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Biomedical subjects

G P Mannaerts

Publications and source records attributed to G P Mannaerts.

At least 127 records · Page 7Linked to original sources
PubMed1989

Deficient oxidation of trihydroxycoprostanic acid in liver homogenates from patients with peroxisomal diseases.

The activation of palmitate and trihydroxycoprostanic acid and the peroxisomal oxidation of palmitate, trihydroxycoprostanic acid and their CoA esters were measured in homogenates prepared from fresh liver tissue of patients undergoing hepatic surgery and from frozen postmortem liver specimens of controls, patients with Zellweger syndrome and a patient with pseudo-Zellweger syndrome, a deficiency of peroxisomal 3-oxoacyl-CoA thiolase. In contrast to the findings in control livers, peroxisomal beta-oxidation of palmitate and of palmitoyl-CoA was severely impaired, and oxidation of trihydroxycoprostanic acid and its CoA ester could not be detected in the livers of the patients affected by peroxisomal diseases. The finding in this paper, that the oxidation of trihydroxycoprostanoyl-CoA can be measured reliably in small amounts of human liver, will be of valuable help in the differential diagnosis and classification of peroxisomal disorders and will help to elucidate the exact nature of some of the defects present in these disorders.

Acetyl-CoA C-Acetyltransferase↗
PubMed1989

Activation and peroxisomal beta-oxidation of fatty acids and bile acid intermediates in liver from Bombina orientalis and from the rat.

1. Bombina orientalis excretes mainly C27 bile acids: trihydroxycoprostanic and varanic acids. More than 90% of the trihydroxycoprostanic acid (THCA) present in the bile, was conjugated with taurine; varanic acid was present in the unconjugated form. 2. Trihydroxycoprostanoyl-CoA (THC-CoA) synthetase activity, required for the formation of the taurine conjugate, was present in the liver of Bombina orientalis. 3. Peroxisomal beta-oxidation, which catalyzes the oxidation of fatty acids as well as the conversion of C27 bile acids into C24 bile acids in rat and human liver, could be detected in liver of Bombina orientalis when palmitoyl-CoA was used as substrate, but not when trihydroxycoprostanoyl-CoA (THC-CoA) was used.

Animals↗
PubMed1988

Inhibition of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid oxidation and of bile acid secretion in rat liver by fatty acids.

In isolated rat hepatocytes, fatty acids inhibited the side chain oxidation, but not the uptake, of exogenously added 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid (THCA). THCA did not inhibit fatty acid oxidation. In liver homogenates, fatty acids inhibited THCA activation to its CoA ester (THC-CoA) and THCA oxidation. THCA did not influence fatty acid activation or oxidation. Comparison of the THC-CoA concentrations present in the incubation mixtures during THCA oxidation, with substrate concentration curves determined for THC-CoA oxidation, indicated that the inhibition of THCA oxidation by fatty acids was at least partly exerted at the activation step. The inhibition of THCA activation by fatty acids was noncompetitive. Palmitoyl-CoA at concentrations found in the incubation mixtures during THCA oxidation in the presence of palmitate inhibited THC-CoA oxidation, but not sufficiently to fully explain the fatty acid-induced inhibition of THCA oxidation. The inhibition of THC-CoA oxidation by palmitoyl-CoA did not seem to be competitive. Acyl-CoA oxidase, the first enzyme of peroxisomal beta-oxidation (which catalyzes the side chain oxidation of THCA), was enhanced 15-fold in liver homogenates from clofibrate-treated rats when palmitoyl-CoA was the substrate, but the oxidase activity remained unaltered when THC-CoA was the substrate. In the perfused liver, oleate, infused after a wash-out period of 60 min, markedly inhibited bile acid secretion. The results 1) suggest that fatty acids inhibit THCA metabolism both at the activation step and at the peroxisomal beta-oxidation sequence and that separate enzymes may be involved in both the activation and peroxisomal beta-oxidation of fatty acids and THCA and 2) raise the question whether fatty acids might (indirectly?) affect overall bile acid synthesis via their inhibitory effect on THCA metabolism.

