Purification and characterization of lipoprotein lipase from pig myocardium.

1. Lipoprotein lipase was purified from pig myocardium by a two-step purification procedure involving (a) the formation of an enzyme-substrate complex and (b) affinity chromatography on Sepharose which contained covalently linked heparin. The purified enzyme gave in sodium dodecyl sulphate-polyacrylamide-gel electrophoresis one main band with an apparent molecular weight of 73 000. The enzyme, which was purified 70 000-fold, had a specific activity of 860 mumol of unesterified fatty acid liberated/h per mg of protein. 2. The purified enzyme hydrolysed [14C]triolein emulsions in the absence of added cofactors but its activity was increased fivefold by adding normal human serum. Of the low-density lipoprotein apoproteins only apolipoprotein CII could be substituted for serum in activating the enzyme. This lipase had maximum activity at 0.05-0.15 M-NaCl. Heparin increased the activity of the purified enzyme twofold at low concentrations, but high concentrations inhibited. The triglyceride lipase of pig myocardium thus resembles lipoprotein lipase purified from adipose tissue and from plasma, but is clearly different from pig hepatic triglyceride lipase.     

A new approach to the measurement of phospholipase A2 in tissue homogenates and its application to human skin

We describe a new approach to the quantification of tissue phospholipase A2 based on the observation that the Vmax is increased by several orders of magnitude in the presence of high concentrations of dimethyl sulphoxide. The enzyme from human skin showed optimal activity at 30% dimethyl sulphoxide and pH 6-7.5, and had an absolute dependence on Ca2+ (apparent Km = 1.4 mmol/l). The relationship between reaction velocity and substrate concentration did not follow Michaelis-Menten kinetics. The enzyme was inhibited by p-bromophenacyl bromide, half-maximal inhibition occurring at 2.4 mumol/l. Surprisingly, assay of specimens of various human tissues indicated that, with the exception of pancreas, skin contained higher levels of phospholipase A2 activity than any other tissue investigated. Equally surprising was that primate skin (in particular human) seems considerably more active than that of other mammals. Speculatively, these findings may be related to the high levels of free fatty acids occurring on the skin surface.     

Hyperlipoproteinemia type I in a patient with active lipoprotein lipase in adipose tissue and indications of defective transport of the enzyme

This paper presents a case of typical hyperlipoproteinemia type I in a young woman. Her serum triglycerides varied between 2 and 90 mmol/l and she had substantial amounts of apolipoprotein B-48 in fasting plasma. She had no detectable lipoprotein lipase (LPL) activity in post-heparin plasma (less than 0.2 percent of normal). Southern blot analysis suggested no major defect in her LPL gene and Northern blot analysis of adipose tissue RNA showed normal-sized LPL-mRNA. A 2-h [35S]methionine incorporation experiment with adipose tissue pieces in vitro showed that she produced normal-sized LPL and had LPL catalytic activity in the tissue. The amounts were, however, only 5-10% of control. No detectable LPL radioactivity or catalytic activity was released from patient tissue even in the presence of heparin in the incubations. Immunofluorescent staining of adipose tissue biopsies from the patient showed LPL immunoreactivity only in adipocytes and little or none within the capillaries. Treatment of immunoprecipitated labeled LPL with endoglycosidase H showed that the oligosaccharide chains on her enzyme were of the high-mannose type and not processed as in controls. Taken together the data suggest that the patient synthesizes a relatively normal LPL protein which is core-glycosylated and folded into active enzyme as in normal subjects, but is not effectively transported via the Golgi to the cell surface.     

Purification and properties of the membrane-bound by hydrogenase from Desulfovibrio desulfuricans.

The membrane-bound hydrogenase from the anaerobic sulphate-reducing bacterium Desulfovibrio desulfuricans (Norway strain) has been purified to homogeneity, with an overall 80-fold purification and a specific activity of 70 mumol of H2 evolved/min per mg of protein. The hydrogenase had a relative molecular mass of 58 000 as determined by gel filtration and was estimated to contain six iron atoms and six acid-labile sulphur groups per molecule. The absorption spectrum of the enzyme was characteristic of an iron-sulphur protein. The E400 and E280 were 28 500 and 109 000 M-1.cm-1 respectively. The e.s.r. of the oxidized protein indicated the presence of [4Fe-4S]3+ or [3Fe-3S]3+, and another paramagnetic centre, probably Ni(III). The hydrogenase was inhibited by heavy-metal salts, carbon monoxide and high ionic strength. However, it was resistant to inhibition by thiol-blocking and metal-complexing reagents. N-Bromosuccinimide totally inhibited the enzyme activity at low concentrations. The enzyme was stable to O2 over long periods and to high temperatures. It catalyses both H2-evolution and H2-uptake with a variety of artificial electron carriers. D. desulfuricans cytochrome C3, its natural electron carrier, had a high affinity for the enzyme (Km = 2 microns). Rate enhancement was observed when cytochrome C3 was added to Methyl Viologen in the H2-evolution assay. The pH optimum for H2-evolution was 6.5.     

