Relationship between translocation of long-chain acyl-CoA hydrolase,phosphatidate phosphohydrolase and CTP:Phosphocholine cytidylyltransferase and the synthesis of triglycerides and phosphatidylcholine in rat liver

Translocation of long-chain acyl-coenzyme A hydrolase from the microsomal fraction to the cytosolic fraction was promoted in cell-free extracts of rat liver by palmitic acid, oleic acid, tetradecylthioacetic acid, and tetradecylthiopropionic acid, and by their CoA esters. The CoA esters were more effective than the non-esterified acids in the translocation of the enzyme. Treatment of normolipidemic rats with sulfur-substituted non-β-oxidizable fatty acid analogues resulted in a transitory increase in hepatic concentration of long-chain acyl-CoA. Longer feeding times almost normalized the hepatic long-chain acyl-CoA content. Microsomal long-chain acyl-CoA hydrolase activity was inhibited, whereas the activity of the cytosolic form was stimulated. The rise in enzyme activity coincided with a reduction in liver content of triglyceride and an increase in hepatic phospholipid content. The results suggest that the activity of long-chain acyl-CoA hydrolase in the cytosol may control the amount of acyl-CoA thioesters in the liver. Esterified and non-esterified fatty acids causedin vitro translocation of phosphatidate phosphohydrolase and cytidine 5′-triphosphate (CTP):phosphocholine cytidylyltransferase from the cytosolic fraction to the microsomal fraction. However, the translocation of these two enzyme systems was not obtainedin vivo. The activity of phosphatidate phosphohydrolase decreased in microsomal and cytosolic fractions while the activity of cytidylyltransferase in these fractions increased. The activities of soluble phosphatidate phosphohydrolase and long-chain acyl-CoA hydrolase appeared to be inversely correlated. The results imply that in cytoplasm, long-chain acyl-CoA hydrolase may compete with the biosynthetic enzymes for the acyl-CoA substrate, thus influencing the rate of lipid synthesis. The reduced hepatic triglyceride content observed in tetradecylthioacetic acid-treated rats is probably due to reduced triglyceride synthesis, which is mediated by an inhibition of phosphatidate phosphohydrolase accompanied with translocation and stimulation of long-chain acyl-CoA hydrolase. Development of fatty liver as an effect of tetradecylthiopropionic acid is probably due to accelerated triglyceride biosynthesis, which is mediated by a stimulation of phosphatidate phosphohydrolase and a decrease in cytosolic palmitoyl-CoA hydrolase activity.     

Effect of hyperpnea on enzymes of the CDPcholine path for phosphatidylcholine synthesis in rat lung

We have examined the activity of three enzymes in pulmonary surfactant phosphatidylcholine synthesis following the hyperpnea induced by having rats either inspire 5% CO₂/13%O₂/82% N₂ for 24 hr or swim in thermoneutral water for 30 min. Both stimuli markedly increase frequency and tidal volume of breathing and promote the release of surfactant. Lungs were perfused to remove blood, lavaged, and then homogenized in 1 mM Hepes, 0.15M KCl at pH 7.0. The homogenate was centrifuged at 9,000 g (av) for 10 min to sediment the mitochondria and lamellar bodies and at 100,000 g (av) for 60 min to obtain the microsomal and cytosol fractions. Incubations were carried out under determined optimal conditions and zero order kinetics. Choline kinase (CK), cholinephosphate cytidylytransferase (CP-cyT) and choline phosphotransferase (CPT) were assayed by the incorporation of [methyl-¹⁴C] choline chloride into phosphocholine, [methyl-¹⁴C]phosphocholine into CDPcholine, and [¹⁴C]CDPcholine into phosphatidylcholine, respectively. The incubation products were separated by thin-layer chromatography. Whereas both forms of hyperpnea increased the activity of CP-cyT in the microsomal fraction, they had no effect on the activity of either cytosolic CP-cyT and CK, or microsomal CPT. A similar increase in tidal volume in an isolated perfused rat lung had no effect. We conclude that,in vivo, hyperpnea increases the activity of CP-cyT, the rate-limiting enzyme in phosphatidylcholine synthesis. Whether this is due to an increase in the amount of enzyme, or of a cofactor, is unknown.     