Animals↗
PubMed1988

Beta-oxidation of the carboxyl side chain of prostaglandin E2 in rat liver peroxisomes and mitochondria.

Rat liver and kidney homogenates, fortified with the appropriate cofactors, produced H2O2 when incubated with prostaglandin (PG) E2 or its CoA ester (PGE2-CoA), indicating that PGE2-CoA served as substrate for acyl-CoA oxidase, the first enzyme of peroxisomal beta-oxidation. PGE2-CoA oxidase was stimulated to the same extent as palmitoyl-CoA oxidase by treatment of rats with the peroxisome proliferator clofibrate. Subcellular fractionation confirmed that both oxidase activities were located in peroxisomes. When highly purified peroxisomes were incubated with [1-14C]PGE2, radioactive oxidation products were released, demonstrating that peroxisomes were capable of catalyzing the complete beta-oxidation sequence. However, PGE2 beta-oxidizing activity was expressed only when isolated microsomes were added to the purified peroxisomes, indicating that PGE2-CoA synthetase is located in the endoplasmic reticulum. Cofactor requirements for peroxisomal [1-14C]PGE2 and [1-14C]palmitate oxidation were identical, and oxidation was not inhibited by cyanide. [1-14C]PGE2 was also beta-oxidized by highly purified mitochondrial fractions, devoid of peroxisomes, when microsomes were added. Mitochondrial [1-14C]PGE2 and [1-14C]palmitate oxidation was CoA- and ATP-dependent and inhibited by cyanide. Palmitate oxidation was carnitine-dependent, but PGE2 oxidation was not. Acyl-CoA dehydrogenase activity, but not carnitine acyltransferase activity, was detected with PGE2-CoA as substrate. [1-14C]PGE2 oxidation in whole liver homogenates was only slightly inhibited by cyanide, indicating that peroxisomes oxidized the major portion of PGE2. The concentrations of PGE2 employed in these experiments exceeded the in vivo concentrations by several orders of magnitude. Therefore, we suggest that the urinary PG metabolite excretion be measured in patients with hereditary diseases in which peroxisomal or mitochondrial beta-oxidation is not functioning in order to clarify the role of each organelle in PG oxidation in vivo.

Animals↗
PubMed1988

Phospholipid composition and amphiphile content of isolated sarcolemma from normal and autolytic rat myocardium.

Sarcolemmal vesicles were purified to a similar extent, 50- to 60-fold on a protein basis, from normal rat hearts and hearts subjected to 30 or 60 min of autolysis at 37 degrees C (total ischemia in vitro). Electron microscopic examination of the autolytic hearts revealed sarcolemmal discontinuities and other morphological characteristics typical of irreversible cell injury. Total contents and percentage composition of phospholipid classes did not differ between normal and autolytic hearts or between sarcolemmal preparations from these hearts. There was no increase in lysophospholipid contents of whole hearts or of purified sarcolemma after autolysis. Long chain acyl-CoAs or acylcarnitines did not accumulate in autolytic hearts under our experimental conditions. The molar long chain acyl-CoA: phospholipid ratio in isolated sarcolemma was extremely low (1:100,000). It increased 3-fold after autolysis but the increase was most probably the result of an increase in mitochondrial contamination of the sarcolemmal preparations from autolytic hearts. The molar long chain acylcarnitine: phospholipid ratio of isolated sarcolemma was much larger (1:100), but it did not change after autolysis. Experiments, in which radioactive amphiphiles were incorporated in isolated sarcolemma that was subsequently repeatedly washed, indicated that the lysophospholipid and acylcarnitine contents of isolated sarcolemma reflect the contents of sarcolemma in situ, but that sarcolemmal acyl-CoA is used for re-acylation reactions during purification, explaining the low acyl-CoA content of isolated sarcolemma. Na/K-ATPase and Na/Ca-exchange activities were markedly depressed in isolated sarcolemma from autolytic hearts. Our results suggest that sarcolemmal phospholipid breakdown and sarcolemmal amphiphile accumulation are not responsible for the structural and functional defects of the sarcolemma after autolysis.

