Excessive vitamin A toxicity in mice genetically deficient in either alcohol dehydrogenase Adh1 or Adh3.

Alcohol dehydrogenase (ADH) deficiency results in decreased retinol utilization, but it is unclear what physiological roles the several known ADHs play in retinoid signaling. Here, Adh1, Adh3, and Adh4 null mutant mice have been examined following acute and chronic vitamin A excess. Following an acute dose of retinol (50 mg.kg(-1)), metabolism of retinol to retinoic acid in liver was reduced 10-fold in Adh1 mutants and 3.8-fold in Adh3 mutants, but was not significantly reduced in Adh4 mutants. Acute retinol toxicity, assessed by determination of the LD(50) value, was greatly increased in Adh1 mutants and moderately increased in Adh3 mutants, but only a minor effect was observed in Adh4 mutants. When mice were propagated for one generation on a retinol-supplemented diet containing 10-fold higher vitamin A than normal, Adh3 and Adh4 mutants had essentially the same postnatal survival to adulthood as wild-type (92-95%), but only 36% of Adh1 mutants survived to adulthood with the remainder dying by postnatal day 3. Adh1 mutants surviving to adulthood on the retinol- supplemented diet had elevated serum retinol signifying a clearance defect and elevated aspartate aminotransferase indicative of increased liver damage. These findings indicate that ADH1 functions as the primary enzyme responsible for efficient oxidative clearance of excess retinol, thus providing protection and increased survival during vitamin A toxicity. ADH3 plays a secondary role. Our results also show that retinoic acid is not the toxic moiety during vitamin A excess, as Adh1 mutants have less retinoic acid production while experiencing increased toxicity.     

Impaired retinol utilization in Adh4 alcohol dehydrogenase mutant mice

Adh4, a member of the mouse alcohol dehydrogenase (ADH) gene family, encodes an enzyme that functions in vitro as a retinol dehydrogenase in the conversion of retinol to retinoic acid, an important developmental signaling molecule. To explore the role of Adh4 in retinoid signaling in vivo, gene targeting was used to create a null mutation at the Adh4 locus. Homozygous Adh4 mutant mice were viable and fertile and demonstrated no obvious defects when maintained on a standard mouse diet. However, when subjected to vitamin A deficiency during gestation, Adh4 mutant mice demonstrated a higher number of stillbirths than did wild‐type mice. The proportion of liveborn second generation vitamin A‐deficient newborn mice was only 15% for Adh4 mutant mice but 49% for wild‐type mice. After retinol administration to vitamin A‐deficient dams in order to rescue embryonic development, Adh4 mutant mice demonstrated a higher resorption rate at stage E12.5 (69%), compared with wild‐type mice (30%). The relative ability of Adh4 mutant and wild‐type mice to metabolize retinol to retinoic acid was measured after administration of a 100‐mg/kg dose of retinol. Whereas kidney retinoic acid levels were below the level of detection in all vehicle‐treated mice (<1 pmol/g), retinol treatment resulted in very high kidney retinoic acid levels in wild‐type mice (273 pmol/g) but 8‐fold lower levels in Adh4 mutant mice (32 pmol/g), indicating a defect in metabolism of retinol to retinoic acid. These findings demonstrate that another retinol dehydrogenase can compensate for a lack of Adh4 when vitamin A is sufficient, but that Adh4 helps optimize retinol utilization under conditions of both retinol deficiency and excess. Dev. Genet. 25:1–10, 1999. © 1999 Wiley‐Liss, Inc.     

