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Galanin is a neuroendocrine peptide widely distributed in the central and peripheral nervous systems and in endocrine tissues. Using radioimmunoassays, chromatographic separations, and tandem mass spectrometry, we have identified a series of five variant forms of galanin purified from porcine upper intestine. The modified variants include three β-aspartyl-shifted forms, an oxidized form containing Trp monooxide, and an N-terminally truncated form. All were found to be C-terminally amidated. At least the β-aspartyl-shifted forms could be of native occurrence. Mechanistic explanations of the mass spectrometric fragmentation patterns of the different forms are suggested. Copyright © 2004 John Wiley & Sons, Ltd.

Mushroom alcohol dehydrogenase (ADH) from Agaricus bisporus (common mushroom, champignon) was purified to apparent homogeneity. One set of ADH isozymes was found, with specificity against formaldehyde/glutathione. It had two highly similar subunits arranged in a three-member isozyme set of dimers with indistinguishable activity. Determination of the primary structure by a combination of chemical, mass spectrometric and cDNA sequence analyses, correlated with molecular modeling towards human ADHs, showed that the active site residues are of class III ADH type, and that the subunit differences affect other residues. Class I and III forms of ADHs characterized define conserved substrate-binding residues (three and eight, respectively) useful for recognition of these enzymes in any organism.

The coding region of amphioxus alcohol dehydrogenase class 3 (ADH3) has been characterized from two species, Branchiostoma lanceolatum and Branchiostoma floridae. The species variants have residue differences at positions that result in only marginal functional distinctions. Activity measurements show a class 3 glutathione-dependent formaldehyde dehydrogenase, with kcat/Km values about threefold those of the human class 3 ADH enzyme. Only a single ADH3 form is identified in each of the two amphioxus species, and no ethanol activity ascribed to other classes is detectable, supporting the conclusion that evolution of ethanol-active ADH classes by gene duplications occurred at early vertebrate radiation after the formation of the amphioxus lineage. Similarly, Southern blot analysis indicated that amphioxus ADH3 is encoded by a single gene present in the methylated fraction of the amphioxus genome and northern blots revealed a single 1.4-kb transcript. In situ experiments showed that amphioxus Adh3 expression is restricted to particular cell types in the embryos. Transcripts were first evident at the neurula stage and then located at the larval ventral region, in the intestinal epithelium. This tissue-specific pattern contrasts with the ubiquitous Adh3 expression in mammals.

Polymorphism at the ADH2 and ADH3 loci of alcohol dehydrogenase (ADH) has been shown to have an effect on the predisposition to alcoholism in Asian individuals. However, the results are not conclusive for white individuals. We have analyzed the ADH genotype of 876 white individuals from Spain (n = 251), France (n = 160), Germany (n = 184), Sweden (n = 88), and Poland (n = 193). Peripheral blood samples from healthy controls and groups of patients with viral cirrhosis and alcohol-induced cirrhosis, as well as alcoholics with no liver disease, were collected on filter paper. Genotyping of the ADH2 and ADH3 loci was performed using polymerase chain reaction-restriction fragment length polymorphism methods on white cell DNA. In healthy controls, ADH2*2 frequencies ranged from 0% (France) to 5.4% (Spain), whereas ADH3*1 frequencies ranged from 47. 6% (Germany) to 62.5% (Sweden). Statistically significant differences were not found, however, between controls from different countries, nor between patients with alcoholism and/or liver disease. When all individuals were grouped in nonalcoholics (n = 451) and alcoholics (n = 425), ADH2*2 frequency was higher in nonalcoholics (3.8%) than in alcoholics (1.3%) (P =.0016), whereas the ADH3 alleles did not show differences. Linkage disequilibrium was found between ADH2 and ADH3, resulting in an association of the alleles ADH2*2 and ADH3*1, both coding for the most active enzymatic forms. In conclusion, the ADH2*2 allele decreases the risk for alcoholism, whereas the ADH2*2 and ADH3*1 alleles are found to be associated in the European population.