Acetylcarnitine↗
PubMed1987

Inhibition of peroxisomal fatty acyl-CoA oxidase by antimycin A.

Peroxisomal fatty acyl-CoA oxidase was inhibited by micromolar concentrations of antimycin A, an inhibitor of mitochondrial respiration. The inhibition was observed with all three substrates tested, i.e. palmitoyl-CoA, trihydroxycoprostanoyl-CoA and hexadecanedioyl-CoA. The peroxisomal D-amino acid oxidase was also inhibited by antimycin, but the peroxisomal L-alpha-hydroxyacid oxidase and uric acid oxidase and the mitochondrial monoamine oxidase were not. The degree of inhibition of acyl-CoA oxidase by antimycin was strongly dependent on the amount of cellular protein present in the assay mixture: at a fixed antimycin concentration, the inhibition was gradually lost with increasing protein concentrations. At a fixed cellular protein concentration in the assay mixtures, the mitochondrial oxidation of glutamate or palmitoylcarnitine was inhibited at antimycin concentrations that were much lower than those required for the inhibition of fatty acyl-CoA oxidase. Our results, nevertheless, demonstrate that antimycin A must be used with caution, when it is added to homogenates or subcellular fractions in order to distinguish between mitochondrial and peroxisomal fatty acid oxidation.

Acyl-CoA Oxidase↗
PubMed1987

Permeability of the peroxisomal membrane to cofactors of beta-oxidation. Evidence for the presence of a pore-forming protein.

Peroxisomes were purified from livers of clofibrate-treated rats. Permeability measurements on the isolated organelles revealed that peroxisomes are permeable to small solutes, including sucrose and the cofactors for fatty acid oxidation NAD+, CoA, ATP, and carnitine. The intraperoxisomal distribution volume was equal for all solutes. Peroxisomal solute uptake was rapid, not saturable and not visibly influenced by temperature. NAD+ and carnitine uptake in the solute accessible volume was not diminished by a variety of analogs and inhibitors. Subfractionation of peroxisomes and reconstitution of the subfractions into liposomes preloaded with solutes made the liposomes reconstituted with the integral membrane protein fraction, but not those reconstituted with the other subperoxisomal protein fractions, permeable to the same solutes that entered intact peroxisomes. Solute leakage from the preloaded liposomes was rapid and not visibly influenced by temperature. Leakage activity was destroyed by heat treatment of the integral membrane protein fraction and was not present in lipid extracts of the membrane. Separation of the integral membrane proteins on sucrose density gradients and reconstitution of the gradient fractions into liposomes indicated that the leakage activity was caused by a polypeptide of rather low molecular weight. The gradient distribution of leakage activity corresponded most closely to the presence of a 22- and a 28-kDa polypeptide. Our experiments indicate that the nonspecific permeability of the peroxisomal membrane to small solutes is based on the presence in the membrane of a nonselective pore-forming protein.

Adenosine Triphosphate↗
PubMed1987

Inorganic and organic phosphate measurements in the nanomolar range.

A procedure, based on the complex formation of malachite green with phosphomolybdate under acidic conditions, to measure inorganic orthophosphate in the nanomolar range is described. The addition of polyvinyl alcohol is required to stabilize the dye-phosphomolybdate complex. The advantages of the assay are simplicity, stability of the reagents, and high sensitivity. Due to the high permissible acidity in the assay (0.9 N H2SO4), the method can be adapted easily to measure nanomolar amounts of phosphate, liberated from organic compounds like phosphoproteins and phospholipids after wet digestion.

Microchemistry↗
PubMed1986

Coenzyme A in purified peroxisomes is not freely soluble in the matrix but firmly bound to a matrix protein.