Genetic dissection of retinoid dehydrogenases

Biochemical studies indicate that alcohol dehydrogenase (ADH) metabolizes retinol to retinal, and that aldehyde dehydrogenase (ALDH) metabolizes retinal to retinoic acid, a molecule essential for growth and development. Summarized herein are several genetic studies supporting in vivo functions for ADH and ALDH in retinoic acid synthesis. Gene targeting was used to create knockout mice for either Adh1 or Adh4. Both knockout mice were viable and fertile without obvious defects. However, when wild-type and Adh4 knockout mice were subjected to vitamin A deficiency during gestation, the survival rate at birth was 3.3-fold lower for Adh4 knockout mice. When adult mice were examined for production of retinoic acid following retinol administration, Adh1 knockout mice exhibited 10-fold lower retinoic acid levels in liver compared with wild-type, whereas Adh4 knockout mice differed from wild-type by less than 2-fold. Thus, Adh1 plays a major role in the metabolism of a large dose of retinol to retinoic acid in adults, whereas Adh4 plays a role in maintaining sufficient retinol metabolism for development during retinol deficiency. ALDHs were examined by overexpression studies in frog embryos. Injection of mRNAs for either mouse Raldh1 or Raldh2 stimulated retinoic acid synthesis in frog embryos at the blastula stage when retinoic acid is normally undetectable. Overexpression of human ALDH2, human ALDH3, and mouse Aldh-pb did not stimulate retinoic acid production. In addition, Raldh2 knockout mice exhibit embryonic lethality with defects in retinoid-dependent tissues. Overall, these studies provide genetic evidence that Adh1, Adh4, Raldh1, and Raldh2 encode retinoid dehydrogenases involved in retinoic acid synthesis in vivo.     

Metabolic deficiencies in alcohol dehydrogenase Adh1, Adh3, and Adh4 null mutant mice. Overlapping roles of Adh1 and Adh4 in ethanol clearance and metabolism of retinol to retinoic acid.

Targeting of mouse alcohol dehydrogenase genes Adh1, Adh3, and Adh4 resulted in null mutant mice that all developed and reproduced apparently normally but differed markedly in clearance of ethanol and formaldehyde plus metabolism of retinol to the signaling molecule retinoic acid. Following administration of an intoxicating dose of ethanol, Adh1 -/- mice, and to a lesser extent Adh4 -/- mice, but not Adh3 -/- mice, displayed significant reductions in blood ethanol clearance. Ethanol-induced sleep was significantly longer only in Adh1 -/- mice. The incidence of embryonic resorption following ethanol administration was increased 3-fold in Adh1 -/- mice and 1.5-fold in Adh4 -/- mice but was unchanged in Adh3 -/- mice. Formaldehyde toxicity studies revealed that only Adh3 -/- mice had a significantly reduced LD50 value. Retinoic acid production following retinol administration was reduced 4.8-fold in Adh1 -/- mice and 8.5-fold in Adh4 -/- mice. Thus, Adh1 and Adh4 demonstrate overlapping functions in ethanol and retinol metabolism in vivo, whereas Adh3 plays no role with these substrates but instead functions in formaldehyde metabolism. Redundant roles for Adh1 and Adh4 in retinoic acid production may explain the apparent normal development of mutant mice.     

Genetic dissection of retinoid dehydrogenases

Biochemical studies indicate that alcohol dehydrogenase (ADH) metabolizes retinol to retinal, and that aldehyde dehydrogenase (ALDH) metabolizes retinal to retinoic acid, a molecule essential for growth and development. Summarized herein are several genetic studies supporting in vivo functions for ADH and ALDH in retinoic acid synthesis. Gene targeting was used to create knockout mice for either Adh1 or Adh4. Both knockout mice were viable and fertile without obvious defects. However, when wild-type and Adh4 knockout mice were subjected to vitamin A deficiency during gestation, the survival rate at birth was 3.3-fold lower for Adh4 knockout mice. When adult mice were examined for production of retinoic acid following retinol administration, Adh1 knockout mice exhibited 10-fold lower retinoic acid levels in liver compared with wild-type, whereas Adh4 knockout mice differed from wild-type by less than 2-fold. Thus, Adh1 plays a major role in the metabolism of a large dose of retinol to retinoic acid in adults, whereas Adh4 plays a role in maintaining sufficient retinol metabolism for development during retinol deficiency. ALDHs were examined by overexpression studies in frog embryos. Injection of mRNAs for either mouse Raldh1 or Raldh2 stimulated retinoic acid synthesis in frog embryos at the blastula stage when retinoic acid is normally undetectable. Overexpression of human ALDH2, human ALDH3, and mouse Aldh-pb did not stimulate retinoic acid production. In addition, Raldh2 knockout mice exhibit embryonic lethality with defects in retinoid-dependent tissues. Overall, these studies provide genetic evidence that Adh1, Adh4, Raldh1, and Raldh2 encode retinoid dehydrogenases involved in retinoic acid synthesis in vivo.     