Liver phosphorylase b kinase. Cyclic-AMP-mediated activation and properties of the partially purified rat-liver enzyme.

Phosphorylase b kinase was extensively purified from rat liver. It was located in a form which could be activated 20--30-fold by a preincubation with adenosine 3':5'-monophosphate (cyclic AMP) and ATP-Mg. This activation was time-dependent, and was paralleled by a simultaneous incorporation of 32P from [gamma-32P]ATP into two polypeptides which comigrated in sodium dodecyl sulfate gel electrophoresis with the alpha and beta subunits of rabbit skeletal muscle phosphorylase b kinase. The liver enzyme was eluted from Sepharose 4B and Bio-Gel A-50m columns at the same place as muscle phosphorylase b kinase, which is indicative of a molecular weight of 1.3 x 10(6). After activation, the most purified liver preparation had a specific activity about 10-fold less than the homogeneous muscle enzyme at pH 8.2. The inactive enzyme form had a pronounced pH optimum around pH 6.0, whereas the activated form was mostly active above neutral pH. The activation of the enzyme reduced the Km for its substrate phosphorylase b severalfold. Liver phosphorylase b kinase was shown to be partially dependent on Ca2+ ions for its activity: addition of 0.5 mM [ethylenebis-(oxoethylenenitrilo)]tetraacetic acid (EGTA) to the phosphorylase b kinase assay increased the Km for phosphorylase b about twofold for both the inactive and the activated form of liver phosphorylase b kinase, but affected the V of the inactive species only.     

Ontogeny and kinetics of carnitine palmitoyltransferase in liver and skeletal muscle of the domestic felid (Felis domestica)

The ontogeny of carnitine palmitoyltransferase (CPT) was examined in liver and muscle throughout growth and development of the domestic felid. Homogenates from animals in six age categories (newborn, 24-h, 3-, 6- and 9-week-old, and adult) were examined. Hepatic CPT specific activity increased progressively from birth to 6 weeks and then declined slightly into adulthood, with maximal values for animals greater than 24 h of age [171 nmol/(min g wet tissue)] being 70% higher than for newborns [99 nmol/(min g wet tissue)] (P<.05). Specific activity in adults was similar to that in 6- and 9-week-old juveniles. Total hepatic CPT activity [nmol/(min liver)] increased linearly with age, but the activity expressed per kg body weight [nmol/(min kg BW)] declined after 3 weeks. In contrast, skeletal muscle CPT-specific activity remained unchanged from birth to 3 weeks and then increased significantly, with maximal values at 9 weeks being 90% greater than those for young animals (newborn to 3 weeks; P<.05), whereas specific activity in adults was 50% lower than that observed in 9-week-old animals (P<.05). Hepatic and muscle apparent Km's for carnitine averaged 440 microM and did not vary with age. Hepatic carnitine concentrations remained relatively constant during development, but were lower in adult lactating females, whereas skeletal muscle concentrations increased markedly with age. Hepatic concentrations were 20-50% higher than apparent Km's for carnitine in young and growing animals, but concentrations were similar to the apparent Km at 6 weeks and significantly lower than the apparent Km in adults. Carnitine concentrations in skeletal muscle were 37% lower than apparent Km during the neonatal period, but significantly higher in cats >3 weeks of age. We conclude that postnatal increases in CPT activity support increased capacity for fatty acid oxidation in the developing felid and that dietary carnitine may be required to maximize enzyme activity.     