Mechanism of lysophosphatidylcholine accumulation in the ischemic canine heart

The metabolism of lysophosphatidylcholine (LPC) in non-ischemic and ischemic canine heart was investigated byin vitro enzyme analysis. Selected subcellular fractions were assayed for the LPC-producing enzyme phospholipase A and the LPC-eliminating enzymes LPC:acyl-CoA acyltransferase, LPC:LPC transacylase and lysophospholipase. The canine heart was found to contain all enzymes differing, however, in subcellular distribution and specific activity. Phospholipase A activity did not change significantly in any of the fractions prepared from the ischemic tissue of hearts rendered ischemic for 1, 3 or 5 hr when compared to non-ischemic tissue. Changes in the activity of the microsomal LPC:acyl-CoA acyltransferase over the course of 5 hr of ischemia were observed. Significant decreases in the activity of the cytosolic and microsomal lysophospholipases were detected especially after 3 and 5 hr of ischemia. Similarly, a decrease in the activity of the microsomal LPC:LPC transacylase was noted after 3 and 5 hr of ischemia. Our results suggest that impaired catabolism of LPC rather than an enhanced production of LPC is the principal mechanism for the increase in LPC levels in the ischemic canine heart.     

Resistance of cereals to aphids: The interaction between hydroxamic acids and UDP-glucose transferases in the aphidSitobion avenue (Homoptera: Aphididae)

UPD-glucose transferases are found in the cytosolic and microsomal fractions of the grain aphidSitobion avenae F. Gel filtration and SDSPAGE revealed that the microsomal fraction contained several forms of the enzyme. The molecular weights of the three most active fractions might be 68,000, 66,000, and 36,500. There was a negative correlation between the enzymes' activity in extracts of aphids and the concentration of DIMBOAaglucone in the winter wheat variety fed on by the aphid. A strong inhibition of the activity of the UPD-glucose transferases was observedin vitro at a concentration of DIMBOA as low as 0.01 mM. There was a greater activity of the enzymes in aphids fed on seedlings of susceptible than on moderately resistant wheat cultivars. Prolonged feeding on resistant cultivars resulted in a further reduction in the activity of the aphid's enzymes. The significance for cereal aphids of the role of their UDP-glucose tranferases in the detoxification of plant allelochemicals and adaptation to resistant varieties of cereals is discussed.     

Cholesterol metabolism in the rat lactating mammary gland: The role of cholesteryl ester hydrolase

An acid cholesteryl ester hydrolase activity associated with a fraction containing mitochondria and lysosomes from rat lactating mammary glands was found to have a pH optimum of 5.0. Its sedimentation pattern was closely related to that of the lysosomal enzyme markers acid phosphatase and β-glucuronidase, suggesting that the activity is associated with the lysosomes. The enzyme was strongly inhibited by Cu²⁺, but was inhibited little by other divalent metal ions. Acid cholesteryl ester hydrolase activity was almost completely abolished byp-hydroxymercuribenzoate, but this effect was reversed in the presence of an equimolar concentration of reduced glutathione (GSH), indicating that the enzyme requires free sulfhydryl groups for activity. These properties are similar to those of acid, lysosomal cholesteryl ester hydrolases found in other tissues. Acid cholesteryl ester hydrolase activity was 8–14 fold higher in mammary tissue from lactating as compared to virgin rats. Neutral cholesteryl ester hydrolase activities associated with the microsomal and cytosolic subcellular fractions were also increased in lactating glands, but to a lesser extent. In addition, a 2-fold increase in the activities of both the acid and microsomal neutral enzymes was seen during the first few days of lactation, while the cytosolic neutral activity remained constant. These results suggest that mammary gland cholesteryl ester hydrolases have a role in the regulation of cholesterol metabolism in mammary cells, and in the provision of cholesterol for secretion into milk.     

