Cellular Retinol-binding Protein-supported Retinoic Acid Synthesis

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 (IC(50)=~1 μM), 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) (IC(50)= 20 μM) 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. 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 (IC(50)=~1 μM), 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) (IC(50)= 20 μM) 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. INTRODUCTIONThe metabolism of retinol (vitamin A) generates RA, ( 1The abbreviations used are: RAretinoic acidADHalcohol dehydrogenaseCRBPcellular retinol-binding protein, type IDHdehydrogenaseHPLChigh performance liquid chromatography10kS10,000 × g supernatant of a rat liver homogenate.) a humoral factor critical to vertebrate development(1.Napoli J.L. Posch K.C. Fiorella P.D. Boerman M.H.E.M. Biomed. Pharmacother. 1991; 45: 131-143Crossref PubMed Scopus (100) Google Scholar, 2.Blomhoff R. Green M.H. Green J.B. Berg T. Norum K.R. Physiol. Rev. 1991; 71: 951-990Crossref PubMed Scopus (343) Google Scholar). In the developing embryo, RA transcriptionally regulates genes that specify body axis pattern and may help program limb formation(3.Maden M. Acta Biotheor. 1993; 41: 425-445Crossref PubMed Scopus (16) Google Scholar). In mature vertebrates, RA maintains epithelial tissues (prevents squamous cell metaplasia), contributes to bone remodeling, and sustains reproductive processes, including the estrus cycle, spermatogenesis, and placental growth. RA aberrant in concentration, locus, or developmental stage causes teratism and/or toxicity of the central nervous system and skeleton (4.Morriss-Kay G. Ward S. Sokolova N. Arch. Toxicol. 1994; 16: 112-117Google Scholar, 5.Kochhar D.M. Acta Pathol. Microbiol. Scand. 1967; 70: 398-404Crossref PubMed Scopus (188) Google Scholar, 6.Soprano D.R. Harnish D.C. Soprano K.J. Kochhar D.M. Jiang H. J. Nutr. 1993; 123: 367-371Crossref PubMed Scopus (11) Google Scholar, 7.Harnish D.C. Barua A.B. Soprano K.J. Soprano D.R. Differentiation. 1990; 45: 103-108Crossref PubMed Scopus (28) Google Scholar). RA acts through ligand-activated receptors that comprise two distinct subfamilies of the steroid hormone superfamily of receptors (8.Pfahl M. Skin Pharmacol. 1993; 6: 8-16Crossref PubMed Scopus (37) Google Scholar, 9.Chambon P. Gene (Amst.). 1993; 135: 223-228Crossref PubMed Scopus (75) Google Scholar, 10.Mangelsdorf D.J. Kliewer S.A. Kakizuka A. Umesono K. Evans R.M. Recent Prog. Horm. Res. 1993; 48: 99-121Crossref PubMed Google Scholar, 11.Giguere V. Endocr. Rev. 1994; 15: 61-79Crossref PubMed Google Scholar). These receptors modify transcription as homodimers or by modifying the effects of other receptors through heterodimerization. These pervasive and fundamental effects of RA, as well as the consequences of its aberrant distribution, imply that its biosynthesis must be regulated closely.In many tissues, unesterified retinol occurs bound to CRBP. Thus, holo-CRBP may provide the most abundant substrate for RA biosynthesis. Direct transfer of retinol between holo-CRBP and enzymes that catalyze RA synthesis would circumvent uncontrolled diffusion of retinol through the aqueous phase and would participate in controlling the pathways of RA synthesis by protecting retinol from opportunistic reactions catalyzed by enzymes that do not recognize the high affinity, high specificity CRBP. 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.Posch K.C. Boerman M.H.E.M. Burns R.D. Napoli J.L. Biochemistry. 1991; 30: 6224-6230Crossref PubMed Scopus (97) Google Scholar, 13.Boerman M.H.E.M. Napoli J.L. Biochemistry. 1995; 34: 7027-7037Crossref PubMed Scopus (72) Google Scholar, 14.Chai X. Boerman M.H.E.M. Zhai Y. Napoli J.L. J. Biol. Chem. 1995; 270: 3900-3904Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 15.Chai X. Zhai Y. Popescu G. Napoli J.L. J. Biol. Chem. 1995; 270: 28408-28412Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Microsomes have low retinal DH activity and do not convert the retinal produced from holo-CRBP into RA at high rates. Rather, microsome-produced retinal undergoes conversion into RA by cytosolic retinal DHs, which can interact with CRBP-retinal complexes(16.Posch K.C. Burns R.D. Napoli J.L. J. Biol. Chem. 1992; Full Text PDF PubMed Google of microsomal retinol DH, also observed cytosolic retinol DH activity that inhibited by apo-CRBP and that the not from of in the for K.C. Boerman M.H.E.M. Burns R.D. Napoli J.L. Biochemistry. 