Browsing by Author "BRONFMAN, M"

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    ACTIVATION OF HYPOLIPEMIC DRUGS TO ACYL-COENZYME-A THIOESTERS
    (1986) BRONFMAN, M; AMIGO, L; MORALES, MN
    Compounds possessing the characteristics of CoA thioesters of the hypolipidaemic peroxisome proliferators clofibric acid, nafenopin and ciprofibrate were formed on incubation of the drugs with rat liver microsomal fractions, ATP and CoA. The reactivity of the drugs correlated with their pharmacological potency. It is proposed that the active species of these compounds are their acyl-CoA thioesters.
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    ACYL-COA SYNTHETASE AND THE PEROXISOMAL ENZYMES OF BETA-OXIDATION IN HUMAN-LIVER - QUANTITATIVE-ANALYSIS OF THEIR SUBCELLULAR-LOCALIZATION
    (1984) BRONFMAN, M; INESTROSA, NC; NERVI, FO; LEIGHTON, F
    The presence of acyl CoA synthetase (EC 6.2.1.3) in peroxisomes and the subcellular distribution of .beta.-oxidation enzymes in human liver were investigated by using a single-step fractionation method of whole liver homogenates in metrizamide continuous density gradients and a novel procedure of computer analysis of results. Peroxisomes contain 16% of the liver palmitoyl CoA synthetase activity, and 21% and 60% of the enzyme activity was localized in mitochondria and microsomal fractions, respectively. Fatty acyl CoA oxidase was localized exclusively in peroxisomes, confirming previous results. Human liver peroxisomes contribute 13%, 17% and 11% of the liver activities of crotonase, .beta.-hydroxyacyl CoA dehdyrogenase and thiolase, respectively. The absolute activities found in peroxisomes for the enzymes investigated suggest that in human liver fatty acyl CoA oxidase is the rate-limiting enzyme of the peroxisomal .beta.-oxidation pathway, when palmitic acid is the substrate.
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    CARNITINE ACYLTRANSFERASE AND ACYL-COENZYME-A HYDROLASE ACTIVITIES IN HUMAN-LIVER - QUANTITATIVE-ANALYSIS OF THEIR SUBCELLULAR-LOCALIZATION
    (1984) BRONFMAN, M; LEIGHTON, F
    The subcellular localizations of carnitine acyltransferase and acyl CoA hydrolase activities with different chain-length substrates were quantitatively evaluated in human liver by fractionation of total homogenates in metrizamide density gradients and by differential centrifugation. Peroxisomes contain 8-37% of the liver acyltransferase activity, the relative amount depending on the chain length of the substrate. The remaining activity was ascribed to mitochondria, except for carnitine octanoyltransferase, for which 25% of the activity was present in microsomal fractions. In contrast with rat liver, where the activity in peroxisomes is very low or absent, human liver peroxisomes contain about 20% of the carnitine palmitoyltransferase. Short-chain acyl CoA hydrolase activity was localized mainly in the mitochondrial and soluble compartments; the long-chain activity was present in both microsomal fractions and the soluble compartment. Particle-bound acyl CoA hydrolase activity for medium-chain substrates exhibited an intermediate distribution, in mictochondria and microsomal fractions, with 30-40% of the activity in the soluble fraction. No acyl CoA hydrolase activity appears to be present in human liver peroxisomes.
