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Involvement of microsomal vesicles in part of the sensitivity of carnitine palmitoyltransferase I to malonyl-CoA inhibition in mitochondrial fractions of rat liver.

ABSTRACT: Liver mitochondrial fractions as normally isolated contain only 10-20% of total mitochondria and may not be representative of the whole mitochondrial population. This study was designed to evaluate the dependence of the sensitivity of carnitine palmitoyl-transferase I (CPT I) to malonyl-CoA inhibition in mitochondrial fractions that are not normally studied. Four fractions prepared from rat liver were found to be contaminated to different extents by microsome vesicles, on the basis of marker-enzyme activities and micrographic data. Purification of mitochondrial fractions on a Percoll gradient decreased to some extent the microsomal contamination, which was due in part to the existence of close bonds between microsomes and the outer membranes of mitochondria. A greater degree of contamination of mitochondrial fractions by microsomes was correlated with a greater sensitivity of CPT I to malonyl-CoA inhibition. Attempts were made to enhance the sensitivity of CPT I to malonyl-CoA with the use of microsomes. Measurements performed by adding mitochondria and microsomes in the same CPT I assay failed to demonstrate any significant enhancement of malonyl-CoA inhibition. However, addition of ATP to a mixture of mitochondria and microsomes was shown to trigger the binding of both particles, as assessed by enzymic and micrographic data, and to increase the sensitivity of CPT I to malonyl-CoA inhibition. These results demonstrated that the binding of microsomes to mitochondria, unlike the simple mixing of both particles, was capable of altering the sensitivity of CPT I to malonyl-CoA. The data also suggest that this process could be of physiological importance, owing to the frequency of contiguous zones between mitochondria and endoplasmic reticulum observed in sections of intact liver cells.

SUBMITTER: Niot I

PROVIDER: S-EPMC1137531 | biostudies-other | 1994 Dec

REPOSITORIES: biostudies-other

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Project description:Carnitine palmitoyltransferase I (CPT I) catalyses the initial step of fatty acid import into the mitochondrial matrix, the site of beta-oxidation, and its inhibition by malonyl-CoA is a primary control point for this process. The enzyme exists in at least two isoforms, denoted L-CPT I (liver type) and M-CPT I (skeletal-muscle type), which differ in their kinetic characteristics and tissue distributions. A property apparently unique to L-CPT I is that its sensitivity to malonyl-CoA decreases in vivo with fasting or experimentally induced diabetes. The mechanism of this important regulatory effect is unknown and has aroused much interest. CPT I is an integral outer-membrane protein and displays little activity after removal from the membrane by detergents, precluding direct purification of active protein by conventional means. Here we describe the expression of a 6 x His-tagged rat L-CPT I in Pichia pastoris and purification of the detergent-solubilized enzyme in milligram quantities. Reconstitution of the purified product into a liposomal environment yielded a 200--400-fold increase in enzymic activity and restored malonyl-CoA sensitivity. This is the first time that a CPT I protein has been available for study in a form that is both pure and active. Comparison of the kinetic properties of the reconstituted material with those of L-CPT I as it exists in mitochondria prepared from yeast over-expressing the enzyme and in livers from fed or fasted rats permitted novel insight into several aspects of the enzyme's behaviour. The malonyl-CoA response of the liposomal enzyme was found to be greater when the reconstitution procedure was carried out at 22 degrees C compared with 4 degrees C (IC(50) approximately 11 microM versus 30 microM, respectively). When the sensitivities of L-CPT I in each of the different environments were compared, they were found to decrease in the following order: fed liver>fasted liver approximately liposomes prepared at 22 degrees C approximately P. pastoris mitochondria>liposomes prepared at 4 degrees C. In addition, pre-treatment of L-CPT I liposomes with the membrane-fluidizing reagent benzyl alcohol caused densensitization to the inhibitor. In contrast with the variable response to malonyl-CoA, the liposomal L-CPT I displayed a pH profile and kinetics with regard to the carnitine and acyl-CoA substrates similar to those of the enzyme in fed or fasted liver mitochondria. However, despite a normal sensitivity to malonyl-CoA, L-CPT I in P. pastoris mitochondria displayed aberrant behaviour with regard to each of these other parameters. The kinetic data establish several novel points. First, even after stringent purification procedures in the presence of detergent, recombinant L-CPT I could be reconstituted in active, malonyl-CoA sensitive form. Second, the kinetics of the reconstituted, 6 x His-tagged L-CPT I with regard to substrate and pH responses were similar to what is observed with rat liver mitochondria (whereas in P. pastoris mitochondria the enzyme behaved anomalously), confirming that the purified preparation is a suitable model for studying the functional properties of the enzyme. Third, wide variation in the response to the inhibitor, malonyl-CoA, was observed depending only on the enzyme's membrane environment and independent of interaction with other proteins. In particular, the fluidity of the membrane had a direct influence on this parameter. These observations may help to explain the mechanism of the physiological changes in the properties of L-CPT I that occur in vivo and are consistent with the current topographical model of the enzyme.

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Involvement of microsomal vesicles in part of the sensitivity of carnitine palmitoyltransferase I to malonyl-CoA inhibition in mitochondrial fractions of rat liver.

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