Genetic evidence that retinaldehyde dehydrogenase Raldh1 (Aldh1a1) functions downstream of alcohol dehydrogenase Adh1 in metabolism of retinol to retinoic acid

Vitamin A (retinol) is a nutrient that is essential for developmental regulation but toxic in large amounts. Previous genetic studies have revealed that alcohol dehydrogenase Adh1 is required for efficient clearance of excess retinol to prevent toxicity, thus demonstrating that the mechanism involves oxidation of excess retinol to retinoic acid (RA). Whereas Adh1 plays a dominant role in the first step of the clearance pathway (oxidation of retinol to retinaldehyde), it is unknown what controls the second step (oxidation of retinaldehyde to RA). We now present genetic evidence that aldehyde dehydrogenase Aldh1a1, also known as retinaldehyde dehydrogenase Raldh1, plays a dominant role in the second step of retinol clearance in adult mice. Serum RA levels following a 50 mg/kg dose of retinol were reduced 72% in Raldh1-/- mice and 82% in Adh1-/- mice. This represented reductions in RA synthesis of 77-78% for each mutant after corrections for altered RA degradation in each. After retinol dosing, serum retinaldehyde was increased 2.5-fold in Raldh1-/- mice (indicating defective retinaldehyde clearance) and decreased 3-fold in Adh1-/- mice (indicating defective retinaldehyde synthesis). Serum retinol clearance following retinol administration was decreased 7% in Raldh1-/- mice and 69% in Adh1-/- mice. LD50 studies indicated a small increase in retinol toxicity in Raldh1-/- mice and a large increase in Adh1-/- mice. These observations demonstrate that Raldh1 functions downstream of Adh1 in the oxidative metabolism of excess retinol and that toxicity correlates primarily with accumulating retinol rather than retinaldehyde.     

Families of retinoid dehydrogenases regulating vitamin A function: production of visual pigment and retinoic acid.

Vitamin A (retinol) and provitamin A (beta-carotene) are metabolized to specific retinoid derivatives which function in either vision or growth and development. The metabolite 11-cis-retinal functions in light absorption for vision in chordate and nonchordate animals, whereas all-trans-retinoic acid and 9-cis-retinoic acid function as ligands for nuclear retinoic acid receptors that regulate gene expression only in chordate animals. Investigation of retinoid metabolic pathways has resulted in the identification of numerous retinoid dehydrogenases that potentially contribute to metabolism of various retinoid isomers to produce active forms. These enzymes fall into three major families. Dehydrogenases catalyzing the reversible oxidation/reduction of retinol and retinal are members of either the alcohol dehydrogenase (ADH) or short-chain dehydrogenase/reductase (SDR) enzyme families, whereas dehydrogenases catalyzing the oxidation of retinal to retinoic acid are members of the aldehyde dehydrogenase (ALDH) family. Compilation of the known retinoid dehydrogenases indicates the existence of 17 nonorthologous forms: five ADHs, eight SDRs, and four ALDHs, eight of which are conserved in both mouse and human. Genetic studies indicate in vivo roles for two ADHs (ADH1 and ADH4), one SDR (RDH5), and two ALDHs (ALDH1 and RALDH2) all of which are conserved between humans and rodents. For several SDRs (RoDH1, RoDH4, CRAD1, and CRAD2) androgens rather than retinoids are the predominant substrates suggesting a function in androgen metabolism as well as retinoid metabolism.     

Molecular docking studies on interaction of diverse retinol structures with human alcohol dehydrogenases predict a broad role in retinoid ligand synthesis.

