scholarly journals The soluble carnitine palmitoyltransferase from bovine liver. A comparison with the enzymes from peroxisomes and from the mitochondrial inner membrane

1988 ◽  
Vol 249 (1) ◽  
pp. 239-245 ◽  
Author(s):  
R R Ramsay
Keyword(s):  
Mouse Liver ◽  
Bovine Liver ◽  
Thiol Group ◽  
Carnitine Palmitoyltransferase ◽  
Native Enzyme ◽  
Thiol Groups ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Chain Acyl ◽  
Carnitine Acyltransferases

The properties of two carnitine acyltransferases (CPT) purified from bovine liver are compared to confirm that they are different proteins. The soluble CPT and the inner CPT from mitochondria differ in subunit Mr, native Mr, pI and reactivity with thiol reagents. All eight free thiol groups in soluble CPT react with 5,5′-dithiobis-(2-nitrobenzoate) in the absence of any unfolding reagent, and activity is gradually lost. The inner CPT activity is completely stable in the presence of 5,5′-dithiobis-(2-nitrobenzoate), and only one thiol group per molecule of subunit is modified in the native enzyme. Antisera to each enzyme inhibit that enzyme, but do not cross-react. CPT activity in subcellular fractions can now be identified by titration with these antibodies. The soluble CPT from bovine liver is probably peroxisomal in origin, but, although antigenically similar, it differs from the peroxisomal carnitine octanoyltransferase found in rat and mouse liver in its specificity for the longer-chain acyl-CoA substrates.

scholarly journals Half-sites oxidation of bovine liver uridine diphosphate glucose dehydrogenase

1978 ◽  
Vol 173 (2) ◽  
pp. 701-704 ◽  
Author(s):  
J S Franzen ◽  
P Marchetti ◽  
R Ishman ◽  
J Ashcom
Keyword(s):  
Catalytic Activity ◽  
Glucose Dehydrogenase ◽  
Bovine Liver ◽  
Thiol Group ◽  
Catalytic Site ◽  
Uridine Diphosphate ◽  
Thiol Groups ◽  
Irreversible Denaturation ◽  
Diphosphate Glucose ◽  
Uridine Diphosphate Glucose Dehydrogenase

6,6-Dithiodinicotinate shows half-of-the-sites reactivity towards the six catalytic-site thiol groups of bovine liver UDP-glucose dehydrogenase. The reagent introduces three intrasubunit disulphide linkages between catalytic-site thiol groups and non-catalytic-site thiol groups and abrogates 60% of the catalytic activity of the hexameric enzyme; excess 2-mercaptoethanol rapidly restores full catalytic activity. These results show the half-of-the-sites behaviour of the enzyme with the reagent and the presence of a non-catalytic-site thiol group capable of forming a disulphide linkage with a catalytic-site thiol group on the same subunit without irreversible denaturation.

scholarly journals Hepatic mitochondrial inner-membrane properties, β-oxidation and carnitine palmitoyltransferases A and B. Effects of genetic obesity and starvation

1986 ◽  
Vol 233 (2) ◽  
pp. 427-433 ◽  
Author(s):  
L J Brady ◽  
C L Hoppel ◽  
P S Brady
Keyword(s):  
Membrane Fluidity ◽  
Carnitine Palmitoyltransferase ◽  
Membrane Properties ◽  
Zucker Rats ◽  
Beta Oxidation ◽  
Inner Membrane ◽  
Membrane Polarization ◽  
Mitochondrial Inner Membrane ◽  
Malonyl Coa ◽  
Obese Rats

Hepatic mitochondrial carnitine palmitoyltransferase (CPT) properties, beta-oxidation of palmitoyl-CoA and membrane polarization were measured in lean and obese Zucker rats. The Vmax. of the ‘outer’ carnitine palmitoyltransferase (‘CPT-A‘) increased with starvation, with no change in the Km for either carnitine or palmitoyl-CoA. The Ki for malonyl-CoA increased with starvation in lean rats, but not in obese rats. The Vmax. of the ‘inner’ enzyme (‘CPT-B‘), as measured by using inverted submitochondrial vesicles, increased with starvation in obese rats only, with no change in the Km for either carnitine or palmitoyl-CoA. The Ki for malonyl-CoA was 2-5-fold higher in inverted vesicles than in intact mitochondria, and showed no alteration with starvation. The activities of both enzymes correlated positively with each other and with beta-oxidation, and inversely with membrane polarization. Malonyl-CoA had little effect on gross membrane fluidity in the Zucker rat, as reflected by diphenylhexatriene fluorescence polarization. The results indicate that both enzymes are related and respond similarly to alterations in membrane fluidity. Membrane fluidity may provide a mechanism for co-ordinated control of CPT activity on both sides of the mitochondrial inner membrane.

scholarly journals Characterization of hepatic carnitine palmitoyltransferase. Use of bromoacyl derivatives and antibodies

1987 ◽  
Vol 241 (3) ◽  
pp. 751-757 ◽  
Author(s):  
P S Brady ◽  
A K Dunker ◽  
L J Brady
Keyword(s):  
Carnitine Palmitoyltransferase ◽  
Dimethyl Sulphoxide ◽  
Inner Membrane ◽  
Phosphatidylcholine Liposomes ◽  
Aqueous Buffer ◽  
Mitochondrial Inner Membrane ◽  
Membrane Enzyme ◽  
Malonyl Coa ◽  
Assay Conditions