The Drosophila melanogaster sorbitol dehydrogenase (SDH) is characterized as a two-enzyme system of the medium chain dehydrogenase/reductase family (MDR). The SDH-1 enzyme has an enzymology with Km and kcat values an order of magnitude higher than those for the human enzyme but with a similar kcat/Km ratio. It is a tetramer with identical subunits of approximately 38 kDa. At the genomic level, two genes, Sdh-1 and Sdh-2, have a single transcriptional start site and no functional TATA box. Expression is greater in larvae and adults than in pupae, where it is very low. At all three stages, Sdh-1 constitutes the major transcript. Sdh-1 and Sdh-2 genes were located at positions 84E-F and 86D in polytene chromosomes. The deduced amino acid sequences of the two genes show 90% residue identity. Evaluation of the sequence and modeling of the structure toward that of class I alcohol dehydrogenase (ADH) show altered loop and gap arrangements as in mammalian SDH and establishes that SDH, despite gene multiplicity and larger variability than the "constant" ADH of class III, is an enzyme conserved over wide ranges.

The three-dimensional structure of betaine aldehyde dehydrogenase, the most abundant aldehyde dehydrogenase (ALDH) of cod liver, has been determined at 2.1 A resolution by the X-ray crystallographic method of molecular replacement. This enzyme represents a novel structure of the highly multiple ALDH, with at least 12 distinct classes in humans. This betaine ALDH of class 9 is different from the two recently determined ALDH structures (classes 2 and 3). Like these, the betaine ALDH structure has three domains, one coenzyme binding domain, one catalytic domain, and one oligomerization domain. Crystals grown in the presence or absence of NAD+ have very similar structures and no significant conformational change occurs upon coenzyme binding. This is probably due to the tight interactions between domains within the subunit and between subunits in the tetramer. The oligomerization domains link the catalytic domains together into two 20-stranded pleated sheet structures. The overall structure is similar to that of the tetrameric bovine class 2 and dimeric rat class 3 ALDH, but the coenzyme binding with the nicotinamide in anti conformation, resembles that of class 2 rather than of class 3.

Mitochondrial (class 2) hamster aldehyde dehydrogenase has been purified and characterized. Its primary structure has been determined and correlated with the tertiary structure recently established for this class from another species. The protein is found to represent a constant class within a complex family of multiple forms. Variable segments that occur in different species correlate with non-functional segments, in the same manner as in the case of the constant class of alcohol dehydrogenases (class III type) of another protein family, but distinct from the pattern of the corresponding variable enzymes. Hence, in both these protein families, overall variability and segment architectures behave similarly, with at least one `constant' form in each case, class III in the case of alcohol dehydrogenases, and at least class 2 in the case of aldehyde dehydrogenases.

The major form of glutathione transferase from the marsupial Antechinus stuartii has been purified and characterized as an Alpha class enzyme (Ast GST A1-1) with distant sequence relationships to other class Alpha sublines, compatible with the early origin of marsupials. Amino acid replacements toward the closest enzyme characterized (chicken, form A3) involve no less than 79 positions (36%). At the active site, as deduced from comparisons with the known tertiary structure of the corresponding human enzyme, over half of the residues (8 of 15) ascribed to substrate binding interactions are exchanged although the general character of that site is conserved, while only 1 of 11 positions ascribed to interactions with GSH is exchanged. Class variability and species variability appear to coincide, with divergent segments centering around positions 33–49, 103–130 and 205–222. The pattern is reminiscent of that in similarly multiple MDR alcohol dehydrogenases. Both these enzyme families involved in cellular defense reactions have diverged considerably.

Purpose To present the American Brachytherapy Society (ABS) guidelines for plaque brachytherapy of choroidal melanoma and retinoblastoma. Methods and Materials An international multicenter Ophthalmic Oncology Task Force (OOTF) was assembled to include 47 radiation oncologists, medical physicists, and ophthalmic oncologists from 10 countries. The ABS-OOTF produced collaborative guidelines, based on their eye cancer–specific clinical experience and knowledge of the literature. This work was reviewed and approved by the ABS Board of Directors as well as within the journal's peer-reivew process. Results The ABS-OOTF reached consensus that ophthalmic plaque radiation therapy is best performed in subspecialty brachytherapy centers. Quality assurance, methods of plaque construction, and dosimetry should be consistent with the 2012 joint guidelines of the American Association of Physicists in Medicine and ABS. Implantation of plaque sources should be performed by subspecialty-trained surgeons. Although there exist select restrictions related to tumor size and location, the ABS-OOTF agreed that most melanomas of the iris, ciliary body, and choroid could be treated with plaque brachytherapy. The ABS-OOTF reached consensus that tumors with gross orbital extension and blind painful eyes and those with no light perception vision are unsuitable for brachytherapy. In contrast, only select retinoblastomas are eligible for plaque brachytherapy. Prescription doses, dose rates, treatment durations, and clinical methods are described. Conclusions Plaque brachytherapy is an effective eye and vision-sparing method to treat patients with intraocular tumors. Practitioners are encouraged to use ABS-OOTF guidelines to enhance their practice.

