacterium.

Vitamin K requirement and the concentration of vitamin K in rat liver.

The concentration of vitamin K was determined in the liver of different strains of rats, and in male and female warfarin-resistant rats by feeding 3H-vitamin K in a purified diet. In each case, the level of vitamin K in the liver correlated approximately with the amount of vitamin K fed. The results indicate that differences in the requirement for vitamin K between the sexes and between strains of rats are due principally to different required concentrations of vitamin K in liver and not to differences in absorption or turnover of the vitamin. The results of the determination of vitamin K epoxide levels in male and female warfarin-resistant rats, and other data, suggest that the amount of vitamin K required in liver may be in part due to differences in the activity of the enzyme, vitamin K epoxide reductase.

Metabolism and biological activity of cis- and trans-phylloquinone in the rat.

The separation of sufficient cis and trans forms of vitamin K for feeding and metabolic studies was accomplished on silica gel columns eluted with solvent containing n-butyl ether. The lack of biological activity of the cis isomer of phylloquinone was observed. The cis isomer was retained longer in liver, particularly in mitochondria, but had low retention in that portion of the endoplasmic reticulum isolated as the rough membrane fraction. The cis isomer of phylloquinone was a poor substrate for 2,3-epoxidation in vivo and in vitro. These data are consistent with the hypothesis that epoxidation of vitamin K is coupled to the biological activity of the vitamin, and that microsomes are the site of metabolism and function of vitamin K.