Phospholipase D and phosphatidate phosphohydrolase activities in rat cerebellum during aging

Aging is a process that affects different organs, of which the brain is particularly susceptible. PA and DAG are central intermediates in the phosphoglyceride as well as in the neutral lipid biosynthetic pathway, and they have also been implicated in signal transduction. Phospholipase D (PLD) and phosphatidate phosphohydrolase (PAP) are the enzymes that generate PA and DAG. The latter can be transformed into MAG by diacylglycerol lipase (DGL). In the present study, we examine how aging modulates the PLD, PAP, and DGL isoforms in cerebellar subcellular fractions from 4-(adult),28-, and 33-mon-old (aged) rats. Pl-4,5-bisphosphonate (PIP₂)-dependent PLD, PAP1, and DGL1 were distributed in different percentages in all cerebellum subcellular fractions. On the other hand, PAP2 and DGL2 activities were observed in all subcellular fractions except in the cytosolic fraction. Aging modified the enzyme distribution pattern. In addition, aging decreased nuclear (45%), mitochondrial-synaptosomal (55%), and cytosolic (71%) PAP1 activity and increased (28%) microsomal PAP1 activity. DGL1 activity was decreased in nuclear (85%) and mitochondrial-synaptosomal (63%) fractions by aging. On the other hand, PIP₂-dependent PLD activities were increased in the mitochondrial-synaptosomal fraction. PAP2 and DGL2 were increased in the microsomal fraction by 87 and 114%, respectively, and they were decreased in the nuclear fraction. The changes observed in cerebellum PAP1 and DGL1 activities from aged rats with respect to adult rats could be related to modifications in lipid metabolism. Differential PA metabolization during aging through PIP₂-dependent PLD/PAP2/DGL2 activities could be related to alterations in the neural signal transduction mechanisms.     

Factors enhancing diacylglycerol acyltransferase activity in microsomes from cell-suspension cultures of oilseed rape

Several factors, including an unidentified endogenous component, were found to stimulate microsomal diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) from a microspore-derived cell-suspension culture of oilseed rape (Brassica napus L. cv. Jet Neuf). At a concentration of 25mM, MgSO₄ and MgCl₂ stimulated microsomal DGAT 25- and 10-fold, respectively. AIP and CoA at concentrations of 2 and 1 mM stimulated the enzyme 2.4- and 12-fold, respectively, although the effects were lessened in the presence of higher Mg²⁺ concentrations. Although microsomal DGAT activity was increased only slightly by the addition of exogenous sn-1,2-diacylglycerol to the reaction mixture, it was increased substantially by the addition of exogenous phosphatidate. sn-Glycerol-3-phosphate and other phospholipids tested did not have this stimulatory effect. DGAT activity did not decrease when microsomes were incubated with ATP in the presence of the cytosolic fraction. This fraction, however, contained a small organic compound(s) that stimulated microsomal DGAT activity.     

Characterization of calcium-independent cytosolic phospholipase A2 activity in the submucosal regions of rat stomach and small intestine

