Abstract
This study shows that microsomal retinol dehydrogenases, versus cytosolic retinol dehydrogenases, provide the quantitatively major share of retinal for retinoic acid (RA) biogenesis in rat tissues from the predominant substrate available physiologically, holo-cellular retinol-binding protein, type I (CRBP). With holo-CRBP as substrate in the absence of apo-CRBP microsomal retinol dehydrogenases have the higher specific activity and capacity to generate retinal used for RA synthesis by cytosolic retinal dehydrogenases. In the presence of apo-CRBP, a potent inhibitor of cytosolic retinol dehydrogenases (IC50 = approximately 1 microM), liver microsomes provide 93% of the total retinal synthesized in a combination of microsomes and cytosol. Cytosolic retinol dehydrogenase(s) and the isozymes of alcohol dehydrogenase expressed in rat liver had distinct enzymatic properties; yet ethanol inhibited cytosolic retinol dehydrogenase(s) (IC50 = 20 microM) while stimulating RA synthesis in a combination of microsomes and cytosol. At least two discrete forms of cytosolic retinol dehydrogenase were observed: NAD- and NADP-dependent forms. Multiple retinal dehydrogenases also were observed and were inhibited partially by apo-CRBP. These results provide new insights into pathways of RA biogenesis and provide further evidence that they consist of multiple enzymes that recognize both liganded and nonliganded states of CRBP.
Highlights
This study shows that microsomal retinol dehydrogenases, versus cytosolic retinol dehydrogenases, provide the quantitatively major share of retinal for retinoic acid (RA) biogenesis in rat tissues from the predominant substrate available physiologically, holo-cellular retinol-binding protein, type I (CRBP)
Effects of apo-CRBP on Retinol DH Activities in Cytosol and in the 10kS—apo-CRBP markedly inhibited cytosolic retinol DH activity supported by holo-CRBP, measured by RA synthesis (Fig. 1)
Apo-CRBP inhibition of cytosolic RA synthesis was noncompetitive in nature, with both decreased apparent Vmax and increased apparent Km values
Summary
(Received for publication, July 27, 1995, and in revised form, December 28, 1995). From the Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York 14214. With holo-CRBP as substrate in the absence of apo-CRBP microsomal retinol dehydrogenases have the higher specific activity and capacity to generate retinal used for RA synthesis by cytosolic retinal dehydrogenases. Recent work has outlined a pathway of RA biosynthesis with the first step catalyzed by a NADP-dependent microsomal retinol DH, expressed in liver and in extrahepatic tissues, that recognizes holo-CRBP as substrate [12,13,14,15]. With holo-CRBP as substrate in the absence of apo-CRBP, microsomal retinol DH has the higher specific activity and capacity (60 – 83% of the total of cytosol plus microsomes, individually) to generate retinal for RA synthesis in the four tissues assayed. We establish here that multiple paths and complex interactions contribute to RA biosynthesis
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