1991; 30: 6224-6230Crossref PubMed Scopus (97) Google Scholar, J.L. R. and of in and Scholar). from apo-CRBP with a cytosolic that holo-CRBP as This the presence of at least two pathways for RA from retinal synthesis in microsomes and the other retinal synthesis in cytosol. In conversion of the retinal into RA occurs in the cytosol. and have cytosolic retinol DH activity further and that RA synthesis from holo-CRBP in the absence of apo-CRBP S. G. G. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). of microsomes and to RA synthesis as well as the between two pathways and/or effects work the of microsomal and cytosolic retinol DH to RA synthesis and that RA biosynthesis between and With holo-CRBP as substrate in the absence of apo-CRBP, microsomal retinol DH has the higher specific activity and capacity of the total of to generate retinal for RA synthesis in the tissues In the presence of apo-CRBP, the in the microsomal to of the total retinal In the activity of rat liver cytosolic retinol DH from that of the isozymes expressed in rat and further that ethanol cytosolic retinol DH also that at least two forms of cytosolic retinol DH and that apo-CRBP not cytosolic retinol DH activity also conversion of retinal into Thus, that multiple and to RA were from and were by J.L. 123: PubMed Scopus Google Scholar, J.L. 1990; PubMed Scopus Google Scholar). from rat and were from as K.C. Boerman M.H.E.M. Burns R.D. Napoli J.L. Biochemistry. 1991; 30: 6224-6230Crossref PubMed Scopus (97) Google Scholar). by the with as PubMed Scopus Google Scholar). were to with the program with and from in with the J. Biol. Chem. Full Text PDF PubMed Google as K.C. Boerman M.H.E.M. Burns R.D. Napoli J.L. Biochemistry. 1991; 30: 6224-6230Crossref PubMed Scopus (97) Google Scholar, K.C. Burns R.D. Napoli J.L. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). holo-CRBP had of and in 20 and at and as with of apo-CRBP by with and bound retinol by and the DH were in were of at in of and with for microsomes and of and for the combination of microsomes and and for the × g supernatant for the in the of holo-CRBP and apo-CRBP were and in rat were with of with of RA, the phase to with and with of from and were a of in of and by K.C. Burns R.D. Napoli J.L. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, J.L. 1990; PubMed Scopus Google Scholar). in and RA in were with a × a from to in for by of at in and at RA, the phase acid at RA in and at were in with holo-CRBP and of for microsomal retinol DH or of for cytosolic retinol and were to with microsomal retinol DH and as in for cytosolic retinol and were to in the in to with microsomal retinol DH and with between and for cytosolic retinol with between and for both microsomal and cytosolic retinol and were with of cytosolic protein, and ethanol or in of at for P. J. X. J. PubMed Scopus Google Scholar). at of apo-CRBP DH in and in the inhibited cytosolic retinol DH activity by by RA synthesis ( retinal convert retinal from holo-CRBP by retinol DH activity into cytosolic RA retinal as apo-CRBP of a by apo-CRBP and of cytosolic RA synthesis in not with both These were with of holo-CRBP between and to be for and apo-CRBP, of for and apo-CRBP, of apo-CRBP RA synthesis in and the or the and of protein, were with holo-CRBP for with the of RA synthesis by or shows the axis to apo-CRBP. In the absence of apo-CRBP, and of RA were by and the to apo-CRBP not RA synthesis by the which and and of the apo-CRBP, a to that of the holo-CRBP of of RA synthesis in the by apo-CRBP, a that inhibited cytosolic RA synthesis that cytosolic retinol DH activity not to RA synthesis in the in the presence of microsomal retinol DH, must have retinal for RA synthesis by the cytosolic retinal DH of the of RA synthesis in the by higher of apo-CRBP may from a combination of of the microsomal retinol DH K.C. Boerman M.H.E.M. Burns R.D. Napoli J.L. Biochemistry. 