Retinol bound to cellular retinol-binding protein is a substrate for cytosolic retinoic acid synthesis.
Retinol bound to cellular retinol-binding protein (CRBP) was found to be oxidized to retinoic acid by a soluble activity from calf liver. Cytosolic retinoic acid synthesis from retinol-CRBP was strictly dependent on the exogenous supply of either NAD or NADP. NAD-supported reactions carried out in the presence or in the absence of dimethyl sulfoxide yielded apparent Km and Vmax values for the retinol-CRBP complex of 3.5 +/- 0.6 microM, 611 +/- 49 pmol h-1 (mg of protein)-1, and 0.84 +/- 0.12 microM, 601 +/- 38 pmol h-1 (mg of protein)-1, respectively. The corresponding values for the oxidation of free retinol, dissolved in dimethyl sulfoxide, were 7.1 +/- 0.3 microM and 948 +/- 47 pmol h-1 (mg of protein)-1. Since the dissociation constant of the bovine retinol-CRBP complex is less than 10(-8) M, whereas the Km for retinol-CRBP is of the same order as the Km for free retinol, synthesis of retinoic acid from retinol-CRBP does not rely on prior dissociation of retinol. ApoCRBP proved to be a specific inhibitor of retinoic acid synthesis from CRBP-bound retinol. Its inhibitory effect was indistinguishable from the dilution of the radioactive retinol-CRBP substrate that was obtained by the addition of unlabeled holoCRBP. In contrast, the oxidation of CRBP-bound retinol was not inhibited by the addition of other retinoid binding proteins nor by the addition of either free retinol or retinol complexed with proteins distinct from CRBP. These results indicate that the protein moiety of holoCRBP is specifically recognized by the cytosolic enzyme system that catalyzes retinoic acid synthesis from CRBP-bound retinol.
Ottonello
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S
Institute of Biochemical Sciences, University of Parma, Italy.
Scita
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G
Mantovani
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Cavazzini
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D
Rossi
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GL
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003600:Cytosol / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D011485:Protein Binding; D012177:Retinol-Binding Proteins / Q000378:metabolism*; D054840:Retinol-Binding Proteins, Cellular; D014212:Tretinoin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014801:Vitamin A / Q000378:metabolism*
D012177:Retinol-Binding Proteins; D054840:Retinol-Binding Proteins, Cellular; D014801:Vitamin A; D014212:Tretinoin