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    CIPROFIBRATE, A CARCINOGENIC PEROXISOME PROLIFERATOR, INCREASES THE PHOSPHORYLATION OF EPIDERMAL-GROWTH-FACTOR RECEPTOR IN ISOLATED RAT HEPATOCYTES
    (1993) ORELLANA, A; HOLUIGUE, L; HIDALGO, PC; FAUNDEZ, V; GONZALEZ, A; BRONFMAN, M
    Ciprofibrate, a hypolipidaemic drug with carcinogenic and peroxisome-proliferation effects in rat liver, was found to increase the phosphorylation of epidermal-growth-factor receptor in P-32-labeled isolated rat hepatocytes. This effect was suppressed by protein-kinase-C inhibitors, and was accompanied by an almost complete inhibition of the receptor autophosphorylation normally induced by its ligand. However, in vitro experiments showed that protein-kinase-C phosphorylation of purified epidermal-growth-factor receptor was activated by ciprofibroyl-CoA, the acyl-CoA derivative of the drug, but not by the unmodified drug. Neither compound affected the ligand induction of epidermal-growth-factor-receptor autophosphorylation in isolated liver membranes. These results suggest that metabolically produced ciprofibroyl-CoA in liver cells would activate protein-kinase-C and produce changes in epidermal-growth-factor-receptor function.
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    FATTY-ACID OXIDATION BY HUMAN-LIVER PEROXISOMES
    (1979) BRONFMAN, M; INESTROSA, NC; LEIGHTON, F
    A cyanide insensitive fatty acid oxidation system was detected in human liver by subcellular fractionation in Metrizamide continuous density gradients and was localized in peroxisomes. Fatty acyl-CoA oxidase, its characteristic enzyme, acts maximally on C12-C18 saturated fatty acids and oleoyl-CoA and requires FAD. These results, and the already established properties of the system in rat liver support its potential contribution to lipid metabolism and to the hypolipidemic effect of Clofibrate and related drugs in humans.
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    HYPOLIPEMIC DRUGS ARE ACTIVATED TO ACYL-COA ESTERS IN ISOLATED RAT HEPATOCYTES - DETECTION OF DRUG ACTIVATION BY HUMAN LIVER HOMOGENATES AND BY HUMAN PLATELETS
    (1992) BRONFMAN, M; MORALES, MN; AMIGO, L; ORELLANA, A; NUNEZ, L; CARDENAS, L; HIDALGO, PC
    The formation of acyl-CoA esters of the hypolipidaemic peroxisome proliferators clofibric acid, ciprofibrate and nafenopin was studied in isolated rat hepatocytes. The concentration of ciprofibroyl-CoA in the liver of ciprofibrate-treated rats was in the range of 10-30-mu-M. The three drugs formed acyl-CoA esters when incubated with isolated hepatocytes. Their formation was saturable and reached a plateau after 30 min incubation. Maximal intracellular concentrations of ciprofibroyl-CoA and clofibroyl-CoA (100-mu-M and 55-mu-M respectively) were attained at 0.5 mM of the free drugs in the incubation medium, whereas for nafenopin-CoA, the maximal intracellular concentration (9-mu-M) was reached at 1 mM-nafenopin. At low concentrations of the hypolipidaemic compounds in the incubation medium a significant proportion of the total intracellular drug was present as its acyl-CoA ester (25-35% for ciprofibrate). When isolated hepatocytes were incubated with a ciprofibrate concentration comparable with that observed in the blood of drug-treated rats (0.1 mM), ciprofibroyl-CoA attained an intracellular concentration similar to that previously observed in the liver of treated rats. The formation of ciprofibroyl-CoA by isolated rat hepatocytes was stimulated by the addition of carnitine and partially inhibited by the addition of palmitate. Further, it was shown that human liver homogenates synthesized ciprofibroyl-CoA at a rate similar to that observed for rat liver homogenates. Solubilized human platelets also formed ciprofibroyl-CoA, although at a rate two orders of magnitude lower than that of liver. The results support the view that acyl-CoA esters of hypolipidaemic peroxisome proliferators may be the pharmacologically active species of the drugs.
