Molecular cloning of a full-length cDNA for human alcohol dehydrogenase.

We have cloned a full-length cDNA coding for human alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) from a human liver cDNA library constructed in phage lambda gt11. The library was screened by using a rabbit antibody against human ADH as a first probe, by the modified method of Young and Davis [Young, R. A. & Davis, R. W. (1983) Proc. Natl. Acad. Sci. USA 80, 1194-1198]. Mixed 14-mer synthetic oligonucleotides encoding Asp-Asp-His-Val-Val and Gln-Cys-Gly-Lys-Cys were used as a second probe. These amino acid sequences are considered to be common in all three subunits (alpha, beta, and gamma) controlled by the ADH1, ADH2, and ADH3 loci. Ten lambda gt11 recombinants of 35 positive plaques obtained by antibody screening contained inserted cDNAs of 1.5-2.4 kilobase pairs and were found to exhibit positive signals by hybridization with synthetic probes. One of them, with an inserted cDNA of 1631 base pairs, contained a sequence that encodes 374 amino acid residues of the human beta 1 subunit, a chain initiation codon, a chain termination codon, and additional 3' and 5' untranslated regions. A complete amino acid sequence of the human beta 1 subunit was deduced from the cDNA.     

New molecular forms of human liver alcohol dehydrogenase: isolation and characterization of ADHIndianapolis.

The biochemical determinants of alcoholism and genetic correlates for the variability in man's response to alcohol have remained obscure until recently. The identification of genetically determined isoenzymes of alcohol dehydrogenase with different catalytic properties may bear importantly upon this problem. New molecular forms of human liver alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) have recently been identified in 16% of the liver specimens from an urban population from Indianapolis, Indiana [Bosron, W. F., Li, T.-K. & Vallee, B. L. (1979) Biochem. Biophys. Res. Commun. 91, 1549-1555]. The distinguishing features of these specimens were (i) they showed activity optima for ethanol oxidation at both pH 7.0 and 10.0 and (ii) they formed electrophoretic bands cathodic to the beta beta isoenzyme. From such livers, three new ADH forms have now been isolated, one of which has a single pH optimum at 7.0 and two of which have dual optima at pH 7.0 and 10.0. These new forms were designated ADHIndianapolis forms 1,2, and 3, respectively. They can be differentiated from previously described ADH isoenzymes, including the so-called "atypical" isoenzyme, by their electrophoretic mobility, pH optima, and Km for ethanol (approximately 60 mM at pH 7.5). Based upon the electrophoretic pattern of livers containing ADHIndianapolis and the mobility of the three isolated molecular forms, ADHIndianapolis may be the result of polymorphism at the ADH2 gene locus, which codes for the beta subunit.     

Hypoxically inducible barley lactate dehydrogenase: cDNA cloning and molecular analysis.

In the roots of barley and other cereals, hypoxia induces a set of five isozymes of L-lactate dehydrogenase [LDH; (S)-lactate:NADH oxidoreductase, EC 1.1.1.27]. Biochemical and genetic data indicate that the five LDH isozymes are tetramers that arise from random association of the products of two Ldh loci. To investigate this system, cDNA clones of LDH were isolated from a lambda gt11 cDNA library derived from hypoxically treated barley roots. The library was screened with antiserum raised against barley LDH purified approximately 3000-fold by an improved three-step procedure. Immunopositive clones were rescreened with a cDNA probe synthesized by the polymerase chain reaction using primers modeled from the amino acid sequences of two tryptic LDH peptides. Two types of LDH clones were found. Nucleotide sequence analysis of one representative insert of each type (respectively, 1305 and 1166 base pairs) revealed open reading frames encoding 10 peptide fragments of LDH. The 1305-base-pair insert included the entire coding region of a 356-residue LDH monomer. The nucleotide sequences of the two LDH cDNAs were 92% identical in the coding region, but highly divergent in the 3' noncoding region, and thus probably correspond to the two postulated Ldh loci. The deduced amino acid sequences of the two barley LDHs were 96% identical to each other and very similar to those from vertebrate and bacterial LDHs. RNA blot hybridization showed a single mRNA band of 1.5 kilobases whose level rose about 8-fold in roots during hypoxic induction, as did the level of translatable LDH message.     