N-terminal acetylation of polypeptides is a common feature preventing direct Edman degradation. We describe a method for the removal of the acetyl group, with only a low extent of internal peptide bond cleavage, also in large proteins, by treatment at room temperature with trifluoroacetic acid and methanol. The alcohol is essential for selective deacetylation, and it is proposed that the deblocking mechanism consists of an acid-catalyzed nucleophilic substitution involving methanol. The extent of deacetylation is limited, but the initial yield in the sequence analysis can be up to 10%. Deblocking of samples spotted or blotted onto sequencer filters is equally possible as the use of isolated samples from column separations. Deblocking on sequencer filters is also possible directly after negative results on initial sequencer attempts with samples proving to be blocked.

The primary structure of rabbit liver fructose-1,6-bisphosphatase was determined by peptide analysis of digests with different proteases. The results establish the primary structure, complete data bank entries, and show that this enzyme variant is indeed homologous with other liver fructose-1,6-bisphosphatases. Residue differences with the enzymes from other mammals are 9–15%, with those from plants and yeasts about 50%, and with those from characterized prokaryotes up to 70%, showing an enzyme variability intermediate between those of ‘variable’ and ‘constant’ oligomeric dehydrogenases. Structural relationships, conformations and catalytic mechanisms are consistent within the family of fructose-1,6-bisphosphatases, and the rabbit protein is a typical rather than an aberrant form of the enzyme.

A mixed-class alcohol dehydrogenase has been characterized from avian liver. Its functional properties resemble the classical class I type enzyme in livers of humans and animals by exhibiting low Km and kcat values with alcohols (Km = 0.7 mM with ethanol) and low Ki values with 4-methylpyrazole (4 microM). These values are markedly different from corresponding parameters of class II and III enzymes. In contrast, the primary structure of this avian liver alcohol dehydrogenase reveals an overall relationship closer to class II and to some extent class III (69 and 65% residue identities, respectively) than to class I or the other classes of the human alcohol dehydrogenases (52-61%), the presence of an insertion (four positions in a segment close to position 120) as in class II but in no other class of the human enzymes, and the presence of several active site residues considered typical of the class II enzyme. Hence, the avian enzyme has mixed-class properties, being functionally similar to class I, yet structurally similar to class II, with which it also clusters in phylogenetic trees of characterized vertebrate alcohol dehydrogenases. Comparisons reveal that the class II enzyme is approximately 25% more variable than the "variable" class I enzyme, which itself is more variable than the "constant" class III enzyme. The overall extreme, and the unusual chromatographic behavior may explain why the class II enzyme has previously not been found outside mammals. The properties define a consistent pattern with apparently repeated generation of novel enzyme activities after separate gene duplications.

Human stomach mucosa contains a characteristic alcohol dehydrogenase (ADH) enzyme, sigma sigma-ADH. Its cDNA has been cloned from a human stomach library and sequenced. The deduced amino acid sequence shows 59-70% identities with the other human ADH classes, demonstrating that the stomach enzyme represents a distinct structure, constituting class IV, coded by a separate gene, ADH7. The amino acid identity with the rat stomach class IV ADH is 88%, which is intermediate between constant and variable dehydrogenases. This value reflects higher conservation than for the classical liver enzymes of class I, compatible with a separate functional significance of the class IV enzyme. Its enzymic features can be correlated with its structural characteristics. The residues lining the substrate-binding cleft are bulky and hydrophobic, similar to those of the class I enzyme; this explains the similar specificity of both classes, compatible with the origin of class IV from class I. Position 47 has Arg, in contrast to Gly in the rat class IV enzyme, but this Arg is still associated with an extremely high activity (kcat = 1510 min-1) and weak coenzyme binding (KiaNAD+ = 1.6 mM). Thus, the strong interaction with coenzyme imposed by Arg47 in class I is probably compensated for in class IV by changes that may negatively affect coenzyme binding: Glu230, His271, Asn260, Asn261, Asn363. The still higher activity and weaker coenzyme binding of rat class IV (kcat = 2600 min-1, KiaNAD = 4 mM) can be correlated to the exchanges to Gly47, Gln230 and Tyr363. An important change at position 294, with Val in human and Ala in rat class IV, is probably responsible for the dramatic difference in Km values for ethanol between human (37 mM) and rat (2.4 M) class IV enzymes.