Some members of the human alcohol dehydrogenase (ADH) family possess retinol dehydrogenase activity and may thus function in production of the active nuclear receptor ligand retinoic acid. Many diverse natural forms of retinol exist including all-trans-retinol (vitamin A(1)), 9-cis-retinol, 3,4-didehydroretinol (vitamin A(2)), 4-oxo-retinol, and 4-hydroxy-retinol as well as their respective carboxylic acid derivatives which are active ligands for retinoid receptors. This raises the question of whether ADHs can accommodate all these different retinols and thus participate in the activation of several retinoid ligands. The crystal structures of human ADH1B and ADH4 provide the opportunity to examine their active sites for potential binding to many diverse retinol structures using molecular docking algorithms. The criteria used to score successful docking included achievement of distances of 1.9-2.4 A between the catalytic zinc and the hydroxyl oxygen of retinol and 3.2-3.6 A between C-4 of the coenzyme NAD and C-15 of retinol. These distances are sufficient to enable hydride transfer during the oxidation of an alcohol to an aldehyde. By these criteria, all-trans-retinol, 4-oxo-retinol, and 4-hydroxy-retinol were successfully docked to both ADH1B and ADH4. However, 9-cis-retinol and 3,4-didehydroretinol, which have more restrictive conformations, were successfully docked to only ADH4 which possesses a wider active site than ADH1B and more easily accommodates the C-19 methyl group. Furthermore, docking of all retinols was more favorable in the active site of ADH4 rather than ADH1B as measured by force field and contact scores. These findings suggest that ADH1B has a limited capacity to metabolize retinols, but that ADH4 is well suited to function in the metabolism of many diverse retinols and is predicted to participate in the synthesis of the active ligands all-trans-retinoic acid, 9-cis-retinoic acid, 3, 4-didehydroretinoic acid, 4-oxo-retinoic acid, and 4-hydroxy-retinoic acid.     

Hypoxic Induction of Anoxia Tolerance in Roots of Adh1 Null Zea mays L

Seedlings of alcohol dehydrogenase 1 null mutants (Adh1-) of Zea mays L., which fail to synthesize alcohol dehydrogenase 1 (ADH1) isozymes, were hypoxically acclimated by 18 h of exposure to an atmosphere of 4% (v/v) O2 in N2 at 25[deg]C. Their ability to tolerate subsequent anoxia by exposure to anaerobic (O2-free) conditions was compared with that of unacclimated seedlings that were transferred immediately from an atmosphere of 40% (v/v) O2 to anaerobic conditions. Only 10% of the root tips of unacclimated seminal roots survived 6 h of anoxia, whereas 70% of the hypoxically acclimated root tips were viable at 24 h. During anoxia, acclimated root tips had enhanced ADH activity compared with unacclimated root tips, through induction of Adh2. Despite this, enzyme activity was still only about 5% that of acclimated, wild-type root tips and about half that of unacclimated, wild-type root tips. During anoxia, acclimated Adh1- root tips showed a higher rate of anaerobic respiration and ethanol production, greater concentrations of ATP and total adenylates, and a greater adenylate energy charge compared with unacclimated root tips. These results suggest that although enhanced ADH activity may have raised fermentation rates in acclimated Adh1- tissues and thereby contributed to energy metabolism and viability, the high levels of ADH activity inducible in acclimated, wild-type maize root tips appear to be in excess of that required to increase rates of fermentation.     

Relationship between alcohol dehydrogenase activity and low-temperature in two maize genotypes, Silverado F1 and Adh1-Adh2- doubly null.

We have examined the role of alcohol dehydrogenase (ADH, E.C.1.1.1.1) in chilling tolerance using maize (Zea mays L.) Adh1(-)Adh2(-) doubly null mutant. Adh1(-)Adh2(-) doubly null seedlings were found to have lowered survival rates compared to non-doubly null maize seedlings (Silverado F(1)) when held at 2 degrees C for varying periods. Exposure to ethanol did not increase the chilling tolerance in either Silverado F(1) or Adh1(-)Adh2(-) doubly null. ADH activity in Silverado F(1) remained steady when held at 2 degrees C for up to 3 d. ADH1 protein accumulation in chilled Silverado F(1) seedlings remained unchanged throughout the period of cold exposure. Chilling led to a significant inhibition of the P-H(+)-ATPase (E.C. 3.6.3.6) activity in Adh1(-)Adh2(-)doubly null, but minimal inhibition was seen in Silverado F(1). Though P-H(+)-ATPase activity in Adh1(-)Adh2(-) decreased, P-H(+)-ATPase protein levels remained constant during the chilling period. Levels of ATP slightly fluctuated in both types of seedlings during the duration of chilling. Lipid peroxidation levels in Adh1(-)Adh2(-) doubly null increased with chilling exposure, but not in the Silverado F(1). We suggest that ADH activity may play a role in chilling tolerance that is not related to maintenance of glycolysis and ATP production as has been observed during oxygen depravation.     