Molecular cloning and characterization of a cDNA for the beta subunit of human alcohol dehydrogenase.

Human alcohol dehydrogenase (ADH) is encoded by at least five genes that fall into three classes. The class I ADH genes encode the three closely related alpha, beta, and gamma polypeptides. Molecular genetic analysis of class I ADH genes has been initiated by isolating a cDNA clone from a human adult liver cDNA library. A synthetic oligonucleotide mixture encoding a portion of the beta subunit of ADH was used as an in situ hybridization probe for the cDNA library. One positively hybridizing clone, pADH12, which contained an 1100-base-pair cDNA insert, was subjected to DNA sequence analysis. The sequence indicated that the cDNA encoded information for the carboxyl-terminal 91 amino acids of a class I ADH and a 3' untranslated region of 593 nucleotides. Comparisons with the carboxyl terminus of the human ADH beta subunit indicated that the cDNA encoded the beta polypeptide. This probe may facilitate genetic studies of various human alcohol-related syndromes, as well as enable basic molecular studies on human ADH gene expression.     

Fundamental molecular differences between alcohol dehydrogenase classes.

Two types of alcohol dehydrogenase in separate protein families are the "medium-chain" zinc enzymes (including the classical liver and yeast forms) and the "short-chain" enzymes (including the insect form). Although the medium-chain family has been characterized in prokaryotes and many eukaryotes (fungi, plants, cephalopods, and vertebrates), insects have seemed to possess only the short-chain enzyme. We have now also characterized a medium-chain alcohol dehydrogenase in Drosophila. The enzyme is identical to insect octanol dehydrogenase. It is a typical class III alcohol dehydrogenase, similar to the corresponding human form (70% residue identity), with mostly the same residues involved in substrate and coenzyme interactions. Changes that do occur are conservative, but Phe-51 is of functional interest in relation to decreased coenzyme binding and increased overall activity. Extra residues versus the human enzyme near position 250 affect the coenzyme-binding domain. Enzymatic properties are similar--i.e., very low activity toward ethanol (Km beyond measurement) and high selectivity for formaldehyde/glutathione (S-hydroxymethylglutathione; kcat/Km = 160,000 min-1.mM-1). Between the present class III and the ethanol-active class I enzymes, however, patterns of variability differ greatly, highlighting fundamentally separate molecular properties of these two alcohol dehydrogenases, with class III resembling enzymes in general and class I showing high variation. The gene coding for the Drosophila class III enzyme produces an mRNA of about 1.36 kb that is present at all developmental stages of the fly, compatible with the constitutive nature of the vertebrate enzyme. Taken together, the results bridge a previously apparent gap in the distribution of medium-chain alcohol dehydrogenases and establish a strictly conserved class III enzyme, consistent with an important role for this enzyme in cellular metabolism.     

Human glucose-6-phosphate dehydrogenase: primary structure and cDNA cloning.

The X-chromosome-linked glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ oxidoreductase, EC 1.1.1.49) of humans and other mammals consists of a subunit with a molecular weight of about 58,000. The enzyme plays a key role in the generation of NADPH, particularly in matured erythrocytes, and the genetic deficiency of the enzyme is associated with chronic and drug- or food-induced hemolytic anemia in humans. The enzyme was purified to homogeneity from human erythrocytes. The complete amino acid sequence of the subunit, consisting of 531 amino acid residues, was determined by automated and manual Edman degradation of tryptic, chymotryptic, thermolytic, and cyanogen bromide peptides obtained from the enzyme. Based on the amino acid sequence data thus obtained, a 41-mer oligonucleotide with unique sequence was prepared. Two cDNA libraries constructed in phage lambda gt11--i.e., a human liver cDNA library and a human hepatoma Li-7 cDNA library--were screened with the synthetic nucleotide probe. Two positive clones, lambda G6PD-19 and lambda G6PD-25, were obtained from the hepatoma library. lambda G6PD-19 contained an insertion of 2.0 kilobase pairs (kbp), and encoded 204 amino acid residues that were completely compatible with the COOH-terminal portion of the enzyme. The insertion of the clone had a 3' noncoding region of 1.36 kbp. The other clone, lambda G6PD-25, had an insertion of 1.8 kbp and encoded 362 amino acid residues of G6PD. Southern blot analysis of DNA samples obtained from cells with and without the human X chromosome indicated that the cDNA hybridizes with a sequence in the X chromosome.     