Phospholipase C activity in human polymorphonuclear leukocytes: partial characterization and effect of indomethacin

Several hormones act at the cellular level to increase diacylglycerol via increased catabolism of phosphatidylinositol by phospholipase C. Diacylglycerol stimulates protein kinase C, leading to protein phosphorylation and hormone action. Since phospholipase C activity has not been well studied in man, we have established an assay for phospholipase C in human neutrophils. In this assay sonicates of neutrophils were incubated with L-3-phosphatidyl-[U 14C]-inositol and the incubation mixture extracted with chloroform/methanol. Following the additions of 2 mol/l KCl and chloroform, phospholipase C activity was determined by counting [14C] in the aqueous phase. The phospholipase C activity was linear with respect to time and the quantity of added enzyme. Optimum substrate concentration and pH were 2 mmol/l and 7.0, respectively. Optimal activity was dependent on Ca2+ (2 mmol/l) and deoxycholate (2 mmol/l). Naloxone, and PGD2, which affect various aspects of leucocyte function, had no significant effects on neutrophil PLC activity. The effects of various compounds with phospholipase A2 inhibitory activity were also tested on this enzyme. Of these, mepacrine, lidocaine and indomethacin inhibited the enzyme activity. The inhibition by indomethacin was of the noncompetitive type with an apparent Km of 0.17 X 10(-6) mol/l and apparent Ki of 3.6 X 10(-6) mol/l. From these data we conclude that indomethacin is capable of inhibiting phospholipase C activity in neutrophils at clinically significant levels and that this may be relevant in the therapeutic action of this drug.     

Effects of alveolar pressure on lung angiotensin-converting enzyme function in vivo

Angiotensin-converting enzyme lines the luminal surface of pulmonary capillary endothelial cells. The metabolism of its synthetic substrate, 3H-benzoyl-L-phenylalanyl-L-alanyl-L-proline ([3H]BPAP) has been used as an indicator of pulmonary microvascular function. Because the flow-volume status of the pulmonary capillaries is dependent on intra-alveolar pressure, we have studied the effects of airway pressure on endothelial plasmalemmal angiotensin-converting enzyme function in rabbit lungs in vivo. Static inflation of the lungs to a pressure of 0 or 5 Torr did not change percent transpulmonary metabolism and Amax/Km ratio (defined as E X Kcat/Km and thus, under normal conditions, an indirect measure of perfused endothelial luminal surface area) compared with control measurements during conventional mechanical ventilation. When the inflation pressure was increased to 10 Torr, percent metabolism of [3H]BPAP remained unaltered but Amax/Km decreased to 60% of the control value. This decrease was in close relation to the decrease in pulmonary blood flow. Addition of 5 cmH2O positive end-expiratory pressure (PEEP) to the mechanical ventilation also decreased Amax/Km values and pulmonary blood flow but did not influence percent metabolism of [3H]BPAP. These results suggest that the detected alterations in apparent enzyme kinetics were more likely due to hemodynamic changes than to alterations in angiotensin-converting enzyme function. Thus high static alveolar pressures as well as PEEP probably reduced the fraction of perfused microvessels as reflected in changes in Amax/Km ratios. This information should prove useful in interpreting the response of pulmonary endothelial enzymes to injury.     

Testosterone 5 alpha-reductase activity in neural tissue of fetal rhesus macaques

After development of a 5 alpha-reductase activity (5 alpha-RA) assay based on the capacity of microsomes to convert [3H]testosterone (T) to [3H]dihydrotestosterone (DHT), we analyzed 5 alpha-RA in neural tissues of fetal rhesus macaques at 50, 80 and 150 days of gestation. This method allowed us to collect kinetic data on the properties of the 5 alpha-reductase resident in fetal brain at 150 days of gestation. The Km and Vmax calculated from these data were 4.32 microM and 22.6 nmol.mg protein-1.h-1, respectively. Analyses of 5 alpha-RA in microsomes from the hypothalamic-preoptic area-amygdala (HPA) at dilutions of 1/25 and 1/50 indicated higher enzyme activity with increasing dilution of the microsomes. Measurement of 5 alpha-RA using concentrations of [3H]T which saturated the enzyme in diencephalon (DIEN), brain stem (B.STEM), temporal (TCTX) and frontal cortex (FCTX) of six 50-day old fetuses (3 males and 3 females) revealed no obvious sex differences in 5 alpha-RA, however, a significant difference (P less than 0.05) between tissues was noted. The DIEN and B.STEM contained significantly (P less than 0.05) higher levels 5 alpha-RA than the FCTX while the TCTX contained an intermediate level of activity. Significant increases in 5 alpha-RA were observed in FCTX and TCTX with time of gestation (50, 80 and 150 days). Other tissues, including amygdala, hippocampus, cerebellum, tegmentum and septum also change with fetal age. These data demonstrate the existence of 5 alpha-reductase in the fetal monkey brain. Significant changes in cortical 5 alpha-RA suggest some role for 5 alpha-reductase in development.     