This study was undertaken to compare the calcium-independent phospholipase A₂ (PLA₂) activities in the cytosols of twelve rat tissues and to determine whether their activities were distinct. 1-O-Alk-1′-enyl-2-[¹⁴C]-oleoyl-sn-glycero-3-phosphocho-line (PlsC) and 1-O-Alk-1′-enyl-2-[¹⁴C]oleoyl-sn-glycero-3-phosphethanolamine (PlsE) were synthesized and used as substrates, instead of phosphatidyl compounds, to exclude hydrolysis by cytosolic PLA₁ activity that could be present in some of the cytosolic preparations. For each tissue, we examined substrate specificity, pH optimum, and effect of adenosine triphosphate (ATP) and ATP analogues. PLA₂ activity was detected in eleven out of the twelve issues examined. Based on substrate specificity and pH optimum, cytosolic calcium-independent PLA₂ were classified in three groups. The first group, which included PLA₂ from small intestine, stomach and spleen, had the highest specific activity with PlsC as substrate (1253, 309 and 75 nmol/mg protein/hour, respectively) and an optimal pH at 6.5. Activity with PlsE as substrate was much lower (20–37%) than with PlsC. The second group of PLA₂ activities included the cytosolic activities from thymus, lung, liver and pancreas that showed lower specific activities for both substrates (14–23 nmol/mg protein/hour with PlsC) and had a broader optimal pH range of 6.1 to 7.5. The cytosols from brain, kidney, heart and muscle comprised the third PLA₂ group that was found to have a higher specific activity with PlsE (5–20 nmol/mg protein/hour) than PlsC and an optimal pH range from 7.4 to 7.9. Since the highest specific activity was found in the cytosol from small intestine, this PLA₂ was examined further. PLA₂ activity was found to be equally distributed in the cytosol of the submucosal portion of duodenum, jejunum and ileum with an optimal pH of 6.1 and a 5-fold higher activity with PlsC than PlsE as substrate. Moreover, this PLA₂ activity was inhibited by treatment with detergents. These results indicate the presence in the submucosal portion of the intestine of a calcium-independent cytosolic PLA₂ with a high specific activity toward PlsC and properties distinct from those described for the PLA₂ found in the intestinal brush-border.     

Studies on linoleic acid 8R-dioxygenase and hydroperoxide isomerase of the fungusGaeumannomyces graminis

Linoleic acid is sequentially converted to7S,8S-dihydroxy-9Z,12Z-octadecadienoic acid by the 8R-dioxygenase and hydroperoxide isomerase of the fungusGaeumannomyces graminis, which is a common pathogen of wheat. The objective of this study was to separate and characterize the two enzyme activities. The isomerase activity was found mainly in the microsomal fraction of the mycelia and the 8R-dioxygenase in the cytosol. The 8R-dioxygenase could be partially purified by ammonium sulfate precipitation, gel filtration, ion exchange chromatography or isoelectric focusing. The 8R-dioxygenase was unstable during purification, but it could be stabilized by glutathione, glutathione peroxidase and ethylenediaminetetraacetic acid. Several protease inhibitors reduced the enzyme activity. Gel filtration with Sephacryl S-300 showed that most 8R-dioxygenase activity was eluted with the front with little retention. Isoelectric focusing in the presence of ethylene glycol (20%) indicated an isoelectric point of pl 6.1–6.3. The enzyme was retained on strong anion exchange columns at pH 7.4 and could be eluted with 0.3–0.5 M NaCl. Incubation of the enzyme with 0.1 mM linoleic acid led to partial inactivation, which may indicate product inhibition. Paracetamol and the lipoxygenase inhibitor ICI 230,487 at 30 μM inhibited the 8R-dioxygenase by 44 and 58%, respectively. 8R-hydroperoxy-9Z,12Z-octadecadienoic acid was isolated from incubations of linoleic acid with the partially purified enzyme or with the cytosol in the presence ofp-hydroxymercuribenzoate. The hydroperoxide was rapidly converted by the hydroperoxide isomerase in the microsomal fractions to7S,8S-dihydroxy-9Z,12Z-octadecadienoic acid. The isomerase was neither inhibited by carbon monoxide nor by ketoconazole (100 μM). The isomerase was active over a broad pH range. It could be separated from the 8R-dioxygenase by differential centrifugation, by ammonium sulfate precipitation and by gel filtration. We conclude that the linoleic acid 8R-dioxygenase and the hydroperoxide isomerase are two separate enzymes.     