On subfractionation of purified rat liver peroxisomes in matrical, peripheral membrane, integral membrane and core protein fractions, the endogenous peroxisomal CoA was released together with the matrix proteins. The released CoA could not be measured by an enzymatic cycling assay unless the matrix proteins were denatured by acid treatment or by heating at alkaline pH. The cofactor could not be removed by dialysis of the matrix proteins unless salt was added. It was not displaced by exogenous CoA. It migrated into sucrose density gradients together with a protein of approximately 80 kDa. The results indicate that peroxisomal CoA is firmly bound to a matrix protein and that the presence of CoA inside purified peroxisomes does not necessarily imply that the peroxisomal membrane is impermeable to this cofactor.

Animals↗
PubMed1985

Comparison of the activities of some peroxisomal and extraperoxisomal lipid-metabolizing enzymes in liver and extrahepatic tissues of the rat.

Peroxisomal (acyl-CoA oxidase and peroxisomal dihydroxyacetone-phosphate acyltransferase) and extraperoxisomal (mitochondrial fatty acid oxidation, extraperoxisomal dihydroxyacetone-phosphate acyltransferase, mitochondrial and microsomal glycerophosphate acyltransferases) lipid-metabolizing enzymes were measured in homogenates from rat liver and from seven extrahepatic tissues. Except for jejunal mucosa and kidney, extrahepatic tissues contained very little acyl-CoA oxidase activity. Peroxisomal dihydroxyacetone-phosphate acyltransferase, taken as the activity that was not inhibited by 5 mM-glycerol 3-phosphate, was present in all tissues examined, and its specific activity in liver and extrahepatic tissues was roughly of the same order of magnitude. Clofibrate treatment increased the activity of acyl-CoA oxidase in liver, and to a smaller extent also in kidney, but did not influence the activity of peroxisomal dihydroxyacetone-phosphate acyltransferase. Comparison of the activities of peroxisomal and extraperoxisomal lipid-metabolizing enzymes in extrahepatic tissues and in liver, an organ in which the contribution of peroxisomes to fatty acid oxidation and to glycerolipid synthesis has been estimated previously, suggests that, as in liver, peroxisomal long-chain fatty acid oxidation is of minor quantitative importance in extrahepatic tissues, but that in these tissues (micro)-peroxisomes are responsible for most of the dihydroxyacetone phosphate acylation and, consequently, for initiating ether glycerolipid synthesis.

Acyltransferases↗
PubMed1984

Rat liver dihydroxyacetone-phosphate acyltransferases and their contribution to glycerolipid synthesis.

Differential and isopycnic centrifugation of rat liver homogenates showed that, besides its established localization in peroxisomes and endoplasmic reticulum, dihydroxyacetone-phosphate acyltransferase is also present in mitochondria. The three activities differed in a number of properties (pH optimum, palmitoyl-CoA and dihydroxyacetone-phosphate dependence, and sensitivity toward N-ethylmaleimide) and are therefore likely associated with three distinct proteins. Glycerol 3-phosphate (5 mM) did not inhibit peroxisomal dihydroxyacetone-phosphate acyltransferase but inhibited the extraperoxisomal activities virtually completely. Peroxisomal dihydroxyacetone-phosphate acyltransferase was located at the inner aspect of the peroxisomal membrane, but the enzyme was not latent. Purified microsomes, from which intact peroxisomes had been removed, were still contaminated with peroxisomal membranes as deduced from the presence of two dihydroxyacetone-phosphate acyltransferase activities: a glycerol 3-phosphate-resistant activity with properties similar to those of peroxisomal dihydroxyacetone-phosphate acyltransferase and a glycerol 3-phosphate-sensitive "true" microsomal dihydroxyacetone-phosphate acyltransferase. We propose that, assayed in the presence of 5mM glycerol 3-phosphate, dihydroxyacetone-phosphate acyltransferase can be used as a marker enzyme for peroxisomal membranes. Such a marker enzyme has not hitherto been available. The differential effect of 5 mM glycerol 3-phosphate on peroxisomal and extraperoxisomal dihydroxyacetone-phosphate acyltransferases enabled us to determine the relative contribution of these activities to overall dihydroxyacetone-phosphate acylation in whole liver homogenates. At near-physiological pH and at near-physiological concentrations of unbound palmitoyl-CoA and of dihydroxyacetone-phosphate plus glycerol 3-phosphate, peroxisomes contributed 50-75%. The remaining percentage was mostly accounted for by the microsomal enzyme. At near-physiological concentrations of glycerol 3-phosphate plus dihydroxyacetone-phosphate, glycerolphosphate acyltransferase contributed 93% and dihydroxyacetone-phosphate acyltransferase 7% to overall glycerolipid synthesis in homogenates. This suggests that the dihydroxyacetone-phosphate pathway is of minor quantitative importance in overall hepatic glycerolipid synthesis but that its main function lies in the synthesis of ether lipids, which have acyldihydroxyacetone-phosphate as obligatory precursor.(ABSTRACT TRUNCATED AT 400 WORDS)