Digitalis metabolism and human liver alcohol dehydrogenase.

Human liver alcohol dehydrogenase (alcohol: NAD" oxidoreductase, EC 1.1.1.1) catalyzes the oxidation of the 3 beta-OH group of digitoxigenin, digoxigenin, and gitoxigenin to their 3-keto derivatives, which have been characterized by high performance liquid chromatography and mass spectrometry. These studies have identified human liver alcohol dehydrogenase as the unknown NAD(H)-dependent liver enzyme specific for the free hydroxyl group at C3 of the cardiac genins; this hydroxyl is the critical site of the genins' enzymatic oxidation and concomitant pharmacological inactivation in humans. Several kinetic approaches have demonstrated that ethanol and the pharmacologically active components of the digitalis glycosides are oxidized with closely similar kcat/Km values at the same site on human liver alcohol dehydrogenase, for which they compete. Human liver alcohol dehydrogenase thereby becomes an important biochemical link in the metabolism, pharmacology, and toxicology of ethanol and these glycosides, structurally unrelated agents that are both used widely. Both the competition of ethanol with these cardiac sterols and the narrow margin of safety in the therapeutic use of digitalis derivatives would seem to place at increased risk those individuals who receive digitalis and simultaneously consume large amounts of ethanol or whose alcohol dehydrogenase function is impaired.     

Cloning and sequencing of cDNA encoding the complete mouse liver alcohol dehydrogenase.

The main ethanol-active alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) in mouse liver (ADH-AA) is similar in catalytic and molecular properties to horse liver ADH-EE and to the human class I ADHs. We have isolated cDNA clones encoding the entire mouse liver enzyme plus flanking regions. A mixture of 16 different oligonucleotides, each 14 bases long, was used to screen a liver cDNA library made from a DBA/2J mouse. A strongly hybridizing clone was found and identified as an ADH-encoding cDNA by partial DNA sequencing. This clone was used as a probe to identify others. Two overlapping cDNA clones together contained the entire protein-encoding region plus 100 nucleotides of the 5' noncoding region and 133 nucleotides of the 3' noncoding region culminating in a short poly(dA) tail. The amino acid sequence of the mouse liver enzyme deduced from this cDNA closely resembles that of horse liver ADH-E: 316 of 374 residues are identical, and 29 of the differences are conservative substitutions. The 5' region of this cDNA is interesting: the AUG that initiates the ADH polypeptide is preceded by an AUG that would encode the first amino acid of a tripeptide. Presumably termination of this tripeptide is followed by reinitiation at the AUG immediately preceding the sequence of the mature ADH polypeptide.     

Cloning and cDNA sequence of the dihydrolipoamide dehydrogenase component human alpha-ketoacid dehydrogenase complexes.

cDNA clones comprising the entire coding region for human dihydrolipoamide dehydrogenase (dihydrolipoamide:NAD+ oxidoreductase, EC 1.8.1.4) have been isolated from a human liver cDNA library. The cDNA sequence of the largest clone consisted of 2082 base pairs and contained a 1527-base open reading frame that encodes a precursor dihydrolipoamide dehydrogenase of 509 amino acid residues. The first 35-amino acid residues of the open reading frame probably correspond to a typical mitochondrial import leader sequence. The predicted amino acid sequence of the mature protein, starting at the residue number 36 of the open reading frame, is almost identical (greater than 98% homology) with the known partial amino acid sequence of the pig heart dihydrolipoamide dehydrogenase. The cDNA clone also contains a 3' untranslated region of 505 bases with an unusual polyadenylylation signal (TATAAA) and a short poly(A) track. By blot-hybridization analysis with the cDNA as probe, two mRNAs, 2.2 and 2.4 kilobases in size, have been detected in human tissues and fibroblasts, whereas only one mRNA (2.4 kilobases) was detected in rat tissues.     

Origin of the human alcohol dehydrogenase system: implications from the structure and properties of the octopus protein.