Retinol metabolism in LLC-PK1 Cells. Characterization of retinoic acid synthesis by an established mammalian cell line

Specific assays, based on gas chromatography-mass spectrometry and high-performance liquid chromatography, were used to quantify the conversion of retinol and retinal into retinoic acid by the pig kidney cell line LLC-PK1. Retinoic acid synthesis was linear for 2-4 h as well as with graded amounts of either substrate to at least 50 microM. Retinoic acid concentrations increased through 6-8 h, but decreased thereafter because of substrate depletion (t1/2 of retinol = 13 h) and product metabolism (1/2 = 2.3 h). Retinoic acid metabolism was accelerated by treating cells with 100 nM retinoic acid for 10 h (t1/2 = 1.7 h) and was inhibited by the antimycotic imidazole ketoconazole. Feedback inhibition was not indicated since retinoic acid up to 100 nM did not inhibit its own synthesis. Retinol dehydrogenation was rate-limiting. The reduction and dehydrogenation of retinal were 4-8-fold and 30-60-fold faster, respectively. Greater than 95% of retinol was converted into metabolites other than retinoic acid, whereas the major metabolite of retinal was retinoic acid. The synthetic retinoid 13-cis-N-ethylretinamide inhibited retinoic acid synthesis, but 4-hydroxylphenylretinamide did not. 4'-(9-Acridinylamino)methanesulfon-m-anisidide, an inhibitor of aldehyde oxidase, and ethanol did not inhibit retinoic acid synthesis. 4-Methylpyrazole was a weak inhibitor: disulfiram was a potent inhibitor. These data indicate that retinol dehydrogenase is a sulfhydryl group-dependent enzyme, distinct from ethanol dehydrogenase. Homogenates of LLC-PK1 cells converted retinol into retinoic acid and retinyl palmitate and hydrolyzed retinyl palmitate. This report suggests that substrate availability, relative to enzyme activity/amount, is a primary determinant of the rate of retinoic acid synthesis, identifies inhibitors of retinoic acid synthesis, and places retinoic acid synthesis into perspective with several other known pathways of retinoid metabolism.     

Retinol forms retinoic acid via retinal.

Hepatic cytosol from normal deermice having cytosolic alcohol dehydrogenase (ADH+) also displays retinol dehydrogenase activity and converts retinol to retinoic acid, whereas cytosol from ADH- deermice lacks these enzyme activities and does not produce retinoic acid. Furthermore, microsomes from either strain do not convert retinol to retinoic acid. However, when cytosol from ADH- animals is added to the microsomes, retinoic acid is produced. The obligatory role of retinal as an intermediary step in retinoic acid formation is further shown by isotopic dilution of retinoic acid formed from labeled retinol upon addition of unlabeled retinal. Microsomal retinol dehydrogenase also catalyzes the reduction of retinal to retinol, thereby explaining the decrease in retinoic acid production from retinol in liver cytosol of ADH+ deermice when microsomes are added. Thus, the results of this study indicate that retinal is an obligatory intermediate in the hepatic production of retinoic acid from retinol and that cytosolic and microsomal retinol dehydrogenases play a key role in this process.     

Comparative functional analysis of human medium-chain dehydrogenases, short-chain dehydrogenases/reductases and aldo-keto reductases with retinoids