Calcium deficiency modifies polyunsaturated fatty acid metabolism in growing rats

Fatty acid desaturase activities were determined in liver microsomes from calcium-deficient rats and compared to calcium-sufficient ones. The calcium-deprived diet (0.5 g/kg) administered for 60 d caused a 30% inhibition in the Δ5 desaturase activity and a 45–55% decrease in Δ6 and Δ9, respectively, facts that cannot be attributed to a reduction in food intake. In vitro addition of calcium, ethyleneglycol-bis(β-aminoethyl ether)N,N-tetraacetic acid, and/or cytosol fractions from control or calcium-deficient rats to microsomes from both groups of animals indicates that the reduced desaturase capacities would be the consequence of an indirect effect of calcium deprivation. The present work shows that the reduced unsaturated fatty acid biosynthesis might be the result of modifications in the physicochemical properties of microsomal membranes. Such changes could also be derived from the inhibition of phospholipase A₂ activity induced by calcium deficiency.     

Application of 2-hydroxypropyl-β-cyclodextrin in the assay of acyl-CoA: cholesterol acyltransferase and neutral and acid cholesterol ester hydrolases

The utility of 2-hydroxypropyl-β-cyclodextrin for increasing the sensitivity of assays for the microsomal acyl-CoA:cholesterol acyltransferase, and the acid lysosomal and the neutral microsomal and cytosolic cholesterol ester hydrolase activity was studied in rat hepatocytes. Enzyme assays, at optimal concentrations of cyclodextrin, were validated by assessing: (i) linearity of product formation with incubation time and protein amount, and saturation with substrate, and (ii) the effect of treatments of cells or of subcellular fractions on enzyme activities. Delivery of cholesterol dissolved in 2-hydroxypropyl-β-cyclodextrin to the acyl-CoA:cholesterol acyltransferase assay mixture raised the enzyme activity more than 8-fold and was twice that measured when cholesterol was added in Triton WR-1339. 2-Hydroxypropyl-β-cyclodextrin itself was partially effective, apparently by making endogenous cholesterol more accesible to the enzyme. Inclusion of 2-hydroxypropyl-β-cyclodextrin in cholesterol ester hydrolase assays using standard micellar substrates doubled the activity estimated in lysosome and microsome preparations and enhanced the cytosolic cholesterol esterase activity by about 50%. Differences in the catalytic activity of acyl-CoA:cholesterol acyltransferase and cholesterol ester hydrolases caused by treatment of hepatocytes with compound 58-035 or 25-hydroxycholesterol, or of subcellular fractions with NaF, were maintained when enzymes were assayed with cyclodextrin. The results indicate that 2-hydroxypropyl-β-cyclodextrin is a suitable vehicle for delivering cholesterol to acyl-CoA:cholesterol acyltransferase and enhances the sensitivity of standard assays of the enzymes governing the intrahepatic hydrolysis of cholesteryl esters.     

Enzymic regulation of arachidonate metabolism in brain membrane phosphoglycerides