Rat Brain Adenosine Deaminase After 2''-Deoxycoformycin Administration: Biochemical Properties and Evidence for Reduced Enzyme Levels Detected by 2''-[3H]Deoxycoformycin Ligand Binding

Near total inhibition of brain adenosine deaminase (ADA) activity in rats injected with the potent ADA inhibitor 2'-deoxycoformycin (DCF) was previously shown to reduce enzyme activity for up to 50 days during which time the enzyme exhibited reduced sensitivity to in vivo inhibition by DCF. Here, we investigated the biochemical properties of ADA and the basis for its reduced activity after DCF treatment. It was found that much higher doses of DCF were required to inhibit ADA in DCF-treated compared with drug-naive animals. Fourteen days after DCF administration, reduced ADA activity in brain homogenates was due to a decrease in Vmax, rather than to an altered Km of ADA for adenosine. DCF treatment had no effect on Ki values for erythro-9-(2-hydroxy-3-nonyl)adenine inhibition of ADA. The IC50 value for DCF inhibition of ADA in hypothalamus was unchanged. However, the Ki for DCF inhibition of ADA in whole brain increased by fivefold. Sucrose gradient analysis of brain ADA revealed only one corresponding peak of activity and [3H]DCF-labeled ADA in DCF-treated and control rats. A radioligand filtration assay with [3H]DCF was developed to assess the effects of DCF on ADA protein levels. Over a roughly 200-fold range of ADA activities the binding of [3H]DCF was highly correlated with deaminase activity (r = 0.99). In brain tissues taken 8 and 33 days after treatment of rats with DCF, [3H]DCF binding was reduced to 27% and 48% of control levels, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sphingolipid Long-Chain Base Synthesis in Plants (Characterization of Serine Palmitoyltransferase Activity in Squash Fruit Microsomes)

The activity of serine palmitoyltransferase (palmitoyl-coenzyme A [CoA]:L-serine [Ser]-C-palmitoyltransferase [decarboxylating], EC 2.3.1.50), the enzyme catalyzing the first step in the synthesis of the long-chain base required for sphingolipid assembly, has been characterized in a plant system. Enzyme activity in a microsomal membrane fraction from summer squash fruit (Cucurbita pepo L. cv Early Prolific Straightneck) was assayed by monitoring the incorporation of L-[3H]Ser into the chloroform-soluble product, 3-ketosphinganine. Addition of NADPH to the assay system resulted in the conversion of 3-ketosphinganine to sphinganine. The apparent Km for Ser was approximately 1.8 mM. The enzyme exhibited a strong preference for palmitoyl-CoA, with optimal activity at a substrate concentration of 200 [mu]M. Pyridoxal 5[prime]-phosphate was required as a coenzyme. The pH optimum was 7.6, and the temperature optimum was 36 to 40[deg]C. Enzyme activity was greatest in the microsomal fraction obtained by differential centrifugation and was localized to the endoplasmic reticulum using marker enzymes. Two known mechanism-based inhibitors of the mammalian enzyme, L-cycloserine and [beta]-chloro-L-alanine, were effective inhibitors of enzyme activity in squash microsomes. Changes in enzyme activity with size (age) of squash fruit were observed. The results from this study suggest that the properties and catalytic mechanism of Ser palmitoyltransferase from squash are similar to those of the animal, fungal, and bacterial enzyme in most respects. The specific activity of the enzyme in squash microsomes ranged from 0.57 to 0.84 nmol min-1 mg-1 of protein, values 2- to 20-fold higher than those previously reported for preparations from animal tissues.     