1991; 30: 6224-6230Crossref PubMed Scopus (97) Google and a in the conversion of retinal into RA by of and Cytosolic and RA were in and a combination of and microsomes with to In the absence of apo-CRBP the of retinal produced by microsomes and RA produced by the two of the and were the cytosolic that of the of microsomal retinal synthesis that of cytosolic RA In the combination of and retinal a in of and not and RA retinal at This of the retinal from holo-CRBP by microsomes as substrate for cytosolic RA RA in the combination RA by of synthesis by retinal synthesis by RA synthesis by or by a combination of and microsomes were with holo-CRBP in the absence or the presence of apo-CRBP. In of cytosolic and/or of microsomal were the presence of apo-CRBP the of retinal produced by microsomes were quantitatively the to the retinal produced by microsomes in the absence of apo-CRBP. RA by a which the of the of RA observed in the absence of apo-CRBP In the combination of microsomes and retinal a of and not RA by the combination the and at RA by the microsomal to the combination that of the cytosolic specific of microsomal and cytosolic retinol were also in the absence and the presence of apo-CRBP retinol DH to of and retinal from holo-CRBP at of in the absence of apo-CRBP and in the presence of apo-CRBP. In the absence of apo-CRBP, cytosolic retinol DH activity to of and at a of of protein, the to microsomal retinol In the presence of apo-CRBP, cytosolic retinol DH activity not observed of between and of of or that of microsomal retinol DH in the presence of metabolism by holo-CRBP versus retinal by RA by cytosol. were for with holo-CRBP in the absence or the presence of of Microsomes into of microsomes into for its RA synthesis from holo-CRBP In the absence of apo-CRBP produced 20 of RA at a of of ( used for cytosolic retinol DH 20 of of cytosolic This of used in the the in the of cytosolic the of cytosolic retinol DH used for microsomal retinol DH of microsomes of the of RA produced to of RA by the microsomal retinol DH, of protein, higher that of cytosolic retinol In the presence of apo-CRBP RA not in at least of microsomal were RA synthesis which at a of microsomal in the absence of RA and retinal of microsomes into cytosol. RA retinal were with holo-CRBP in the absence or the presence of apo-CRBP for with of cytosolic and the of microsomal of RA from by the of microsomes into in the absence of retinal not observed the microsomal of microsomal to of protein, retinal that microsomal retinal its cytosolic conversion into In the presence of apo-CRBP the of microsomes in retinal RA observed the with the the of had in the absence of apo-CRBP. retinal in the absence of apo-CRBP. the of that apo-CRBP not retinol DH also the conversion by of retinal into the that apo-CRBP the conversion of retinal into RA, cytosolic RA from retinal bound to in the presence of of apo-CRBP apo-CRBP of the inhibited apo-CRBP to the of the of to in These that as substrate for RA the of retinal and the of RA synthesis not the of retinal synthesis not the of in RA synthesis with apo-CRBP, by of retinal by apo-CRBP. This contributes to the of RA synthesis in the by the higher of apo-CRBP of apo-CRBP the conversion of retinal into RA by cytosol. were with of cytosolic protein, and the of apo-CRBP for RA retinal were a of for the between retinal and S. A. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). of RA were in the absence of of to RA specific of microsomal retinol DH, in the absence of apo-CRBP from to of cytosolic retinol DH in and At least of the total retinol DH were microsomal in tissues, in of the total retinol DH were In the presence of apo-CRBP, the specific activity of microsomal retinol DH from to cytosolic retinol of the were in of the were in a new ethanol cytosolic retinol DH not microsomal retinol DH used to provide further into the of retinal by to RA synthesis inhibited by cytosolic RA synthesis by holo-CRBP and retinal synthesis in microsomes and and in a combination of microsomes and and These that ethanol the cytosolic retinol DH, ethanol its the cytosolic retinal and also that cytosolic retinal do not cytosolic or produced that in the combination of and of cytosolic retinol DH not RA of ethanol retinol metabolism in and a combination of and and of or a combination of microsomes and and and of protein, were for RA synthesis or microsomes were for retinal synthesis and of from holo-CRBP in the absence of ethanol or in the presence of ethanol for of of the were from the ethanol of and cytosolic retinol were by and for alcohol dehydrogenase were potent of