In contrast to the multiplicity of alcohol dehydrogenase in vertebrates, a class III type of the enzyme [i.e., a glutathione-dependent formaldehyde dehydrogenase; formaldehyde; NAD+ oxidoreductase (glutathione-formylating), EC 1.2.1.1.] is the only form detectable in appreciable yield in octopus. It is enzymatically and structurally highly similar to the human class III enzyme, with limited overall residue differences (26%) and only a few conservative residue exchanges at the substrate and coenzyme pockets, reflecting "constant" characteristics of this class over wide time periods. It is distinct from the ethanol-active "variable" class I type of the enzyme (i.e., classical liver alcohol dehydrogenase; alcohol:NAD+ oxidoreductase, EC 1.1.1.1). The residue conservation of class III is also spaced differently from that of class I but is typical of that of proteins in general, emphasizing that class I, with divergence at three functional segments, is the form with deviating properties. In spite of the conservation in class III, surface charges differ considerably. The apparent absence of a class I enzyme in octopus and the constant nature of the class III enzyme support the concept of a duplicative origin of the class I line from the ancient class III form. Still more distant relationships define further enzyme lines that have subunits with other properties.     

Mouse sn-glycerol-3-phosphate dehydrogenase: molecular cloning and genetic mapping of a cDNA sequence.

The isozymes of glycerol-3-phosphate dehydrogenase (GPDH; sn-glycerol-3-phosphate:NAD+2-oxidoreductase, EC 1.1.1.8) in tissues of the mouse are coded for by two structural genes, Gdc-1 and Gdc-2, located on chromosomes 15 and 9, respectively. In order to investigate the expression of these genes, we isolated a GPDH cDNA clone from a mRNA preparation isolated from brown adipose tissue. The GPDH cDNA clone was identified by colony hybridization and hybrid selection of a mRNA that was translated in vitro to produce immunoprecipitable GPDH protein. In blot analysis, the GPDH cDNA hybridized to a single mRNA species that migrated at the position of 23S ribosomal RNA. This GPDH cDNA clone was mapped to the Gdc-1 locus by identification of a restriction enzyme polymorphism present in genomic DNA isolated from Gdc-1 congeneic lines of mice.     

Mammalian class IV alcohol dehydrogenase (stomach alcohol dehydrogenase): structure, origin, and correlation with enzymology.

The structure of a mammalian class IV alcohol dehydrogenase has been determined by peptide analysis of the protein isolated from rat stomach. The structure indicates that the enzyme constitutes a separate alcohol dehydrogenase class, in agreement with the distinct enzymatic properties; the class IV enzyme is somewhat closer to class I (the "classical" liver alcohol dehydrogenase; approximately 68% residue identities) than to the other classes (II, III, and V; approximately 60% residue identities), suggesting that class IV might have originated through duplication of an early vertebrate class I gene. The activity of the class IV protein toward ethanol is even higher than that of the classical liver enzyme. Both Km and kcat values are high, the latter being the highest of any class characterized so far. Structurally, these properties are correlated with replacements at the active site, affecting both substrate and coenzyme binding. In particular, Ala-294 (instead of valine) results in increased space in the middle section of the substrate cleft, Gly-47 (instead of a basic residue) results in decreased charge interactions with the coenzyme pyrophosphate, and Tyr-363 (instead of a basic residue) may also affect coenzyme binding. In combination, these exchanges are compatible with a promotion of the off dissociation and an increased turnover rate. In contrast, residues at the inner part of the substrate cleft are bulky, accounting for low activity toward secondary alcohols and cyclohexanol. Exchanges at positions 259-261 involve minor shifts in glycine residues at a reverse turn in the coenzyme-binding fold. Clearly, class IV is distinct in structure, ethanol turnover, stomach expression, and possible emergence from class I.     