Properties of a Leu-Phe-Cleaving Endopeptidase Activity Putatively Involved in β-Endorphin Metabolism in Rat Brain

Incubation of beta-endorphin with cytosolic and particulate fractions of rat brain resulted in the formation of several peptides, including gamma-endorphin [beta-endorphin-(1-17)] and beta-endorphin-(18-31), indicating the presence of enzyme activity cleaving the Leu17-Phe18 bond of beta-endorphin. An assay for this Leu-Phe cleaving activity, based on the cleavage of the 14C-labeled substrate acetyl-Val-Thr-Leu-Phe-[epsilon-([14C]CH3)2]Lys-NHCH3, was used to examine the properties of this enzyme activity. beta-Endorphin-(1-31) competitively inhibited the Leu-Phe-cleaving enzyme activity on the pentapeptide substrate. Over 90% of activity was recovered in the cytosolic fraction. Leu-Phe-cleaving activity behaved like a thiol endopeptidase because it was inhibited by low concentrations of N-ethylmaleimide, p-chloromercuribenzoate, p-chloromercuribenzoyl sulfate, and low concentrations of Hg2+. Low concentrations of sulfhydryl compounds stimulated Leu-Phe-cleaving activity. The activity was optimal between pH 8.5 and 9.0. The Km of Leu-Phe-cleaving activity in the cytosolic fraction was 35 microM and in the particulate fraction 88 microM with Vmax values of 193 and 15 nmol mg protein-1 h-1, respectively. The apparent molecular mass of the Leu-Phe-cleaving enzyme was estimated by gel filtration to be approximately 200 kilodaltons. These properties of Leu-Phe-cleaving activity indicate that the Leu-Phe-cleaving enzyme is distinct from any known brain endopeptidase.     

Characterization of the forward and reverse reactions catalyzed by CDP-diacylglycerol:inositol transferase in rabbit lung tissue.

CDPdiacylglycerol:inositol transferase activity in rabbit lung tissue has been characterized and the optimum conditions for assaying this enzyme in vitro were determined. Rabbit lung tissue CDPdiacylglycerol:inositol transferase activity was found primarily in the microsomal fraction. The pH optimum of the enzyme activity was between 8.8 and 9.4, and the reaction was dependent on either Mn2+ or Mg2+. Detergents and Ca2+ inhibited the activity of the enzyme. The apparent Km values of the enzyme for CDPdioleoylglycerol and myoinositol were 0.18 mM and 0.10 mM, respectively. The reversibility of the reaction catalyzed by CDPdiacylglycerol:inositol transferase in microsomes prepared from rabbit lung tissue was demonstrated by the synthesis of [3H]CMPdiacylglycerol when [3H]CMP and phosphatidylinositol were present in the incubation mixture. The reverse reaction was characterized and its importance in the regulation of the acidic phospholipid composition of surfactant during lung development is discussed. The pH optimum for the reverse reaction was 6.2, and the reverse reaction was also dependent on Mn2+ or Mg2+. The apparent Km value of CDPdiacylglycerol:inositol transferase for CMP was found to be 2.8 mM.     

Human placental glutathione transferase: interactions with steroids

Glutathione transferases exhibit both isomerase and transferase activity. The acceptance of steroids as substrates for or inhibitors of these activities was studied using a 350-fold enriched preparation of the enzyme from human placenta. As an isomerase, the enzyme preparation catalyzed the conversion of pregn-5-ene-3,20-dione (Km 0.03 mmol/l) and androst-5-ene-3,17-dione (Km 0.05 mmol/l) to the respective 4-ene-3-oxosteroids (specific activity 0.8 U/mg protein). This isomerase activity strictly depended on the presence of glutathione (Km 0.04 mmol/l). As a transferase, the enzyme preparation catalyzed the conjugation of glutathione (Km 0.5 mmol/l) with 1-chloro-2,4-dinitrobenzene (Km 1.0 mmol/l) (specific activity 100 U/mg protein). This transferase activity was inhibited by all phenolic (KI values 0.2-1.5 mmol/l) and some of the neutral steroids (KI values 1.4-3.5 mmol/l) tested. Phenolic steroids inhibited the enzyme activity competitively to 1-chloro-2,4-dinitrobenzene and non-competitively to both substrates. The results indicate that steroids can interact with the placental glutathione transferase in vitro both as substrates and as inhibitors. Since, however, the observed Km and KI values of the steroids are far above the values of their concentrations in the placenta, these interactions are of only minor physiological relevance.     