Retinoic acid biosynthesis in vertebrates occurs in two consecutive steps: the oxidation of retinol to retinaldehyde followed by the oxidation of retinaldehyde to retinoic acid. Enzymes of the MDR (medium-chain dehydrogenase/reductase), SDR (short-chain dehydrogenase/reductase) and AKR (aldo-keto reductase) superfamilies have been reported to catalyse the conversion between retinol and retinaldehyde. Estimation of the relative contribution of enzymes of each type was difficult since kinetics were performed with different methodologies, but SDRs would supposedly play a major role because of their low K(m) values, and because they were found to be active with retinol bound to CRBPI (cellular retinol binding protein type I). In the present study we employed detergent-free assays and HPLC-based methodology to characterize side-by-side the retinoid-converting activities of human MDR [ADH (alcohol dehydrogenase) 1B2 and ADH4), SDR (RoDH (retinol dehydrogenase)-4 and RDH11] and AKR (AKR1B1 and AKR1B10) enzymes. Our results demonstrate that none of the enzymes, including the SDR members, are active with CRBPI-bound retinoids, which questions the previously suggested role of CRBPI as a retinol supplier in the retinoic acid synthesis pathway. The members of all three superfamilies exhibit similar and low K(m) values for retinoids (0.12-1.1 microM), whilst they strongly differ in their kcat values, which range from 0.35 min(-1) for AKR1B1 to 302 min(-1) for ADH4. ADHs appear to be more effective retinol dehydrogenases than SDRs because of their higher kcat values, whereas RDH11 and AKR1B10 are efficient retinaldehyde reductases. Cell culture studies support a role for RoDH-4 as a retinol dehydrogenase and for AKR1B1 as a retinaldehyde reductase in vivo.     

A deletion adjacent to the maize transposable element Mu-1 accompanies loss of Adh1 expression.

Insertion of the maize transposable element Mu-1 into the first intron of the alcohol dehydrogenase locus (Adh1) of maize produced mutant Adh1-S3034 with 40% of the wild-type level of protein and mRNA. Continued instability at this locus resulted in secondary mutations with lower levels of protein expression. One of these, Adh1-S3034a, has no detectable ADH1 expression. This paper describes the precise nature of the changes in the Adh1 gene that gave rise to the S3034a allele. The Mu-1 element is still present in the mutant, but Adh1 sequences immediately adjacent to the element are deleted. The deletion starts precisely at the Mu-1 insertion site and extends 74 bp leftward removing part of the first intron, the intron:exon junction and 2 bp of the eleventh amino acid codon in the first exon of the gene. Tests for reversion within the somatic tissue of plants show that mutant S3034a, unlike its progenitor, is stably null for ADH1 activity.     

Comparative expression analysis of Adh3 during arthropod,urochordate, cephalochordate,and vertebrate development challenges its predicted housekeeping role

Gene and genome duplications in the vertebrate lineage explain the complexity of extant gene families. Among these, the medium-chain alcohol dehydrogenase (ADH), which expanded by tandem duplications after the cephalochordate-vertebrate split, is a good model with which to analyze the evolution of gene function. Although the ancestral member of this family, ADH3, has been strictly conserved throughout animal evolution, its physiological role is still controversial. Previous evidence indicates that it contributes to formaldehyde cytoprotection, retinoic acid metabolism, and nitric oxide homeostasis. We performed in situ hybridization during Drosophila, ascidian (Ciona intestinalis), and zebrafish (Danio rerio) development. We showed that Adh3 expression was restricted to the fat body in Drosophila embryos at stage 17 and to the anterior endoderm in C. intestinalis tail bud, whereas in the zebrafish 2.5-day larvae the signal appeared widespread. A more comprehensive expression analysis including amphioxus and mice revealed that ancestral Adh3 was tissue specific, whereas a widespread expression was later attained in vertebrates. These variations occurred concomitantly with the expansion of the ADH family and the acquisition of new functions but were unlinked to the genomic changes that led to the transition from fractional to global methylation in vertebrates. Our data challenge the housekeeping role of ADH3 and question its involvement in the prevertebrate retinoic acid pathway.     

Anaerobic tolerant null: a mutant that allows Adh1 nulls to survive anaerobic treatment