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    INDUCTION OF PEROXISOMAL FATTY ACYL-COENZYME A-OXIDASE AND TOTAL CARNITINE ACETYL-COENZYME A-TRANSFERASE IN PRIMARY CULTURES OF RAT HEPATOCYTES BY GARLIC EXTRACTS
    (1992) ORELLANA, A; KAWADA, ME; MORALES, MN; VARGAS, L; BRONFMAN, M
    Garlic has been proposed as a natural hypolipidemic substance. Most hypolipidemic compounds induce peroxisomal proliferation and increase enzyme activities associated with peroxisomal beta-oxidation in rat liver. Here we report that garlic methanol-extracts behave as hypolipidemic drugs, increasing the activity of peroxisomal fatty acyl-coenzyme A oxidase and of total carnitine acetyl-coenzyme A transferase in primary cultures of rat hepatocytes. Both enzymes are considered markers associated with increased peroxisomal beta-oxidation. As in the case of hypolipidemic peroxisome proliferators, garlic extracts partially prevented the decrease in fatty acyl-coenzyme A oxidase as the culture aged. No changes were observed in the activity of microsomal NADPH cytochrome c reductase or of mitochondrial glutamate dehydrogenase.
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    MULTITEST-II - A PROGRAM FOR THE GENERATION, CORRECTION, AND ANALYSIS OF MULTIPLE-CHOICE TESTS
    (1990) LIRA, P; BRONFMAN, M; EYZAGUIRRE, J
    Multitest II is a powerful program for the generation, correction, and analysis of multiple choice examinations. The program is written in Pascal and has been implemented for the IBM-PC and compatible microcomputers, and for the VAX minicomputer. The program allows the random generation of tests from a master test, and supports several types of multiple choice tests. The tests are graded by means of a user-definable numerical grading system. Certain statistical analyses can also be performed on the tests results.
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    PALMITOYL-COA AND THE ACYL-COA THIOESTER OF THE CARCINOGENIC PEROXISOME-PROLIFERATOR CIPROFIBRATE POTENTIATE DIACYLGLYCEROL-ACTIVATED PROTEIN KINASE-C BY DECREASING THE PHOSPHATIDYLSERINE REQUIREMENT OF THE ENZYME
    (1990) ORELLANA, A; HIDALGO, PC; MORALES, MN; MEZZANO, D; BRONFMAN, M
    To gain insight into the mechanism by which long-chain acyl-CoA thioesters potentiate diacylglycerol-activated protein kinase C, the cofactor dependence of this activating effect was studied with purified rat brain enzyme and histone H1 as substrate. Using two different assay systems, palmitoyl-CoA was found to decrease greatly the amount of phosphatidylserine required to activate the kinase. No relative changes were observed in the dependence of the enzyme for other cofactors (diacylglycerol, ATP, and Ca2+) in the presence of palmitoyl-CoA. The potentiating effect of palmitoyl-CoA and the decrease in phosphatidylserine requirement of the kinase was also demonstrated using the 47-kDa protein of human platelets as substrate and platelet protein kinase C as source of enzyme. The acyl-CoA thioester of the carcinogenic peroxisome-proliferator ciprofibrate was also found to decrease the phosphatidylserine requirement of protein kinase C. The data suggest that acyl-CoAs may play a role in the regulation of protein kinase C activity.
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    POTENTIATION OF DIACYLGLYCEROL-ACTIVATED PROTEIN KINASE-C BY ACYL-COENZYME A THIOESTERS OF HYPOLIPEMIC DRUGS
    (1989) BRONFMAN, M; ORELLANA, A; MORALES, MN; BIERI, F; WAECHTER, F; STAUBLI, W; BENTLEY, P
    Acyl-Coenzyme A thioesters of the hypolipidaemic and cancerinogenic peroxisome proliferators clofibric acid, nafenopin, ciprofibrate, benzafibrate and tibric acid were found to greatly increase the activity of rat brain protein kinase C. Maximal activation required the simultaneous presence of Ca2+, phosphatidylserine and diolein, thus differentiating their action from that of other tumor promoters such as phorbol esters. Under similar conditions the unesterified drugs were comparatively ineffective. Similar results were obtained using the rat liver enzyme. The data suggest that acyl-coenzyme A thioesters of hypolipidaemic drugs, may play a role in the induction of liver tumors by these compounds, through the potentiation of protein kinase C.