Age-associated changes in fat metabolism in the rat and its relation to sympathetic activity

In this study, to determine if age associated changes in fat metabolism in skeletal muscle and liver were related with sympathetic activity, we measured sympathetic activity and palmitate oxidation rate, carnitine palmitoyltransferase-1 (CPT-1) activity, and triglyceride concentration in skeletal muscle and liver of rats at 8, 30 and 60 weeks of age. Body weight, intra-abdominal percent of fat mass, and plasma level of insulin, leptin, and triglyceride were all significantly increased with age. Tissue triglyceride concentration was increased with age in liver and skeletal muscle. The palmitate oxidation rate in liver and skeletal muscle was reduced with age in rats and inversely correlated with tissue triglyceride concentration. CPT-1 activity was not altered with age. Plasma catecholamine concentration and sympathetic activity, as measured by spectral analysis of heart rate variability, were increased with age. Plasma norepinephrine or epinephrine and tissue triglyceride had a positive correlation in liver and skeletal muscle. Plasma norepinephrine or epinephrine to tissue triglyceride ratio was similar according to age. In summary, in spite of increased sympathetic activity with age, the tissue triglyceride concentration was increased. Increased sympathetic activity may be the compensatory response and the reduced capacity of fatty acid oxidation is a main cause of obesity.     

Inhibition of the methylcoenzyme M methylreductase system by NAD+ and NADP+ in cell-extracts of Methanosarcina barkeri

In cell extracts of Methanosarcina barkeri, the methylcoenzyme M methylreductase system with H2 as the electron donor was inhibited by NAD+ and NADP+, but NADH and NADPH had no effect on enzyme activity. NAD+ (4 and 8 mM) shifted the saturation curve for methylcoenzyme M from hyperbolic (Hill coefficient [nH] = 1.0; concentration of substrate giving half maximal velocity [Km] = 0.21 mM) to sigmoidal (nH = 1.5 and 2.0), increased Km (Km = 0.25 and 0.34 mM), and slightly decreased Vmax. Similarly NADP+ at 4m and 8 mM increased nH to 1.6 and 1.85 respectively, but the Km values (0.3 and 0.56 mM) indicated that NADP+ was a more efficient inhibitor than NAD+.     

Sialidase in Cerebellar Granule Cells Differentiating in Culture

The optimal conditions for the assay of sialidase in cerebellar granule cells cultivated in vitro, established using [3H]GD1a and 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (MUB-NeuNAc) as substrates, were the following: pH optimum for both substrates, 3.9; optimal molarity of sodium acetate/acetic acid buffer, 0.05 M with [3H]GD1a and 0.1 M for MUB-NeuNAc; substrate concentration for apparent maximal activity, 0.5 mM for MUB-NeuNAc and 0.1 mM for [3H]GD1a; enzyme activity linear with time up to 30 min with MUB-NeuNAc and up to 90 min with [3H]GD1a; and enzyme activity linear with enzyme protein content up to 80 micrograms with MUB-NeuNAc and up to 20 micrograms with [3H]GD1a. The assay with [3H]GD1a required the presence of Triton X-100 in a molar ratio to GD1a of 15:1. Poly-L-lysine, which was used for plating the cells, was capable of decreasing sialidase activity against [3H]GD1a/Triton X-100 when added to the incubation mixture. However, it had no effect on the enzyme working on MUB-NeuNAc. Using no more than 20 micrograms of cellular protein, the contamination, if any, by poly-L-lysine released from the dish was below the concentration limit exhibiting inhibition. Using the above optimal conditions, sialidase activity was measured during cerebellar granule cell differentiation in culture. From day 0 to day 7-8 in culture, the enzyme activity rose from 20 to 130 nmol of product released/h/mg of protein with MUB-NeuNAc and from 1 to 100 nmol of product released/h/mg of protein with [3H]GD1a.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective inhibition of acyl coenzyme A:cholesterol acyltransferase by compound 58-035