Intraacinar profiles of alcohol dehydrogenase and aldehyde dehydrogenase activities in human liver

The intraacinar activity profiles of alcohol dehydrogenase and the aldehyde dehydrogenases (I, I plus II, and total) were determined, using liver biopsy samples from eight male and eight female patients. Microchemical assays were performed in microdissected tissue samples from the whole length of the sinusoid. Alcohol dehydrogenase activity in men less than 53 years of age showed a maximum in the intermediate zone, whereas in women less than 50 years of age an increase in the gradient toward the perivenous zone was observed. Furthermore, alcohol dehydrogenase activity in the livers of women was significantly higher than in men. After the age of 53 in men and 50 in women, the sex specificity of the distribution profiles was no longer apparent. The intraacinar profiles of aldehyde dehydrogenase isoenzymes showed only minor variations in the different groups; they were not statistically significant. This is also true for low-Michaelis constant (Km) aldehyde dehydrogenase, which is most important for acetaldehyde oxidation in vivo. Thus, of the variations in zonal heterogeneity of ethanol-degrading enzymes, it is mainly the activity of alcohol dehydrogenase that may contribute to the sex- and age-related susceptibility of liver parenchyma.     

Cloning and sequencing of cDNAs encoding alpha and beta subunits of human pyruvate dehydrogenase.

The cDNAs encoding fragments of the alpha and beta subunits (PDH alpha and PDH beta) of human pyruvate dehydrogenase (PDH, EC 1.2.4.1) were isolated from a HeLa cell cDNA library in the lambda gt11 expression vector by immunoscreening. Phage cDNA fragments were subsequently used to screen a human foreskin fibroblast cDNA library by colony hybridization. Nucleotide sequence analyses of the positive plasmid clones (pHPDA and pHPDB) revealed an insert of 1.36 kilobases (kb) for PDH alpha and one of 1.69 kb for PDH beta, respectively, allowing us to predict the complete amino acid sequences of the precursor and mature proteins of these two subunits. A putative leader sequence of 29 amino acid residues was identified in pHPDA, resulting in a precursor protein of 392 amino acid residues (Mr 43,414) and a mature protein of 363 residues (Mr 40,334). A similar leader sequence of 30 amino acid residues in pHPDB was also identified, resulting in a precursor protein of 359 amino acid residues (Mr 39,046) and a mature protein of 329 residues (Mr 35,911). The amino acid sequences of NH2-terminal regions of the two subunits of human PDH were highly homologous with those of mature porcine PDH. The amino acid sequences of phosphorylation sites determined in PDH alpha of bovine and porcine enzymes were also conserved in the human PDH alpha. Blot analysis of HeLa cell poly(A)+ RNA showed a single mRNA of 1.8 kb for PDH alpha and 1.7 kb for PDH beta, respectively. The precursor proteins of PDH alpha and PDH beta were detected by immunoprecipitation from an 35S-labeled cell-free translation system.     

Epitopes of human testis-specific lactate dehydrogenase deduced from a cDNA sequence.

The sequence and structure of human testis-specific L-lactate dehydrogenase [LDHC4, LDHX; (L)-lactate: NAD+ oxidoreductase, EC 1.1.1.27] has been derived from analysis of a complementary DNA (cDNA) clone comprising the complete protein coding region of the enzyme. From the deduced amino acid sequence, human LDHC4 is as different from rodent LDHC4 (73% homology) as it is from human LDHA4 (76% homology) and porcine LDHB4 (68% homology). Subunit homologies are consistent with the conclusion that the LDHC gene arose by at least two independent duplication events. Furthermore, the lower degree of homology between mouse and human LDHC4 and the appearance of this isozyme late in evolution suggests a higher rate of mutation in the mammalian LDHC genes than in the LDHA and -B genes. Comparison of exposed amino acid residues of discrete antigenic determinants of mouse and human LDHC4 reveals significant differences. Knowledge of the human LDHC4 sequence will help design human-specific peptides useful in the development of a contraceptive vaccine.     

Isolation of cDNA clones coding for human tissue factor: primary structure of the protein and cDNA.