Class I alcohol dehydrogenase has been characterized from ostrich liver in order to evaluate enzyme variability between two independent lines, mammalian forms of class I alcohol dehydrogenase as a group, and a sufficient number of the enzyme from the most recent animal class (Aves, birds) as another. Between the two enzyme groups, patterns are consistent and mutually similar. This indicates conserved metabolic and catalytic properties of class I alcohol dehydrogenase, suggesting its metabolic role to be distinct, in spite of its protein variability. The new structure has a microheterogeneity (position 112, Arg/Cys) in a variable Zn-binding loop. In addition, it also establishes further native variants at active-site positions, including one thus far unique residue at the inner part of the substrate-binding pocket (Ala141), and a replacement at position 271 (giving His271), which is also the site of a human alcohol dehydrogenase gamma 1/gamma 2 isozyme variability. The data correlate with functional differences in catalytic properties, the ostrich enzyme having a comparatively high Km for ethanol (5.9 mM at pH 10), and emphasize the importance of single positions in substrate and coenzyme binding, paralleling isozyme variability with protein variability within the class I enzymes.

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.

The Swedish Lucentis Quality Registry is a 12-month, open-label, observational, prospective, and retrospective study of ranibizumab administration for wet AMD. Visual acuity (VA) was measured with Snellen or ETDRS chart in 370 patients (66.8% women; age range 46–93 years). In total, a mean of4.7±1.6injections per patient (range 1–10) was given to month 12. Mean VA score was58.3±12.2letters before treatment,63.3±12.5after 3 injections (Δ4.9±10.1letters from baseline), and59.3±16.2at 12 months (Δ1.0±13.6). VA score from baseline to month 12 was stable in 74.4% of patients, improved by 15 letters/3 lines or more in 14.7%, and decreased by≥15 letters/3 lines in 10.9% of patients. With a mean of 4.7 ranibizumab injections per patient per year, mean VA was stabilised but not increased. To maintain the initial gain seen after the first three injections, an average of1.8±1.5additional injections does not appear to be adequate.

Four additional variants of alcohol and aldehyde dehydrogenases have been purified and functionally characterized, and their primary structures have been determined. The results allow conclusions about the structural and evolutionary relationships within the large family of MDR alcohol dehydrogenases from characterizations of the pigeon (Columba livia) and dogfish (Scyliorhinus canicula) major liver alcohol dehydrogenases. The pigeon enzyme turns out to be of class I type and the dogfish enzyme of class III type. This result gives a third type of evidence, based on purifications and enzyme characterization in lower vertebrates, that the classical liver alcohol dehydrogenase originated by a gene duplication early in the evolution of vertebrates. It is discernable as the major liver form at about the level in-between cartilaginous and osseous fish. The results also show early divergence within the avian orders. Structures were determined by Edman degradations, making it appropriate to acknowledge the methodological contributions of Pehr Edman during the 65 years since his thesis at Karolinska Institutet, where also the present analyses were performed.

Alcohol dehydrogenases (ADHs) of the MDR type (medium-chain dehydrogenases/reductases) have diverged into two evolutionary groups in eukaryotes: a set of 'constant' enzymes (class III) typical of basal enzymes, and a set of 'variable' enzymes (remaining classes) suggesting 'evolving' forms. The variable set has larger overall variability, different segment variability, and variability also in functional segments. Using a major aldehyde dehydrogenase (ALDH) from cod liver and fish ALDHs deduced from the draft genome sequence of Fugu rubripes (Japanese puffer fish), we found that ALDHs form more complex patterns than the ADHs. Nevertheless, ALDHs also group into 'constant' and 'variable' sets, have separate segment variabilities, and distinct functions. Betaine ALDH (class 9 ALDH) is 'constant,' has three segments of variability, all non-functional, and a limited fish/human divergence, reminiscent of the ADH class III pattern. Enzymatic properties of fish betaine ALDH were also determined. Although all ALDH patterns are still not known, overall patterns are related to those of ADH, and group separations may be distinguished. The results can be interpreted functionally, support ALDH isozyme distinctions, and assign properties to the multiplicities of the ADH and ALDH enzymes.

