scholarly journals Acyl-CoA synthetase and the peroxisomal enzymes of β-oxidation in human liver. Quantitative analysis of their subcellular localization

1984 ◽  
Vol 224 (3) ◽  
pp. 709-720 ◽  
Author(s):  
M Bronfman ◽  
N C Inestrosa ◽  
F O Nervi ◽  
F Leighton
Keyword(s):  
Human Liver ◽  
Fatty Acyl ◽  
Single Step ◽  
Synthetase Activity ◽  
Beta Oxidation ◽  
Analysis Of Results ◽  
Fractionation Method ◽  
Hydroxyacyl Coa Dehydrogenase ◽  
Acyl Coa Oxidase ◽  
Rate Limiting

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 were found to 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 were found to contribute 13%, 17% and 11% of the liver activities of crotonase, beta-hydroxyacyl-CoA dehydrogenase 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.

scholarly journals Peroxisomal and mitochondrial beta-oxidation in the rat kidney: distribution of fatty acyl-coenzyme A oxidase and 3-hydroxyacyl-coenzyme A dehydrogenase activities along the nephron.

1982 ◽  
Vol 30 (5) ◽  
pp. 441-444 ◽  
Author(s):  
M Le Hir ◽  
U C Dubach
Keyword(s):  
Proximal Tubule ◽  
Coenzyme A ◽  
Rat Kidney ◽  
Mitochondrial Pathway ◽  
Fatty Acyl ◽  
Beta Oxidation ◽  
Collecting Ducts ◽  
Hydroxyacyl Coa Dehydrogenase ◽  
Acyl Coa Oxidase ◽  
Acyl Coenzyme A

In order to determine the localization of the peroxisomal and the mitochondrial pathways of beta-oxidation in the rat kidney, fatty acyl-coenzyme A (CoA) oxidase and 3-hydroxyacyl-CoA dehydrogenase activities were measured in glomeruli and in eight proximal and distal segments of the nephron. Structurally defined segments were dissected and analyzed with microchemical assays. The peroxisomal fatty acyl-CoA oxidase is restricted to the proximal tubule. The 3-hydroxyacyl-CoA dehydrogenase activity represents mainly the mitochondrial pathway and is similarly distributed in all cortical proximal and distal segments. It is much lower in glomeruli and collecting ducts. The distribution patterns of the two enzymes remain the same after 48 hr of starvation, although the activity of fatty acyl-CoA oxidase increases in glomeruli, proximal convolution, and collecting ducts. It is concluded that the capacity for mitochondrial beta-oxidation of fatty acids is similar in the proximal and the distal nephron. The proximal tubule possesses, additionally, a peroxisomal pathway for beta-oxidation with a capacity of the same order of magnitude as in liver cells.

Mammalian Fatty Acid Synthetase II. Modification of Purified Human Liver Complex Activity

1975 ◽  
Vol 53 (2) ◽  
pp. 135-142 ◽  
Author(s):  
Daniel A. K. Roncari
Keyword(s):  
Fatty Acid ◽  
Human Liver ◽  
Fatty Acid Synthetase ◽  
Potassium Phosphate ◽  
Fatty Acyl ◽  
Synthetase Activity ◽  
Prosthetic Group ◽  
Complex Activity ◽  
Coa Thioesters ◽  
Fasted Rats

Highly purified human-liver fatty acid synthetase complex was used to study the effect of several potential modifiers. Adenosine 3′,5′-phosphate did not alter the activity of either purified synthetase or of multienzyme present in 700 × g supernates. Its dibutyryl derivative was also ineffective when incubated with liver slices. Fructose 1,6-diphosphate, fructose 6-phosphate, and glucose 6-phosphate stimulated significantly the activity of the purified enzyme. Fructose 1,6-diphosphate, which was most effective, decreased the Km of the synthetase for NADPH. Phosphoenolpyruvate, rac-glycero-3 -phosphate and potassium phosphate were ineffective. All long-chain fatty acyl-CoA thioesters tested were inhibitory, but this effect was not observed until the regions of their critical micellar concentrations were reached. Free myristate, palmitate, and stearate did not inhibit synthetase activity up to the highest concentration tested (1 mM). An enzyme preparation derived from livers of fasted rats inactivated purified rat-liver 4′-phospho[14C]pantetheine-fatty acid synthetase by releasing its prosthetic group. It also decreased the activity of the purified human-liver complex.

scholarly journals Detection of peroxisomal fatty acyl-coenzyme A oxidase activity

1979 ◽  
Vol 182 (3) ◽  
pp. 779-788 ◽  
Author(s):  
N C Inestrosa ◽  
M Bronfman ◽  
F Leighton
Keyword(s):  
Fatty Acid ◽  
Oxidase Activity ◽  
Subcellular Fractionation ◽  
Fatty Acyl ◽  
O2 Consumption ◽  
H2o2 Generation ◽  
Acyl Coa Oxidase ◽  
Acyl Coenzyme A ◽  
Rate Limiting ◽  
Peroxisomal Fatty Acid

