Abstract
The subcellular localization of l-lactate dehydrogenase (LDH) in rat hepatocytes has been studied by analytical subcellular fractionation combined with the immunodetection of LDH in isolated subcellular fractions and liver sections by immunoblotting and immunoelectron microscopy. The results clearly demonstrate the presence of LDH in the matrix of peroxisomes in addition to the cytosol. Both cytosolic and peroxisomal LDH subunits have the same molecular mass (35.0 kDa) and show comparable cross-reactivity with an anti-cytosolic LDH antibody. As revealed by activity staining or immunoblotting after isoelectric focussing, both intracellular compartments contain the same liver-specific LDH-isoforms (LDH-A4 > LDH-A3B) with the peroxisomes comprising relatively more LDH-A3B than the cytosol. Selective KCl extraction as well as resistance to proteinase K and immunoelectron microscopy revealed that at least 80% of the LDH activity measured in highly purified peroxisomal fractions is due to LDH as a bona fide peroxisomal matrix enzyme. In combination with the data of cell fractionation, this implies that at least 0.5% of the total LDH activity in hepatocytes is present in peroxisomes. Since no other enzymes of the glycolytic pathway (such as phosphoglucomutase, phosphoglucoisomerase, and glyceraldehyde-3-phosphate dehydrogenase) were found in highly purified peroxisomal fractions, it does not seem that LDH in peroxisomes participates in glycolysis. Instead, the marked elevation of LDH in peroxisomes of rats treated with the hypolipidemic drug bezafibrate, concomitantly to the induction of the peroxisomal beta-oxidation enzymes, strongly suggests that intraperoxisomal LDH may be involved in the reoxidation of NADH generated by the beta-oxidation pathway. The interaction of LDH and the peroxisomal palmitoyl-CoA beta-oxidation system could be verified in a modified beta-oxidation assay by adding increasing amounts of pyruvate to the standard assay mixture and recording the change of NADH production rates. A dose-dependent decrease of NADH produced was simulated with the lowest NADH value found at maximal LDH activity. The addition of oxamic acid, a specific inhibitor of LDH, to the system or inhibition of LDH by high pyruvate levels (up to 20 mm) restored the NADH values to control levels. A direct effect of pyruvate on palmitoyl-CoA oxidase and enoyl-CoA hydratase was excluded by measuring those enzymes individually in separate assays. An LDH-based shuttle across the peroxisomal membrane should provide an efficient system to regulate intraperoxisomal NAD+/NADH levels and maintain the flux of fatty acids through the peroxisomal beta-oxidation spiral.
Highlights
The subcellular localization of L-lactate dehydrogenase (LDH) in rat hepatocytes has been studied by analytical subcellular fractionation combined with the immunodetection of LDH in isolated subcellular fractions and liver sections by immunoblotting and immunoelectron microscopy
The bulk of LDH activity was recovered in the cytosolic fraction (S), with only about 1% of the total activity being present in the crude peroxisomal fraction (Fig. 1)
The PO in the heavy and light fractions differed substantially in their enzyme composition, with the light fractions containing significantly higher ratios of palmitoyl-CoA oxidase/catalase. These results are consistent with the data reported previously by Schrader et al [24] from our laboratory and LDH, PGI, PGM, and GAPDH were measured in total liver homogenate and subcellular fractions of untreated rats
Summary
(Received for publication, September 6, 1995, and in revised form, December 4, 1995). Selective KCl extraction as well as resistance to proteinase K and immunoelectron microscopy revealed that at least 80% of the LDH activity measured in highly purified peroxisomal fractions is due to LDH as a bona fide peroxisomal matrix enzyme. Whereas PO in rat liver contain several dehydrogenases utilizing NADϩ as cofactor such as (a) 3-hydroxyacyl-CoA dehydrogenases [12], (b) ␣-glycerol phosphate dehydrogenase [13], and (c) alcohol dehydrogenase [14], a peroxisomal enzyme system for the reoxidation of NADH has not been described. Since the exact mechanism of stimulation of -oxidation by pyruvate remained ambiguous, we speculated that it could be due to the presence of LDH inside the PO, since in our earlier studies this enzyme was consistently found in the highly purified (98%) peroxisomal fractions isolated by metrizamide density gradient centrifugation [18]. The involvement of peroxisomal LDH in the reoxidation of NADH produced by the -oxidation system of this organelle is demonstrated
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