Acyltransferases↗
PubMed1984

Effects of benfluorex and fenofibrate treatment on mitochondrial and peroxisomal marker enzymes in rat liver.

Our results demonstrate that benfluorex at doses that are strongly hypotriglyceridemic does not increase hepatic peroxisomal enzyme activities, whereas fenofibrate at doses that are only slightly hypolipidemic induces a dramatic increase in the activity of these enzymes. Thus, the biochemical approach used in this study reveals that the hypolipidemic drug benfluorex does not belong to the class of hypolipidemic compounds known to induce hepatomegaly, hepatic peroxisome proliferation and hepatocarcinoma in rodents. Morphological studies should confirm the absence of peroxisomal induction.

Animals↗
PubMed1983

Water- and solute-accessible spaces of purified peroxisomes. Evidence that peroxisomes are permeable to NAD+.

Peroxisomes were purified from liver homogenates from rats, treated with the peroxisome proliferator clofibrate, by a combination of differential centrifugation and isopycnic centrifugation in iso-osmotic self-generating Percoll gradients. Structural integrity of the peroxisomes appeared to be preserved as evidenced by a high degree of catalase latency, the absence of catalase release during purification and the exclusion of inulin (mol.wt. +/- 5000). Spaces for water and solutes were measured after incubation of the peroxisomes in iso-osmotic sucrose with radioactive water or solutes and separation of the organelles from their media by centrifugation through an organic layer. Extraperoxisomal water was corrected for by the use of radioactive dextran or inulin. The sucrose, glucose, urea, methanol and acetate-accessible spaces were identical, suggesting that these spaces represent the volume in which molecules that can cross the membrane distribute. This volume equalled 50-65% of the water space. Urate and NAD+, a cofactor of peroxisomal beta-oxidation of fatty acids, also distributed in this volume, but were also partly bound. Urate and NAD+ binding was not abolished by sonication, which released the bulk of matrix catalase activity, but NAD+ binding was seriously diminished. The peroxisomal water and sucrose spaces were estimated to be 107 microliters and 55 microliters per g of liver tissue from a clofibrate-treated rat. From quantitative morphometric data [Anthony, Schmucker, Mooney & Jones (1978) J. Lipid Res. 19, 154-165] and our marker enzyme analyses, as well as from our experimentally determined water spaces of mitochondrial and microsomal fractions, it could be calculated that the volume contamination by lysosomes, mitochondria and microsomes did not exceed 1, 8 and 6% respectively. Our data indicate that apparently intact peroxisomes are permeable to a number of small molecules, including NAD+. Whether the NAD+-binding sites in sonicated peroxisomes mirror the likely existence of a membrane carrier requires further investigation.

Animals↗
PubMed1982

Evidence that peroxisomal acyl-CoA synthetase is located at the cytoplasmic side of the peroxisomal membrane.