The metabolism of arachidonate in brain membrane phosphoglycerides was investigated in vivo by intracerebral injection of labeled arachidonate and by in vitro assay of enzymic systems associated with the metabolism. After intracerebral injection, labeled arachidonate was incorporated rapidly into brain phosphoglycerides with radioactivity distributed mainly in diacyl-sn-glycero-3-phosphoinositols (GPI) and diacyl-sn-glycero-3-phosphocholines (GPC). Some evidence of a metabolic relationship between diacyl-sn-glycerophosphoinositols (diacyl-GPI) and diacylglycerols was observed. Among the phosphoglycerides labeled with [¹⁴C] arachidonoyl groups, diacyl-GPI were most rapidly metabolized in brain microsomal and synaptosomal fractions. The decay of diacyl-GPI in brain synaptosomes may be represented by two pools with half-lives of 5 hr and 5 days. Three types of enzymic systems related to metabolism of the polyunsaturated fatty acids in brain were investigated. The first system involves the cyclic events relating the ATP-dependent activation of polyunsaturated fatty acids (PUFA) to their acylCoA by the acylCoA ligase and subsequent hydrolysis of acylCoA to free fatty acids by the acylCoA hydrolase. It is apparent that fatty acid activation and hydrolysis is under strigent control in order to maitain suitable levels of free fatty acids and acylCoA in the brain tissue for various metabolic use. Factors involved in the regulation may include the level of ATP, divalent cations and the nature of substrates. The second enzymic system pertains to deacylation via phospholipase A₂ and reacylation via the acyltransferase of membrane phosphoglycerides. In brain tissue, activity of the acyl transferase is generally higher than that of the phospholipase A₂. Factors known to affect specificity of the acyltransferase include substrate concentration and the nature of the acyl groups and lysophosphoglycerides. The acyltranferase(s) in brain preferentially transfers arachidonate to 1-acyl-GPI. Activity of the acyltransferase can be inhited by a number of lypophilic compounds including local anesthetics and cell surface agents. Activity of the phospholipase A₂ in brain may depend on the physical form of the substrates, i.e., whether the substrates are in monomeric or micellar form. The third process is associated with the degradation of diacyl-GPI by enzymes present in brain subcellular membranes. Incubation of brain subcellular membranes with 1-acyl-2-[¹⁴C] arachidonoyl-GPI yielded labeled diacylglycerols and arachidonate. The phospholipase C action is specific for hydrolysis of diacyl-GPI. The arachidonate released from incubation of labeled diacyl-GPI may be the result of phospholipase A₂ action which is not specific for diacyl-GPI or the hydrolysis by lipase acting on the diacylglycerols formed from the phospholipase C activity. Enzymic hydrolysis of diacyl-GPI is most active in the microsomal fraction, but uoon disruption of synaptosomes, enzyme in synaptic plasma membranes is also active in degradating this glycerophospholipid. In general, the results of in vitro studies are in good agreement with those observed in vivo and the information yielded has contributed towards understanding the metabolism of polyunsaturated fatty acids in brain subcellular membranes.     

Effects of aurothioglucose on iron-induced rat liver microsomal lipid peroxidation

Aurothioglucose (ATG), an inhibitor of selenium-dependent glutathione peroxidase activity, at a concentration of 100 μM, strongly increases lipid peroxidation of rat liver microsomes exposed to either ferrous ion (10 μM) or the combination of ferric ion (10 μM) and ascorbic acid (500 μM), in the presence of reduced glutathione (GSH, 800 μM). This effect was not achieved using heat-inactivated microsomes and was dependent on the presence of GSH. ATG did not affect the lag period associated with ascorbic acid/ferric ion-induced microsomal lipid peroxidation (previously attributed to an undefined GSH-dependent microsomal agent), but did increase the rate of peroxidation subsequent to the lag period. The potent GSH-dependent inhibition of microsomal lipid peroxidation by cytosol (10% of total volume) was completely reversed by ATG (100 μM). ATG similarly reversed an inhibition of phosphatidyl-choline hydroperoxide-dependent liposomal peroxidation that has been attributed to phospholipid hydroperoxide glutathione peroxidase (PHGPX), an enzyme distinct from the classical glutathione that cannot utilize intact phospholipids. ATG inhibited, in addition to the classifical selenium-dependent glutathione peroxidase, both cytosolic and microsomal (basal and N-ethyl maleimide-stimulated) glutathione S-transferase activities with greater than 80% inhibition achieved at 100 μM ATG. ATG, at concentrations up to 250 μM, did not inhibit PHGPX activity measured by the coupled-enzyme method in the presence of Triton X-100 (0.1%). These data demonstrate the potential of ATG to increase toxicity of lipid peroxidative stimuli by inhibition of microsomal and cytosolic defense mechanisms. Although ATG did not inhibit Triton-enhanced PHGPX activity, overall evidence points toward inhibition of this enzyme as the mechanism for ATG-augmented lipid peroxidation and supports the conclusion that PHGPX plays a major role in the cellular defense mechanism.     