It has been postulated that the peroxisomal fatty acid-oxidizing system [Lazarow & de Duve (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2043–2046; Lazarow (1978) J. Biol. Chem. 253, 1522–1528] resembles that of mitochondria, except for the first oxidative reaction. In this step, O2 would be directly reduced to H2O2 by an oxidase. Two specific procedures developed to detect the activity of the characteristic enzyme fatty acyl-CoA oxidase are presented, namely polarographic detection of palmitoyl-CoA-dependent cyanide-insensitive O2 consumption and palmitoyl-CoA-dependent H2O2 generation coupled to the peroxidation of methanol in an antimycin A-insensitive reaction. Fatty acyl-CoA oxidase activity is stimulated by FAD, which supports the flavoprotein nature postulated for this enzyme. Its activity increases 7-fold per g wet wt. of liver in rats treated with nafenopin, a hypolipidaemic drug. Subcellular fractionation of livers from normal and nafenopin-treated animals provides evidence for its peroxisomal localization. The stoicheiometry for palmitoyl-CoA-dependent O2 consumption, H2O2 generation and NAD+ reduction is 1 : 1 : 1. This suggests that fatty acyl-CoA oxidase is the rate-limiting enzyme of the peroxisomal fatty acid-oxidizing system.

scholarly journals Substrate selectivities differ for hepatic mitochondrial and peroxisomal β-oxidation in an Antarctic fish, Notothenia gibberifrons

1993 ◽  
Vol 289 (2) ◽  
pp. 427-433 ◽  
Author(s):  
E L Crockett ◽  
B D Sidell
Keyword(s):  
Antarctic Fish ◽  
Fatty Acyl ◽  
Beta Oxidation ◽  
Oxidation Rates ◽  
Substrate Preferences ◽  
Antarctic Marine ◽  
Marked Preference ◽  
Aerobic Energy Metabolism ◽  
High Concentrations ◽  
Rate Limiting

Hepatic mitochondrial and peroxisomal beta-oxidation were examined in an Antarctic marine teleost, Notothenia gibberifrons. Enzymic profiles and rates of beta-oxidation by intact organelles were determined by using a range of fatty acyl-CoA substrates to evaluate substrate preferences. Partitioning of beta-oxidation between organelles was estimated. Substrate selectivities are broader for peroxisomal beta-oxidation than for mitochondrial beta-oxidation. Mitochondria show marked preference for the oxidation of a monounsaturated substrate, palmitoleoyl-CoA (C16:1), and two polyunsaturates, eicosapentaenoyl-CoA (C20:5) and docosahexaenoyl-CoA (C22:6). Carnitine palmitoyltransferase activities with palmitoleoyl-CoA (C16:1) are 2.4-fold higher than activities with palmitoyl-CoA (C16:0). Most polyunsaturated acyl-CoA esters measured appear to inhibit by over 40% the oxidation of palmitoyl-CoA by peroxisomes. Our findings suggest that the polyunsaturates, eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6), found in high concentrations in Antarctic fishes [Lund and Sidell (1992) Mar. Biol. 112, 377-382], are utilized as fuels to support aerobic energy metabolism. Metabolic capacities of rate-limiting enzymes and beta-oxidation rates by intact organelles indicate that up to 30% of hepatic beta-oxidation in N. gibberifrons can be initiated by the peroxisomal pathway.

scholarly journals Comparison of metabolic fluxes of cis-5-enoyl-CoA and saturated acyl-CoA through the β-oxidation pathway

1995 ◽  
Vol 307 (1) ◽  
pp. 23-28 ◽  
Author(s):  
K Y Tserng ◽  
L S Chen ◽  
S J Jin
Keyword(s):  
Stable Isotope ◽  
Rat Liver ◽  
Chromatographic Method ◽  
Spectrophotometric Assay ◽  
Beta Oxidation ◽  
Metabolic Fluxes ◽  
Hydroxyacyl Coa Dehydrogenase ◽  
Acyl Coa Oxidase ◽  
Acyl Coa Dehydrogenases ◽  
Gas Chromatographic Method

The metabolic fluxes of cis-5-enoyl-CoAs through the beta-oxidation cycle were studied in solubilized rat liver mitochondrial samples and compared with saturated acyl-CoAs of equal chain length. These studies were accomplished using either spectrophotometric assay of enzyme activities and/or the analysis of metabolites and precursors using a gas chromatographic method after conversion of CoA esters into their free acids. Cis-5-enoyl-CoAs were dehydrogenated by acyl-CoA oxidase or acyl-CoA dehydrogenases at significantly lower rates (10-44%) than saturated acyl-CoAs. However, enoyl-CoA hydratase hydrated trans-2-cis-5-enoyl-CoA at a faster rate (at least 1.5-fold) than trans-2-enoyl-CoA. The combined activities of 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase for 3-hydroxy-cis-5-enoyl-CoAs derived from cis-5-enoyl-CoAs were less than 40% of the activity for the corresponding 3-hydroxyacyl-CoAs prepared from saturated acyl-CoAs. Rat liver mitochondrial beta-oxidation enzymes were capable of metabolizing cis-5-enoyl-CoA via one cycle of beta-oxidation to cis-3-enoyl-CoA with two less carbons. However, the overall rates of one cycle of beta-oxidation, as determined with stable-isotope-labelled tracer, was only 15-25%, for cis-5-enoyl-CoA, of that for saturated acyl-CoA. In the presence of NADPH, the metabolism of cis-5-enoyl-CoAs was switched to the reduction pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Acyl-Coa oxidase and hydratase-dehydrogenase, two enzymes of the peroxisomal beta-oxidation system, are synthesized on free polysomes of clofibrate-treated rat liver.