1. Subfractionation by isopycnic density-gradient centrifugation in self-generating Percoll gradients of peroxisome-rich fractions prepared by differential centrifugation confirmed the presence of acyl-CoA synthetase in peroxisomes. Peroxisomes did not contain nicotinamide or adenine nucleotides other than CoA. 2. The gradient fractions most enriched in peroxisomes were pooled and the peroxisomes sedimented by centrifugation, resulting in a 50-fold-purified peroxisomal preparation as revealed by marker enzyme analysis. 3. Palmitate oxidation by intact purified peroxisomes was CoA-dependent, whereas palmitoyl-CoA oxidation was not, demonstrating that the peroxisomal CoA was available for the thiolase reaction, located in the peroxisomal matrix, but not for acyl-CoA synthetase. This suggests that the latter enzyme is located at the cytoplasmic side of the peroxisomal membrane. 4. Additional evidence for this location of peroxisomal acyl-CoA synthetase was as follows. Mechanical disruption of purified peroxisomes resulted in the release of catalase from the broken organelles, but not of acyl-CoA synthetase, indicating that the enzyme was membrane-bound. Acyl-CoA synthetase was not latent, despite the fact that at least one of its substrates appears to have a limited membrane permeability, as evidenced by the presence of CoA in purified peroxisomes. Finally, Pronase, a proteinase that does not penetrate the peroxisomal membrane, almost completely inactivated the acyl-CoA synthetase of intact peroxisomes.

Adenine Nucleotides↗
PubMed1982

Role of glycerol 3-phosphate and glycerophosphate acyltransferase in the nutritional control of hepatic triacylglycerol synthesis.

1. Glycerol 3-phosphate content of isolated hepatocytes from starved rats and of glycogen-depleted hepatocytes from fed rats was low and severely limited triacylglycerol synthesis. 2. Raising the glycerol 3-phosphate content by addition of precursors to the cells resulted in a hyperbolic-like relationship between triacylglycerol synthesis and cellular glycerol 3-phosphate content. Statistical analysis of the curves showed no significant differences between the nutritional states either at saturating or at subsaturating glycerol 3-phosphate content. 3. V(max.) of glycerophosphate acyltransferase measured in homogenized hepatocytes was decreased by 30-40% in starvation. There was no change in apparent K(m) for glycerol 3-phosphate. Since at saturating glycerol 3-phosphate content esterification rates in hepatocytes of both nutritional states were identical, the enzyme is not limiting esterification under this condition. 4. At subsaturating glycerol 3-phosphate content the flux through glycerophosphate acyltransferase necessarily limits esterification. Therefore one would expect a decrease in esterification in starvation under this condition. This was the case when triacylglycerol synthesis was plotted against intracellular glycerol 3-phosphate concentration, calculated from the cellular glycerol 3-phosphate content and the intracellular water space, which was smaller in hepatocytes from starved rats. 5. The data obtained in hepatocytes were extrapolated to the intact liver by using the number of parenchymal cells per g of liver as determined from marker-enzyme analysis and the liver weight per 100g body weight. The extrapolation suggested that glycerol 3-phosphate is limiting esterification in vivo for contents below 0.3-0.4 and 0.5-0.65mumol/g for livers from fed and starved animals respectively. Also for a given fatty acid load and a glycerol 3-phosphate content below 0.3mumol/g the liver may esterify less in the starved state. However, at the glycerol 3-phosphate contents measured in freeze-clamped livers (0.30 and 0.44mumol/g for the fed and starved state respectively), livers in both nutritional states seemed capable of esterifying similar amounts of fatty acids.

Acyltransferases↗
PubMed1982

Glucagon inhibits triacylglycerol synthesis in isolated hepatocytes by lowering their glycerol 3-phosphate content.

Triacylglycerol synthesis by glycogen-depleted hepatocytes from fed rats that have low glycerol 3-phosphate contents was stimulated by the addition of glycerol 3-phosphate precursors. Glucagon decreased triacylglycerol synthesis only when it also lowered glycerol 3-phosphate content. The hyperbolic-like relationship between glycerol 3-phosphate content and rates of triacylglycerol synthesis was identical in the absence or presence of glucagon, indicating that the glucagon effect on triacylglycerol synthesis was not mediated through changes in enzyme activities of the esterification pathway but through changes in cellular glycerol 3-phosphate content.

Animals↗