Tissue factor is a membrane-bound procoagulant protein that activates the extrinsic pathway of blood coagulation in the presence of factor VII and calcium. lambda Phage containing the tissue factor gene were isolated from a human placental cDNA library. The amino acid sequence deduced from the nucleotide sequence of the cDNAs indicates that tissue factor is synthesized as a higher molecular weight precursor with a leader sequence of 32 amino acids, while the mature protein is a single polypeptide chain composed of 263 residues. The derived primary structure of tissue factor has been confirmed by comparison to protein and peptide sequence data. The sequence of the mature protein suggests that there are three distinct domains: extracellular, residues 1-219; hydrophobic, residues 220-242; and cytoplasmic, residues 243-263. Three potential N-linked carbohydrate attachment sites occur in the extracellular domain. The amino acid sequence of tissue factor shows no significant homology with the vitamin K-dependent serine proteases, coagulation cofactors, or any other protein in the National Biomedical Research Foundation sequence data bank (Washington, DC).     

Primary structure and cDNA cloning of human fibroblast collagenase inhibitor.

We report the primary structure and cDNA cloning of human fibroblast collagenase inhibitor, a glycoprotein that appears to play a central role in modulating the activity of a number of metalloendoproteases of connective tissue origin including collagenase, gelatinase, and proteoglycanase. Secreted human fibroblast collagenase inhibitor was purified and subjected to automated Edman degradation. The secreted protein consists of 184 amino acid residues; it contains two sites of N-linked oligosaccharide linkage and six disulfide bonds. Synthetic oligonucleotide probes based on selected amino acid sequences of the inhibitor were used to screen a lambda gt10 cDNA library from a human fibroblast line. Two overlapping cDNA clones were characterized to determine the complete coding and noncoding sequences of the specific mRNA. The amino acid sequence deduced from the nucleotide sequence agrees with that determined by protein sequencing. One clone appears to contain the complete 5' end and, in addition, the cDNA sequence predicts a 23-amino acid leader peptide. The other clone represents the 3' end of the mature message and includes a short poly(A)+ tract. This 3' sequence is remarkably similar to a reported cDNA encoding part of the protein derived from mouse fibroblast poly(A)+ RNA. However, this inhibitor has no substantial homology with previously sequenced protease inhibitors.     

The alpha and beta subunits of phosphorylase kinase are homologous: cDNA cloning and primary structure of the beta subunit.

We have cloned cDNA molecules encoding the beta subunit of phosphorylase kinase (ATP:phosphorylase-b phosphotransferase; EC 2.7.1.38) from rabbit fast-twitch skeletal muscle and have determined the complete primary structure of the polypeptide by a combination of peptide and DNA sequencing. In the mature beta subunit, the initial methionine is replaced by an acetyl group. The subunit is composed of 1092 amino acids and has a calculated molecular mass of 125,205 Da. Alignment of its sequence with the alpha subunit of phosphorylase kinase reveals extensive regions of homology, but each molecule also possesses unique sequences. Two of the three phosphorylation sites known for the beta subunit and all seven phosphorylation sites known for the alpha subunit are located in these unique domains.     

Molecular structure and evolutionary origin of human cardiac muscle actin gene.

Two recombinant phages that contain cardiac muscle actin gene were isolated from a human DNA library and their structures were determined. Restriction analysis indicates that both clones carry the same EcoRI 13-kilobase fragment where the coding sequence is mapped. The cloned DNA hybridized with polyadenylylated RNA from human fibroblasts, which directs the synthesis of cytoplasmic beta- and gamma-actin in vitro. However, sequence determination of the cloned DNA showed that the entire coding sequence perfectly matched the amino acid sequence of cardiac muscle actin. The initiation codon is followed by a cysteine codon that is not found at the amino-terminal site of any actin isoform, suggesting the necessity of post-translational processing for in vivo actin synthesis. There are five introns interrupting exons at codons 41/42, 150, 204, 267, and 327/328. Surprisingly, these intron locations are exactly the same as those of the rat skeletal muscle actin gene but different from those of nonmuscle beta-actin gene. Nucleotide sequences of all exon/intron boundaries agree with the G-T/A-G rule (G-T at the 5' and A-G at the 3' termini of each intron). The 3'-untranslated sequence has no homology to that of nonmuscle beta- or gamma-actin gene, but Southern blot hybridization has shown that this region has considerable homology to that of one of the other actin genes. These results indicate that the recombinant phages, which we have isolated, contain cardiac muscle actin gene and that cardiac muscle actin gene and skeletal muscle actin genes are derived from their ancestor gene at a relatively recent time in evolutionary development.