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    PURIFICATION OF THE PEROXISOMAL FATTY ACYL-COA OXIDASE FROM RAT-LIVER
    (1980) INESTROSA, NC; BRONFMAN, M; LEIGHTON, F
    Fatty acyl-CoA oxidase, the rate limiting enzyme of the peroxisomal fatty acid oxidizing system, was purified from rat liver to near homogeneity by a procedure involving affinity chromatography of its apoenzyme on FAD-Sepharose. The oxidase presents an absolute requirement for the dinucleotide which is weakly bound to the apoenzyme (KD, 0.6 .mu.M). The highest specific activity obtained was 27 units/mg protein. The purified enzyme was 2 major polypeptides with apparent MW of 45,000 and 22,000. The enzyme is a flavoprotein with non-covalently bound flavin adenin dinucleotide composed of 4 subunits, 2 of 45,000 MW and 2 of 22,000 MW.
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    QUANTITATIVE SUBCELLULAR STUDY OF APICAL POLE MEMBRANES FROM CHICKEN OXYNTIC CELLS IN RESTING AND HCL SECRETORY STATE
    (1987) KOENIG, CS; DABIKE, M; BRONFMAN, M
    Vertebrate oxyntic cells, responsible for gastric HCl production, undergo a remarkable morphological reorganization in relation to their secretory cycle. In resting state, the luminal surface of the cells is smooth; a peculiar system of endocellular membranes, the tubular system, occupies the luminal cytoplasm. Actin filaments frame a cortical network between the tubular system and the luminal plasma membrane. With the onset of HCl secretion, the tubular system becomes incorporated into the luminal plasma membrane. Villous processes containing microfilaments fill the secretory surface. This morphological reorganization of membranes and cytoskeletal matrix could regulate HCl secretion by translocation of membranes containing the proton pump from the endocellular compartment to the secretory surface. In this paper, we describe the isolation of membranes that selectively belong to the tubular system or to the cytoplasmic processes of the secretory surface of chicken oxyntic cells. Chicken oxyntic cells are the main cellular component of the proventricular glands. A resting state was obtained after cimetidine treatment, whereas the HCl-secretory state was induced by histamine. We present a comparative analysis of resting and stimulated chicken gastric glands by quantitative subcellular fractionation. The HCl secretory state was related to specific modifications in membrane fractions derived from the secretory pole of oxyntic cells. Morphological and functional reorganization of oxyntic cells was closely correlated with changes in: the sedimentation pattern of the marker enzyme of the apical pole membrane (K-NPPase), the total activity of K-NPPase and nonmitochondrial Mg-ATPase, the valinomycin dependence of K-ATPase, and polypeptides that cosediment in purified membrane fractions. Changes in the distribution pattern of K-NPPase after fractionation of histamine-stimulated glands were consistent with the replacement of the small vesicles typical of resting glands by dense membrane profiles, analogous to the luminal processes of stimulated oxyntic cells. SDS-PAGE showed that, in purified membrane fractions of stimulated glands, the concentration of 28-, 43-, and 200-kDa polypeptides increased while that of 95- and 250-kD polypeptides decreased. The present results define the tubular system of oxyntic cells as an organelle with properties different from those of endoplasmic reticulum, mitochondria, and plasma membrane. The biochemical and physicochemical properties of this membraneous system changed when the organization of the membranes and the cytoskeletal matrix of the apical pole was modified by the onset of HCl secretion.