Background Annotations of completely sequenced genomes reveal that nearly half of the genes identified are of unknown function, and that some belong to uncharacterized gene families. To help resolve such issues, information can be obtained from the comparative analysis of homologous genes in model organisms. Results While characterizing genes from the retinitis pigmentosa locus RP26 at 2q31-q33, we have identified a new gene, ORMDL1, that belongs to a novel gene family comprising three genes in humans (ORMDL1, ORMDL2 and ORMDL3), and homologs in yeast, microsporidia, plants, Drosophila, urochordates and vertebrates. The human genes are expressed ubiquitously in adult and fetal tissues. The Drosophila ORMDL homolog is also expressed throughout embryonic and larval stages, particularly in ectodermally derived tissues. The ORMDL genes encode transmembrane proteins anchored in the endoplasmic reticulum (ER). Double knockout of the two Saccharomyces cerevisiae homologs leads to decreased growth rate and greater sensitivity to tunicamycin and dithiothreitol. Yeast mutants can be rescued by human ORMDL homologs. Conclusions From protein sequence comparisons we have defined a novel gene family, not previously recognized because of the absence of a characterized functional signature. The sequence conservation of this family from yeast to vertebrates, the maintenance of duplicate copies in different lineages, the ubiquitous pattern of expression in human and Drosophila, the partial functional redundancy of the yeast homologs and phenotypic rescue by the human homologs, strongly support functional conservation. Subcellular localization and the response of yeast mutants to specific agents point to the involvement of ORMDL in protein folding in the ER.

The alcohol dehydrogenase (ADH) family has evolved into at least eight ADH classes during vertebrate evolution. We have characterized three prevertebrate forms of the parent enzyme of this family, including one from an urochordate (Ciona intestinalis) and two from cephalochordates (Branchiostoma floridae and Branchiostoma lanceolatum). An evolutionary analysis of the family was performed gathering data from protein and gene structures, exon-intron distribution, and functional features through chordate lines. Our data strongly support that the ADH family expansion occurred 500 million years ago, after the cephalochordate/vertebrate split, probably in the gnathostome subphylum line of the vertebrates. Evolutionary rates differ between the ancestral, ADH3 (glutathione-dependent formaldehyde dehydrogenase), and the emerging forms, including the classical alcohol dehydrogenase, ADH1, which has an evolutionary rate 3.6-fold that of the ADH3 form. Phylogenetic analysis and chromosomal mapping of the vertebrate Adh gene cluster suggest that family expansion took place by tandem duplications, probably concurrent with the extensive isoform burst observed before the fish/tetrapode split, rather than through the large-scale genome duplications also postulated in early vertebrate evolution. The absence of multifunctionality in lower chordate ADHs and the structures compared argue in favor of the acquisition of new functions in vertebrate ADH classes. Finally, comparison between B. floridae and B. lanceolatum Adhs provides the first estimate for a cephalochordate speciation, 190 million years ago, probably concomitant with the beginning of the drifting of major land masses from the Pangea.

A series of synthetic peptides (3–15 residues), C-terminally derivatized with 4-aminonaphthalenesulfonic acid (ansa), have been analyzed on a hybrid magnetic sector–orthogonal acceleration time-of-flight tandem mass spectrometer, fitted with a nano-electrospray (nano-ES) interface. Deprotonated molecules generated by negative-ion ES were subjected to collision-induced dissociation (CID) using either methane or xenon as the collision gas, at a collision energy of 400 eV (laboratory frame of reference). As a consequence of charge localization on the sulfonate group, only C-terminal fragment ions were formed, presumably by charge-remote fragmentation mechanisms. Interpretable CID spectra were obtained from fmol amounts of the small peptides (up to 6 residues), whereas low pmol amounts were required for the larger peptides. CID spectra were also recorded of derivatized, previously noncharacterised peptides obtained by proteolysis of cytosolic hamster liver aldehyde dehydrogenase. Interpretation of these CID spectra was based on rules established for the fragmentation of the synthetic peptides. This study shows that derivatization with ansa may be useful in the de novo sequencing of peptides.