1984 ◽  
Vol 99 (6) ◽  
pp. 2241-2246 ◽  
Author(s):  
R A Rachubinski ◽  
Y Fujiki ◽  
R M Mortensen ◽  
P B Lazarow
Keyword(s):  
Rat Liver ◽  
Translational Efficiency ◽  
Beta Oxidation ◽  
Sds Page ◽  
Reticulocyte Lysate ◽  
Normal Rat ◽  
Membrane Bound ◽  
Hydroxyacyl Coa Dehydrogenase ◽  
Acyl Coa Oxidase ◽  
Oxidation System

We investigated the site of synthesis of two abundant proteins in clofibrate-induced rat hepatic peroxisomes. RNA was extracted from free and membrane-bound polysomes, heated to improve translational efficiency, and translated in the mRNA-dependent, reticulocyte-lysate-cell-free, protein-synthesizing system. The peroxisomal acyl-CoA oxidase and enoyl-CoA hydratase-beta-hydroxyacyl-CoA dehydrogenase 35S-translation products were isolated immunochemically, analyzed by SDS PAGE and fluorography, and quantitated by densitometric scanning. The RNAs coding for these two peroxisomal proteins were found predominantly on free polysomes, and the translation products co-migrated with the mature proteins. As in normal rat liver, preproalbumin and catalase were synthesized mainly by membrane-bound and by free polysomes, respectively. mRNAs for a number of minor 35S-translation products also retained by the anti-peroxisomal immunoadsorbent were similarly found on free polysomes. These results, together with previous data, allow the generalization that the content proteins of rat liver peroxisomes are synthesized on free polysomes, and the data imply a posttranslational packaging mechanism for these major content proteins.

scholarly journals Evidence for a single non-arachidonic acid-specific fatty acyl-CoA synthetase in heart which is regulated by Mg2+

1996 ◽  
Vol 313 (3) ◽  
pp. 849-853 ◽  
Author(s):  
Christine SAUNDERS ◽  
Jeffrey M. VOIGT ◽  
Margaret T. WEIS
Keyword(s):  
Fatty Acids ◽  
Arachidonic Acid ◽  
Oleic Acid ◽  
Unsaturated Fatty Acids ◽  
Saturated Fatty Acids ◽  
Rabbit Heart ◽  
Fatty Acyl ◽  
Synthetase Activity ◽  
Low Concentrations ◽  
Rate Limiting

Previous reports indicated that arachidonic acid is incorporated into the isolated perfused rabbit heart in preference to other fatty acids, and that incorporation of arachidonic acid, but not other fatty acids, is inhibited during Mg2+ depletion. In this study, we have not been able to demonstrate an arachidonic acid-specific fatty acyl-CoA synthetase in rat or rabbit heart by hydroxyapatite chromatography. Kinetic evidence was consistent with a single enzyme, as the slopes of pseudo-Hill plots were not significantly different from -1. The single fatty acyl-CoA synthetase present appears to prefer C18:0 unsaturated fatty acids to arachidonate, and had about the same affinity for C10:0–C14:0 saturated fatty acids as for arachidonate. At 35 μM arachidonate, enzyme velocity increased as the total Mg2+ was increased from 3 to 80 mM. Calculated [MgATP] indicated that the MgATP complex was not rate-limiting. At low concentrations, Mn2+ and Ni2+ supported activity, but Cu2+ and Zn2+ did not. Low Ca2+ concentrations activated only oleic acid conversion. Kinetic analysis indicated that the Vmax of the enzyme was increased with increasing concentrations of ionized Mg2+ for both oleic acid and arachidonic acid. The data are consistent with the hypothesis that Mg2+ has a direct effect on fatty acyl-CoA synthetase activity, and suggest that preference for oleic acid and arachidonic acid can be influenced by the ionic milieu.

scholarly journals Identification and purification of a peroxisomal branched chain fatty acyl-CoA oxidase.

1991 ◽  
Vol 266 (36) ◽  
pp. 24676-24683
Author(s):  
P.P. Van Veldhoven ◽  
G. Vanhove ◽  
F. Vanhoutte ◽  
G. Dacremont ◽  
G. Parmentier ◽  
...  
Keyword(s):  
Fatty Acyl ◽  
Acyl Coa Oxidase ◽  
Branched Chain
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