Properties of PAF-synthesizing phosphocholinetransferase and evidence for lysoPAF acetyltransferase activity in rat brain

Several reports have indicated that platelet-activating factor (PAF) may play a role in the physiopathology of nervous tissue. We previously have demonstrated the presence, in the microsomal fractions of rat brain, of a phosphocholinetransferase which is able to synthesize PAF by thede novo pathway. The presence of dithiothreitol in the medium increases the rate of PAF biosynthesis, whereas it inhibits the synthesis of long-chain alkylacyl- and diacyl-glycerophosphocholines (GPC), including dioctanoyl-GPC. This and other properties, such as pH dependence and thermal stability, indicate that rat brain may have two distinct enzymes for the synthesis of PAF and other choline phospholipids. The affinity of these enzymes for CDPcholine is similar to that reported for other tissues, the K_m being 42 μm and 55 μm with alkylacetylglycerol and dioctanoylglycerol as lipid substrates, respectively. The V_max values were 3.0 and 2.2 nmol/mg prot/min for PAF and dioctanoyl-GPC, respectively. In addition, it was shown that the microsomal fraction of rat brain contains an acetyltransferase which can convert lysoPAF to PAF. Since it has been reported previously that brain tissue possesses phospholipase A₂ activity that can hydrolyze alkylacyl-GPC to lysoPAF, we conclude that brain tissue has all enzymic activities for the synthesis of PAF by the “remodeling pathway”. The role of the two routes of PAF biosynthesis in nervous tissue remains to be established. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Thiophosphoester analogs of phosphatidic acids: Spectrophotometric substrates for phosphomonoesterases

Thiophosphoester analogs of dioctanoyl and didecanoyl phosphatidic acids were synthesized for use as substrates in spectrophotometric assays. These substrates are easily dispersable in aqueous media and release thiodiacylglycerols after phosphomonoesterase catalyzed hydrolysis. The free sulfhydryl of these thiodiacylglycerols reacts with the colorimetric reagent 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), allowing the reaction to be followed. These analogs were shown to be good substrates for calf intestine alkaline phosphatase (highest activity at alkaline pH) and phosphomonoesterases of partially purified beef brain cytosol (highest activity at physiologic pH). Cationic amphiphilic drugs inhibit the actions of alkaline phosphatase on the dioctanoyl analog, but did not inhibit enzymatic hydrolysis ofp-nitrophenyl phosphate. In contrast, the beef brain cytosolic fractionp-nitrophenyl phosphate hydrolysis was mildly inhibited, and the phosphatidic acid analog hydrolysis was increased slightly. Tetramisole inhibited all enzyme activities withp-nitrophenyl phosphate, but was inhibitory only to the alkaline-phosphatase activity with the phosphatidic acid analog. This research was completed as partial fulfillment of the Ph.D. degree, awarded August, 1987, at Northern Illinois University, DeKalb, IL.     

Metabolism of epoxidized phosphatidylcholine by phospholipase A2 and epoxide hydrolase

The isolation and measurement of phospholipid epoxides as major peroxidation products in biomembrane preparations prompted an investigation of enzymatic mechanisms which may be responsible for their elimination. Analysis of microsomal epoxide hydrolase and phospholipase A₂ activity against a phospholipid epoxide commonly encountered in tissues indicated it to be a poor substrate for epoxide hydrolase, but rapidly hydrolyzed by phospholipase A₂. Microsomal and purified phospholipase A₂ preparations hydrolyzed the phospholipid epoxide at rates 2-fold greater than were observed with a monoenoic phospholipid from which the epoxide would be derived. The product fatty acid epoxide,cis-9,10-epoxystearic acid, was rapidly hydrated by microsomal and cytosolic epoxide hydrolase. On the basis of earlier reports demonstrating increased phospholipase activity against oxidized phospholipids, and on the results of the present study, a model for the metabolism of oxidized membrane phospholipids is proposed.     