Carnitine palmitoyltransferase (CPT) is a mitochondrial-inner-membrane enzyme, with activities located on both the outer and inner sides of the membrane. The inhibition of CPT by bromopalmitate derivatives was studied in intact hepatic mitochondria (representing CPT-A activity, the outer enzyme), in inverted submitochondrial vesicles (representing CPT-B, the inner enzyme), and in purified hepatic CPT. Bromopalmitoyl-CoA had an I50 (concentration giving 50% inhibition of CPT activity) of 0.63 +/- 0.08 microM in intact mitochondria and 2.44 +/- 0.86 microM in inverted vesicles. Preincubation of mitochondria with bromopalmitoyl-CoA decreased V max. for both CPT-A and CPT-B. Sonication decreased sensitivity to bromopalmitoyl-CoA, and solubilization with Triton abolished sensitivity at the concentrations used (0-10 microM). Purified CPT had a bromopalmitoyl-CoA I50 of 353 microM in aqueous buffer, 67 microM in 20% dimethyl sulphoxide, 45 microM in phosphatidylcholine liposomes and 26 microM in cardiolipin liposomes. Increasing [carnitine] at constant bromopalmitoyl-CoA concentrations or increasing [bromopalmitoyl-CoA] in the preincubation resulted in increased inhibition of purified CPT. 2-Tetradecylglycidyl-CoA and malonyl-CoA did not offer measurable protection against bromopalmitoyl-CoA inhibition of the purified CPT, suggesting a different site of interaction of bromopalmitoyl-CoA with CPT. The data suggest that the sensitivity of CPT to bromopalmitoyl-CoA may be modulated by membrane environment and assay conditions.

Very-long-chain acyl-CoA dehydrogenase subunit assembles to the dimer form on mitochondrial inner membrane

FEBS Letters ◽  
1998 ◽  
Vol 426 (2) ◽  
pp. 187-190 ◽  
Author(s):  
Masayoshi Souri ◽  
Toshifumi Aoyama ◽  
George Hoganson ◽  
Takashi Hashimoto
Keyword(s):  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Chain Acyl ◽  
Long Chain

scholarly journals Altered release of carnitine palmitoyltransferase activity by digitonin from liver mitochondria of rats in different physiological states

1985 ◽  
Vol 230 (2) ◽  
pp. 389-394 ◽  
Author(s):  
V A Zammit ◽  
C G Corstorphine
Keyword(s):  
Liver Mitochondria ◽  
Diabetic Rats ◽  
Carnitine Palmitoyltransferase ◽  
Rat Liver Mitochondria ◽  
Intermembrane Space ◽  
Marker Enzyme ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Cpt I ◽  
Carnitine Palmitoyltransferase Activity

The release of carnitine palmitoyltransferase (CPT) activity from rat liver mitochondria by increasing concentrations of digitonin was studied for mitochondrial preparations from fed, 48 h-starved and diabetic animals. A bimodal release of activity was observed only for mitochondria isolated from starved and, to a lesser degree, from diabetic rats, and it appeared to result primarily from the enhanced release of approx. 40% and 60%, respectively, of the total CPT activity. This change in the pattern of release was specific to CPT among the marker enzymes studied. For all three types of mitochondria there was no substantial release of CPT concurrently with that of the marker enzyme for the soluble intermembrane space, adenylate kinase. These results illustrate that the bimodal pattern of release of CPT reported previously for mitochondria from starved rats [Bergstrom & Reitz (1980) Arch. Biochem. Biophys. 204, 71-79] is not an immutable consequence of the localization of CPT activity on either side of the mitochondrial inner membrane. Sequential loss of CPT I (i.e. the overt form) from the mitochondrial inner membrane did not affect the concentration of malonyl-CoA required to effect fractional inhibition of the CPT I that remained associated with the mitochondria. The results are discussed in relation to the possibility that altered enzyme-membrane interactions may account for some of the altered regulatory properties of CPT I in liver mitochondria of animals in different physiological states.

scholarly journals Amino acid sequence of the tryptic peptide containing the catalytic-site thiol group of bovine liver uridine diphosphate glucose dehydrogenase

1981 ◽  
Vol 199 (3) ◽  
pp. 599-602 ◽  
Author(s):  
B Franzen ◽  
C Carrubba ◽  
D S Feingold ◽  
J Ashcom ◽  
J S Franzen
Keyword(s):  
Glucose Dehydrogenase ◽  
Thick Layer ◽  
Bovine Liver ◽  
Thiol Group ◽  
Paper Electrophoresis ◽  
Catalytic Site ◽  
Uridine Diphosphate ◽  
Thiol Groups ◽  
Diphosphate Glucose ◽  
Adsorption Desorption

The catalytic-site thiol groups of UDP-glucose dehydrogenase from bovine liver were carboxymethylated with iodo[2-14C]acetate or with iodoacetamidofluorescein. After the residual thiol groups were carboxymethylated with iodoacetate, the proteins were digested with trypsin. The 14C-labelled peptide from the carboxymethylated enzyme was purified to homogeneity by successive thick-layer chromatography on silica gel, paper electrophoresis and chromatography, and column chromatography on Bio-Gel P-6. Homogeneous fluoresceincarboxamidomethylated peptide was prepared from a tryptic digest of fluoresceincarboxamidomethylated enzyme by specific adsorption--desorption from Sephadex G-25. The sequences of either peptide determined by the manual Edman dansyl procedure is: Ala-Ser-Val-Gly-Phe-Gly-Gly-Ser-Cys-Phe-Glx-Glx-Gly-Lys.

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