Compound 58-035 (3-[decyldimethylsilyl]-N-[2-(4-methylphenyl)-1-phenylethyl]pro panamide) has been found to inhibit the accumulation of cholesteryl esters in both rat hepatoma (Fu5AH) cells and arterial smooth muscle cells in culture. To explore the specificity of 58-035, we have studied the esterification of cholesterol, retinol, and glycerides by the Fu5AH cell and by isolated membranes. Exposure of Fu5AH to cholesterol/phospholipid dispersions and 58-035 (greater than 100 ng/ml) for 24 h resulted in greater than 95% inhibition of cholesterol esterification while cellular free cholesterol increased slightly. Inhibition was also rapid; incorporation of [3H]oleate into cholesteryl [3H]oleate equaled only 12% of control value after 30 min with 58-035 at 5 micrograms/ml. In contrast, there was no decrease in [3H]oleate incorporation into phospholipids or diglycerides, nor was the esterification of [3H]retinol inhibited by 58-035. In microsomal fractions, acyl-CoA:cholesterol acyltransferase could be inhibited completely by 58-035, while activities of acyl-CoA: retinol acyltransferase and triglyceride synthesis proceeded at 75-100% of control values. These observations that 58-035 is highly selective allow the inference that acyl-CoA:cholesterol acyltransferase is a separate microsomal enzyme whose activity can be modulated independently from acyl-CoA:retinol acyltransferase and other cellular acyltransferases.     

Measurement of Rat Brain Kynurenine Aminotransferase at Physiological Kynurenine Concentrations

The production of the neuroinhibitory and neuroprotective metabolite kynurenic acid (KYNA) was investigated in rat brain by examining its biosynthetic enzyme, kynurenine aminotransferase (KAT). By using physiological (low micromolar) concentrations of the substrate L-kynurenine (KYN) and by determining the irreversible conversion of [3H]KYN to [3H]KYNA as a measure of KAT activity, a novel, simple, and sensitive assay was developed which permitted the detailed characterization of the enzyme. Only a single protein, which under routine assay conditions showed approximately equal activity with 2-oxoglutarate and pyruvate as the aminoacceptor, was found in rat brain. The enzyme was distributed heterogeneously between the nine brain regions studied, with the KAT-rich olfactory bulb displaying approximately five times higher activity than the cerebellum, the area with lowest KAT activity. In subcellular fractionation studies, the majority of KAT was recovered in mitochondria. In contrast to many known aminotransferases, partially purified KAT was shown to be highly substrate-specific. Thus, of the amino acids tested, only alpha-aminoadipate and tryptophan displayed moderate competition with KYN. Notably, 3-hydroxykynurenine, reportedly a very good substrate of KAT, competed rather poorly with KYN as well. Aminooxyacetic acid, a nonspecific transaminase inhibitor, blocked KAT activity with an apparent Ki of 5 microM. Kinetic analyses with partially purified rat brain KAT revealed a Km of 17 microM for KYN with 1 mM 2-oxoglutarate, but a much higher Km (910 microM) with 1 mM pyruvate. Km values for 2-oxoglutarate and pyruvate were 150 and 160 microM, respectively. The cellular localization of KAT was examined in striatal homogenates obtained from rats 7 days after an intrastriatal injection of quinolinate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunocytochemical studies of aromatase in early and full-term human placental tissues: comparison with biochemical assays

An immunocytochemical method for visualizing the aromatase P450 enzyme with a specific monoclonal antibody has been developed for use with unfixed, frozen tissue sections. We compared both monoclonal and polyclonal aromatase-specific antibodies and found that placental aromatase was consistently and exclusively located in the syncytiotrophoblast layer of chorionic villi. The monoclonal antibody had the highest affinity, with negligible associated background stain. Fixation was found to impair stain reaction. Examination of first trimester and term placentae revealed identical immunostaining patterns of similar intensity in 9 of 10 samples. The immunostain reactions of first trimester and term placentae were compared with their respective microsomal aromatase activity, determined simultaneously by both indirect radiometric tritiated water (3H2O) assay, and direct product isolation by HPLC, using [1, 2, 6, 7(-3)H] androstenedione as substrate. The two assays were found to be comparable for enzyme activity estimates of term placental specimens. However, when first trimester specimens were analyzed, the direct-product measurements were significantly larger than the corresponding 3H2O assay results. Nonetheless, biochemical aromatase activity was found to correlate positively with immunostain reaction. Although 17 beta-hydroxysteroid dehydrogenase activity was not directly measured, differences in the estradiol:estrone product ratio (2.49 +/- 0.68 first trimester vs. 0.89 +/- 0.15 term) suggest differential control of this enzyme at the two stages of pregnancy. One first trimester specimen with an atypical, patchy immunostain distribution also had extremely low aromatase activity. The results indicate that both antibodies recognize functional aromatase enzyme and suggest that immunocytochemical detection is a sensitive, qualitative technique for investigating this important steroidogenic enzyme.