cytosolic retinol DH activity with at not microsomal retinol and used in were to of cytosolic retinol DH from a both cytosolic retinol DH, and a higher ethanol ethanol of or inhibited microsomal retinol In to the alcohol inhibitor of alcohol V. R. PubMed Scopus Google Scholar, R. J. Biol. 1993; 45: PubMed Scopus Google potent inhibitor of both microsomal and cytosolic in a new of with and the two isozymes of the alcohol dehydrogenases expressed in rat liver were with effects microsomal and cytosolic retinol DH to retinol the of cytosolic retinol DH and to had cytosolic retinol DH and not the with retinol DHs, apo-CRBP also not or activity in to in the of and for and for cytosolic retinol DH further that the of cytosolic retinol DH and Cytosolic and DH retinol DH activity at in the presence of of or S. G. G. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). by distinct of rat liver the of were for RA synthesis from by or With synthesized RA at rates. two produced RA with with and of by two of the had RA and of the by retinal not in that had low the of NADP-dependent cytosolic retinol This with the of at least two cytosolic retinol with the cytosolic retinol DH activity by inhibited to a by ethanol the activity This further for the presence of two retinol in a new of RA in from holo-CRBP with as the that cytosolic retinal with cytosolic the cytosolic retinol DH as well as with the cytosolic retinol DH retinal into RA with as had a or the of RA synthesis from in a new work the quantitatively of microsomal retinol DH to RA biosynthesis to cytosolic retinol DH microsomal retinol DH in specific activity cytosolic retinol DH activity in a of from rat tissues, had a capacity to generate retinal of and to by apo-CRBP. were the that cytosolic retinol DH be inhibited in a of cytosolic retinol DH and microsomal retinol DH the of RA apo-CRBP inhibited cytosolic retinol DH had the of RA produced by the ethanol of cytosolic retinol DH RA in a combination of and to its retinal synthesis in These results with cytosolic retinol DH not in the presence of microsomal retinol DH or microsomal retinol DH for the quantitatively of retinal by cytosolic retinol DH in of cytosolic retinol DH to physiologically, a of holo-CRBP and apo-CRBP with in the of and microsomal retinol DH for of the capacity in of the tissues a RA in the H. P. J. P. J. 1991; PubMed Scopus Google and RA in R. Res. PubMed Scopus Google have that of by RA may provide apo-CRBP as a in a to RA synthesis from cytosolic retinol S. G. G. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). This has may the of retinol by as the of holo-CRBP as as retinol were J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). of not in of apo-CRBP. that RA acts as of in the of CRBP. of and cell retinol RA synthesis would to to generate the humoral substrate were of cytosolic retinol DH apo-CRBP would to be to RA at a RA would be of for cytosolic retinol DH in RA would be inhibited by the apo-CRBP imply cytosolic retinol DH has cytosolic retinol DH retinal by metabolism into a would with to the of cytosolic retinol DH to by apo-CRBP. cytosolic retinol DH were a would provide retinal from for conversion into work and have that microsomal retinol DH not to the K.C. Boerman M.H.E.M. Burns R.D. Napoli J.L. Biochemistry. 1991; 30: 6224-6230Crossref PubMed Scopus (97) Google Scholar, 13.Boerman M.H.E.M. Napoli J.L. Biochemistry. 1995; 34: 7027-7037Crossref PubMed Scopus (72) Google Scholar, 14.Chai X. Boerman M.H.E.M. Zhai Y. Napoli J.L. J. Biol. Chem. 1995; 270: 3900-3904Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 15.Chai X. Zhai Y. Popescu G. Napoli J.L. J. Biol. Chem. 1995; 270: 28408-28412Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). This work has also between the two rat liver isozymes and cytosolic retinol in for apo-CRBP, and cytosolic retinol DH and in for apo-CRBP, and the and cytosolic retinol DH and These results with results to study RA synthesis from retinol not bound with in from rat tissues and from the tissues of and J.L. K.R. Arch. PubMed Scopus Google Scholar, K.C. Napoli J.L. Arch. PubMed Scopus Google Scholar, K.C. Napoli J.L. Pharmacol. 