Anaerobic tolerant null (ATN) is a recessive factor that allows alcohol dehydrogenase-1 (ADH1) null individuals of Zea mays L. to survive 24 h of anaerobic conditions. ADH1 null lines that do not possess this factor survive only a few hours of anoxia. We studied ADH activity levels in protein extracts from the primary root tissue of ATN. ADH levels were similar in ATN and other ADH1 null lines, suggesting that ADH activity does not account for differences in the ability of ATN to survive anaerobic treatment. The ATN survival trait segregated as a single recessive locus in crosses between ATN and double null (Adh1-S5657, Adh2-33). We also made crosses between ATN and 1s2p, an inbred line with ADH1 activity that carries an electrophoretic mutation of Adh2, to determine whether atn increases the number of survivors over that which would be expected from the segregation of Adh1 alone and to use the Adh2P allele to study the cosegregation of Adh2 and atn. The observed number of survivors in that cross exceeded the expected number of survivors by a margin consistent with a single recessive gene adding to the ADH+ survivors. Extracts from the primary root or scutellum of induced F2 seedlings from the above crosses were assayed for ADH activity by native polyacrylamide gel electrophoresis (PAGE) and simultaneously scored for survival to determine whether Adh2 and atn were segregating independently. We screened the (ATN x 1s2p)F2 progeny for ADH1 activity by staining root tips with an ADH-specific stain to select Adh1 null individuals prior to gel assay. Atn was found to be assorting independently of Adh1 and Adh2 in both crosses.     

Analysis of ENU-induced mutations at the Adh locus in Drosophila melanogaster

N-Ethyl-N-nitrosourea (ENU) was used to induce mutations in the Drosophila melanogaster, alcohol dehydrogenase (Adh) gene. Flies were treated with ENU and mated to homozygous intragenic Adh null mutants; Adh null mutations were selected by exposure of the F1 generation to 1-penten-3-ol. Fourteen Adh null mutations were recovered which included 11 from spermatozoa, 2 from oocytes and 1 from a premeiotic spermatocyte. 2 mutations from spermatozoa and 1 of the mutations from oocytes were multilocus deficiencies which included the Adh locus as determined by complementation tests. The remaining 11 intragenic Adh null mutations were sequenced using the Sanger dideoxy method. One Adh null mutation induced in an oocyte was an AT to TA transversion and the mutation induced in a premeiotic spermatocyte was a GC to AT transition, both of which resulted in a single amino acid substitution. The 11 null mutations induced in spermatozoa were a data set in which both the dose of ENU and the treated germ-cell stage were held constant; therefore, only these 11 mutations were used to calculate the mutation frequency and compare the mutations at the Adh locus with those recovered in other studies. The dose of ENU induced a sex-linked recessive lethal frequency approximately 300 times that of the spontaneous frequency; therefore, these mutations were assumed to have been induced by ENU. 2 of the 11 mutations induced in spermatozoa were multilocus deficiencies and 9 were intragenic mutations. 7 of the 9 intragenic mutations were GC to AT transitions which resulted in 5 single amino acid substitutions, 1 premature translation termination codon, and 1 splice site mutation.(ABSTRACT TRUNCATED AT 400 WORDS)     

The specificity of alcohol dehydrogenase with cis-retinoids. Activity with 11-cis-retinol and localization in retina.

Studies in knockout mice support the involvement of alcohol dehydrogenases ADH1 and ADH4 in retinoid metabolism, although kinetics with retinoids are not known for the mouse enzymes. Moreover, a role of alcohol dehydrogenase (ADH) in the eye retinoid interconversions cannot be ascertained due to the lack of information on the kinetics with 11-cis-retinoids. We report here the kinetics of human ADH1B1, ADH1B2, ADH4, and mouse ADH1 and ADH4 with all-trans-, 7-cis-, 9-cis-, 11-cis- and 13-cis-isomers of retinol and retinal. These retinoids are substrates for all enzymes tested, except the 13-cis isomers which are not used by ADH1. In general, human and mouse ADH4 exhibit similar activity, higher than that of ADH1, while mouse ADH1 is more efficient than the homologous human enzymes. All tested ADHs use 11-cis-retinoids efficiently. ADH4 shows much higher k(cat)/K(m) values for 11-cis-retinol oxidation than for 11-cis-retinal reduction, a unique property among mammalian ADHs for any alcohol/aldehyde substrate pair. Docking simulations and the kinetic properties of the human ADH4 M141L mutant demonstrated that residue 141, in the middle region of the active site, is essential for such ADH4 specificity. The distinct kinetics of ADH4 with 11-cis-retinol, its wide specificity with retinol isomers and its immunolocalization in several retinal cell layers, including pigment epithelium, support a role of this enzyme in the various retinol oxidations that occur in the retina. Cytosolic ADH4 activity may complement the isomer-specific microsomal enzymes involved in photopigment regeneration and retinoic acid synthesis.