Influence of hypo- and hyperthyroidism on rat liver glycerophospholipid metabolism

The effects of hyper- and hypothyroidism on enzyme activities involved in phospholipid metabolism in the rat liver were studied. Hyperthyroidism significantly decreases activities of both microsomal acyl-CoA:glycero-3-phosphate acyltransferase (GPAT) (34%, p<0.01) and microsomal acyl-CoA:1-acylglycero-3-phosphocholine acyltransferase (GPCAT) (28–33%, p<0.01). This may contribute to the decreased proportions of certain unsaturated fatty acids found in microsomal phosphoglycerides in hyperthyroidism. Mitochondrial GPAT, phospholipase A₂ and cytosol lysophospholipase are unaffected by hyperthyroidism. In contrast, hypothyroidism stimulates mitochondrial GPAT (38%, p<0.01) and microsomal GPCAT (14–19%) activities but decreases both mitochondrial phospholipase A₂ (36%, p<0.01) and cytosol lysophospholipase (56%, p<0.01) activities. The increased GPCAT activity may contribute to the increased proportions of certain unsaturated fatty acids found in microsomal phosphoglycerides in hypothyroidism. Triiodothyronine (T₃) treatment of the hypothyroid rat (25 μg/100 g body weight/day for four days) corrected phospholipase A₂ and lysophospholipase activities to the level of the control rat, but failed to correct the increased mitochondrial GPAT activity and not only corrected but lowered GPCAT activity to the level of the hyperthyroid rat.     

Hepatic zonation of the formation and hydrolysis of cholestery esters in periportal and perivenous parenchymal cells

The periportal (PP) and perivenous (PV) zones of the liver acinus differ in enzyme complements and capacities for cholesterol and bile acid synthesis and other metabolic processes. The aim of this investigation was to determine the acinar distribution of the catalytic activity of the enzymes governing the formation and hydrolysis of cholesteryl esters using PP and PV hepatocytes from normal or cholestyramine-fed rats. The hepatocyte subpopulations were isolated by centrifugal elutriation, characterized according to the distribution pattern of a number of cell parameters and marker enzymes, and assayed for acyl-CoA: cholesterol acyltransferase (ACAT) and lysosomal, cytosolic and microsomal cholesteryl ester hydrolase (CEH). In normally fed rats, no zonation was found in the activity of lysosomal CEH and ACAT, and the activity of both cytosolic and microsomal CEH zonated toward the PV zone of the acinus. Concentrations of free and esterified cholesterol in homogenates, cytosol, and microsomes of PP and PV cells were, however, similar. Cholestyramine raised significantly the PV/PP ratio of ACAT because of an exclusive PP reduction of activity and abolished the heterogeneity in microsomal CEH because of a greater inhibitory PV response, whereas the PV dominance of cytosolic CEH and the homogeneous distribution of lysosomal CEH were unaffected. These results demonstrated homogeneity within the liver acinus for the enzymatic degradation of endocyted lipoprotein-derived cholesteryl esters, a structural zonation of the cytosolic CEH and a dynamic zonation of ACAT and the microsomal CEH, with a PV dominance of the enzymatic capacity for the degradation of stored cholesteryl esters in normal livers.     

Phosphatidate phosphatase of dermatophytes

Phosphatidate phosphatase (EC 3.1.3.4) was detected in filamentous pathogenic fungi. In both dermatophytes,Microsporum gypseum andEpidermophyton floccosum, the enzyme was located in the mitochondrial and microsomal subcellular fractions with a pH optimum of 6.0. TheE. floccosum enzyme was more active than that ofM. gypseum. Although the enzyme in both fractions of the dermatophytes was susceptible to inhibition by Fe²⁺, Mn²⁺, Cu²⁺, Ba²⁺ and Hg²⁺, theE. floccosum microsomal enzyme was completely inhibited by Mn²⁺ and Fe²⁺. The enzyme ofM. gypseum as well as ofE. floccosum exhibited a Mg²⁺ dependency in presence of EDTA. Sulphydryl reagents did not inhibit either the mitochondrial or the microsomal enzyme of these fungi. Phosphorylcholine and lysophosphatidylcholine markedly inhibited the enzyme in the two dermatophytes; however, the inhibition was less pronounced with phosphatidylcholine.