1992; PubMed Scopus (11) Google Scholar). inhibited the conversion of retinol into RA catalyzed by liver from rat or In from the which of the retinol DH activity of the a Thus, had the presence of a quantitatively retinol DH activity in liver that not by that the cytosolic retinol DH activity that recognizes holo-CRBP as substrate for by S. G. G. J. Biol. Chem. 1993; Full Text PDF PubMed Google had that cytosolic retinol DH distinct from in not cytosolic retinol not P. J. X. J. PubMed Scopus Google Scholar). the other not the V. R. PubMed Scopus Google Scholar, R. J. Biol. 1993; 45: PubMed Scopus Google Scholar). cytosolic retinol DH, microsomal retinol DH, to the superfamily to be has that may to RA in to and and has as evidence has that the for and specificity in RA the of holo-CRBP as the predominant substrate available in the of of the to convert retinol into K.C. Napoli J.L. Arch. PubMed Scopus Google Scholar, K.C. Napoli J.L. Pharmacol. 1992; PubMed Scopus (11) Google and the of distinct enzymes and cytosolic retinol with in they recognize holo-CRBP as substrate(12.Posch K.C. Boerman M.H.E.M. Burns R.D. Napoli J.L. Biochemistry. 1991; 30: 6224-6230Crossref PubMed Scopus (97) Google Scholar, 13.Boerman M.H.E.M. Napoli J.L. Biochemistry. 1995; 34: 7027-7037Crossref PubMed Scopus (72) Google Scholar, 14.Chai X. Boerman M.H.E.M. Zhai Y. Napoli J.L. J. Biol. Chem. 1995; 270: 3900-3904Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 15.Chai X. Zhai Y. Popescu G. Napoli J.L. J. Biol. Chem. 1995; 270: 28408-28412Abstract Full Text Full Text PDF PubMed Scopus (89) Google cytosolic retinol DH between cytosolic retinol DH and the liver Thus, the first of ethanol of a retinol DH, by its for holo-CRBP rat liver cytosolic retinol DH, liver cytosolic retinol DH, S. G. G. J. Biol. Chem. 1993; Full Text PDF PubMed Google of ethanol the humoral system be from ethanol RA biogenesis in microsomes and in a combination of microsomes and cytosol. ethanol Google Scholar, J. R. PubMed Scopus Google and to and of Arch. Pathol. Scholar, J. Full Text PDF PubMed Scopus Google Scholar, J. Pathol. PubMed Scopus Google to J. PubMed Scopus Google Scholar, R. Biol. PubMed Scopus Google Scholar). of the has that the catalyze retinol metabolism in and ethanol may retinol Google Scholar, J. R. PubMed Scopus Google Scholar). the the of in retinol metabolism in that a cytosolic retinol DH, which not a and inhibited by low of the of rat liver in the cytosolic retinol in to the of the This two or cytosolic retinol and a NADP-dependent potent ethanol of the the NADP-dependent activity versus with the of two distinct also that or can the conversion of retinal into isozymes with retinal dehydrogenase activity have by of from rat major of the total liver cytosolic has and not as K.C. Burns R.D. Napoli J.L. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). other and isozymes from and have not with and may not from the NADP-dependent activity observed with the of cytosolic retinol DH the of enzymes and/or pathways in RA new insights have from quantitatively has of microsomal versus cytosolic retinol DH to RA including the of microsomal retinol DH to retinal produced by cytosolic retinol At least two cytosolic retinol have a DH and a NADP-dependent ethanol in cytosolic retinol RA the between cytosolic retinol DH and the and RA synthesis by microsomes or a combination of microsomes and cytosol. the of retinal has and that RA biogenesis pathways that multiple enzymes that recognize both liganded and nonliganded forms of CRBP. INTRODUCTIONThe metabolism of retinol (vitamin A) generates RA, ( 1The abbreviations used are: RAretinoic acidADHalcohol dehydrogenaseCRBPcellular retinol-binding protein, type IDHdehydrogenaseHPLChigh performance liquid chromatography10kS10,000 × g supernatant of a rat liver homogenate.) a humoral factor critical to vertebrate development(1.Napoli J.L. Posch K.C. Fiorella P.D. Boerman M.H.E.M. Biomed. Pharmacother. 1991; 45: 131-143Crossref PubMed Scopus (100) Google Scholar, 2.Blomhoff R. Green M.H. Green J.B. Berg T. Norum K.R. Physiol. Rev. 1991; 71: 951-990Crossref PubMed Scopus (343) Google Scholar). In the developing embryo, RA transcriptionally regulates genes that specify body axis pattern and may help program limb formation(3.Maden M. Acta Biotheor. 