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    REGULATION OF BILIARY CHOLESTEROL SECRETION IN THE RAT - ROLE OF HEPATIC CHOLESTEROL ESTERIFICATION
    (1984) NERVI, F; BRONFMAN, M; ALLALON, W; DEPIEREUX, E; DELPOZO, R
    Although the significance of the enterohepatic circulation of bile salts in the solubilization and biliary excretion of cholesterol is well established, little is known about the intrahepatic determinants of biliary cholesterol output. Studies were undertaken to elucidate some of these determinants in the rat. Feeding 1% diosgenin for 1 wk increased biliary cholesterol output and saturation by 400%. Bile flow, biliary bile salt, phospholipid and protein outputs remained in the normal range. When ethynyl estradiol (EE) was injected into these animals, biliary cholesterol output decreased to almost normal levels under circumstances of minor changes in the rates of biliary bile salt and phospholipid outputs. Similarly, when chylomicron cholesterol was i.v. injected into diosgenin-fed animals, biliary cholesterol output significantly decreased as a function of the dose of chylomicron cholesterol administered. Relative rates of hepatic cholesterol synthesis and esterification were measured in isolated hepatocytes. Although, hepatic cholesterogenesis increased 300% in diosgenin-fed animals, the contribution of newly synthesized cholesterol to total biliary cholesterol output was only 19 .+-. 9%, compared with 12 .+-. 6% in control and 15 .+-. 5% in diosgenin-fed and EE-injected rats. The rate of oleate incorporation into hepatocytic cholesterol esters was 30% inhibited in diosgenin-fed rats. When EE was injected into these animals, the rate of cholesterol esterification increased to almost 300%. To investigate further the interrelationship between hepatic cholesterol esterification and biliary cholesterol output, 21 diosgenin-fed rats were studied. Six of them received in addition EE and 10 received chylomicron cholesterol. The relationships between biliary cholesterol output as a function of both microsomal acyl-CoA:cholesterol acyltransferase (ACAT) activity and hepatic cholesterol ester concentration were significantly correlated in a reciprocal manner. The size of the biliary cholesterol precursor pool can be rapidly modified through changes in the activity of the hepatic ACAT. [Implications with respect to the role of high cholesterol secretion to gallstone disease were presented.].
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    SUB-CELLULAR DISTRIBUTION OF CHOLESTEROL ESTER HYDROLASE IN HUMAN-LIVER
    (1981) DELPOZO, R; BRONFMAN, M; BULL, P; NERVI, F
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    SUBCELLULAR-DISTRIBUTION AND CHARACTERISTICS OF CIPROFIBROYL-COA SYNTHETASE IN RAT-LIVER - ITS POSSIBLE IDENTITY WITH LONG-CHAIN ACYL-COA SYNTHETASE
    (1992) AMIGO, L; MCELROY, MC; MORALES, MN; BRONFMAN, M
    The subcellular distribution and characteristics of ciprofibroyl-CoA synthetase were studied in rat liver and compared with those of long-chain acyl-CoA synthetase (palmitate as substrate) which, as already known, is distributed among mitochondria, microsomes and peroxisomes. Upon differential centrifugation, the subcellular distribution of ciprofibroyl-CoA synthetase followed closely that of palmitoyl-CoA synthetase and was specifically inactivated in the mitochondrial fraction by freezing and thawing, a behaviour already described for palmitoyl-CoA synthetase. Both enzyme activities were found to co-purify through several steps from rat liver microsomes. By using a partially purified enzyme, the activation of ciprofibrate to its acyl-CoA ester followed Michaelis-Menten kinetics with an apparent K(m) of 0.63 +/- 0.1 mM. Ciprofibroyl-CoA synthetase was competitively inhibited by 25 and 50-mu-M-palmitic acid. Higher concentrations of the fatty acid resulted in a mixed type of inhibition. Conversely, ciprofibrate up to 0.5 mM was found to inhibit competitively palmitoyl-CoA synthetase, whereas higher concentrations also resulted in a mixed inhibition. The highest activity of ciprofibroyl-CoA synthetase was found in fat and liver homogenates. The distribution of the enzyme in different rat tissues was similar to that of palmitoyl-CoA synthetase. The present results suggest that long-chain acyl-CoA synthetase and ciprofibroyl-CoA synthetase activities reside in identical or closely related proteins.