1993; 41: 425-445Crossref PubMed Scopus (16) Google Scholar). In mature vertebrates, RA maintains epithelial tissues (prevents squamous cell metaplasia), contributes to bone remodeling, and sustains reproductive processes, including the estrus cycle, spermatogenesis, and placental growth. RA aberrant in concentration, locus, or developmental stage causes teratism and/or toxicity of the central nervous system and skeleton (4.Morriss-Kay G. Ward S. Sokolova N. Arch. Toxicol. 1994; 16: 112-117Google Scholar, 5.Kochhar D.M. Acta Pathol. Microbiol. Scand. 1967; 70: 398-404Crossref PubMed Scopus (188) Google Scholar, 6.Soprano D.R. Harnish D.C. Soprano K.J. Kochhar D.M. Jiang H. J. Nutr. 1993; 123: 367-371Crossref PubMed Scopus (11) Google Scholar, 7.Harnish D.C. Barua A.B. Soprano K.J. Soprano D.R. Differentiation. 1990; 45: 103-108Crossref PubMed Scopus (28) Google Scholar). RA acts through ligand-activated receptors that comprise two distinct subfamilies of the steroid hormone superfamily of receptors (8.Pfahl M. Skin Pharmacol. 1993; 6: 8-16Crossref PubMed Scopus (37) Google Scholar, 9.Chambon P. Gene (Amst.). 1993; 135: 223-228Crossref PubMed Scopus (75) Google Scholar, 10.Mangelsdorf D.J. Kliewer S.A. Kakizuka A. Umesono K. Evans R.M. Recent Prog. Horm. Res. 1993; 48: 99-121Crossref PubMed Google Scholar, 11.Giguere V. Endocr. Rev. 1994; 15: 61-79Crossref PubMed Google Scholar). These receptors modify transcription as homodimers or by modifying the effects of other receptors through heterodimerization. These pervasive and fundamental effects of RA, as well as the consequences of its aberrant distribution, imply that its biosynthesis must be regulated closely.In many tissues, unesterified retinol occurs bound to CRBP. Thus, holo-CRBP may provide the most abundant substrate for RA biosynthesis. Direct transfer of retinol between holo-CRBP and enzymes that catalyze RA synthesis would circumvent uncontrolled diffusion of retinol through the aqueous phase and would participate in controlling the pathways of RA synthesis by protecting retinol from opportunistic reactions catalyzed by enzymes that do not recognize the high affinity, high specificity CRBP. 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.Posch K.C. Boerman M.H.E.M. Burns R.D. Napoli J.L. Biochemistry. 1991; 30: 6224-6230Crossref PubMed Scopus (97) Google Scholar, 13.Boerman M.H.E.M. Napoli J.L. Biochemistry. 1995; 34: 7027-7037Crossref PubMed Scopus (72) Google Scholar, 14.Chai X. Boerman M.H.E.M. Zhai Y. Napoli J.L. J. Biol. Chem. 1995; 270: 3900-3904Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 15.Chai X. Zhai Y. Popescu G. Napoli J.L. J. Biol. Chem. 1995; 270: 28408-28412Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Microsomes have low retinal DH activity and do not convert the retinal produced from holo-CRBP into RA at high rates. Rather, microsome-produced retinal undergoes conversion into RA by cytosolic retinal DHs, which can interact with CRBP-retinal complexes(16.Posch K.C. Burns R.D. Napoli J.L. J. Biol. Chem. 1992; Full Text PDF PubMed Google of microsomal retinol DH, also observed cytosolic retinol DH activity that inhibited by apo-CRBP and that the not from of in the for K.C. Boerman M.H.E.M. Burns R.D. Napoli J.L. Biochemistry. 1991; 30: 6224-6230Crossref PubMed Scopus (97) Google Scholar, J.L. R. and of in and Scholar). from apo-CRBP with a cytosolic that holo-CRBP as This the presence of at least two pathways for RA from retinal synthesis in microsomes and the other retinal synthesis in cytosol. In conversion of the retinal into RA occurs in the cytosol. and have cytosolic retinol DH activity further and that RA synthesis from holo-CRBP in the absence of apo-CRBP S. G. G. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). of microsomes and to RA synthesis as well as the between two pathways and/or effects work the of microsomal and cytosolic retinol DH to RA synthesis and that RA biosynthesis between and With holo-CRBP as substrate in the absence of apo-CRBP, microsomal retinol DH has the higher specific activity and capacity of the total of to generate retinal for RA synthesis in the tissues In the presence of apo-CRBP, the in the microsomal to of the total retinal In the activity of rat liver cytosolic retinol DH from that of the isozymes expressed in rat and further that ethanol cytosolic retinol DH also that at least two forms of cytosolic retinol DH and that apo-CRBP not cytosolic retinol DH activity also conversion of retinal into Thus, that multiple and to RA biosynthesis.