An ethanol-active medium-chain dehydrogenase/reductase (MDR) alcohol dehydrogenase was isolated and characterized from Escherichia coli. It is distinct from the fermentative alcohol dehydrogenase and the class III MDR alcohol dehydrogenase, both already known in E. coli. Instead, it is reminiscent of the MDR liver enzyme forms found in vertebrates and has a K(m) for ethanol of 0.7 mM, similar to that of the class I enzyme in humans, however, it has a very high k(cat), 4050 min(-1). It is also inhibited by pyrazole (K(i) = 0.2 microM) and 4-methylpyrazole (K(i)= 44 microM), but in a ratio that is the inverse of the inhibition of the human enzyme. The enzyme is even more efficient in the reverse direction of acetaldehyde reduction (K(m) = 30 microM and k(cat) = 9800 min(-1)), suggesting a physiological function like that seen for the fermentative non-MDR alcohol dehydrogenase. Growth parameters in complex media with and without ethanol show no difference. The structure corresponds to one of 12 new alcohol dehydrogenase homologs present as ORFs in the E. coli genome. Together with the previously known E. coli MDR forms (class III alcohol dehydrogenase, threonine dehydrogenase, zeta-crystallin, galactitol-1-phosphate dehydrogenase, sensor protein rspB) there is now known to be a minimum of 17 MDR enzymes coded for by the E. coli genome. The presence of this bacterial MDR ethanol dehydrogenase, with a structure compatible with an origin separate from that of yeast, plant and animal ethanol-active MDR forms, supports the view of repeated duplicatory origins of alcohol dehydrogenases and of functional convergence to ethanol/acetaldehyde activity. Furthermore, this enzyme is ethanol inducible in at least one E. coli strain, K12 TG1, with apparently maximal induction at an enthanol concentration of approximately 17 mM. Although present in several strains under different conditions, inducibility may constitute an explanation for the fairly late characterization of this E. coli gene product.

Zinc-containing medium-chain alcohol dehydrogenases/reductases (MDR) are ubiquitous (Danielsson et al., 1994), constituting a group of enzymes with great multiplicity. Natural variants of the family serve to illustrate functional properties, but can also serve as tools for studying evolution of novel forms. Formation of families, enzymes, classes and isozymes reflects these aspects. Class III/glutathione-dependent formaldehyde dehydrogenase, with its constant properties in vertebrates (Jornvall and Hoog, 1995; Hjelmqvist et al., 1995), invertebrates (Danielsson et al., 1994; Kaiser et al., 1993), plants (Shafqat et al., 1996; Martinez et al., 1996), fungi (Sasnaukas et al., 1992; Fernandez et al., 1995), prokaryotes (Gutheil et al., 1992; Ras et al., 1995), and archaeons (Ammendola, 1992) has a distant origin. Class I, with ethanol dehydrogenase activity, has a duplicatory origin from class III (Daniels son and Jornvall, 1992; Jornvall, 1994), as has also the ethanol active ADH in plants (Shafqat et al., 1996). Different regions of the molecules have been assessed for various functional and structural properties, like binding of substrate and coenzyme (Eklund, 1984; Hurley, 1991), metals (Eklund et al., 1976), other subunits (Persson et al., 1993; Danielsson et al., 1994), and further aspects.

Aldehyde and alcohol dehydrogenases (ALDHs and ADHs) are both highly multiple enzymes with many different forms in metabolically linked functional pathways. However, they are derived from separate protein families with distinct properties. Those of the vertebrate ADHs have been well characterized since long, and their different classes belong to the MDR protein family of known structures from several sources. However, the properties and multiplicity of the ALDHs have only recently been partly characterized. Presently, just three of the at least twelve different ALDH classes (Yoshida et al., 1998) are kn in three-dimensional structure (Liu et al., 1997; Steinmetz et al., 1997; Johansson et al.998), several have not been purified and characterized enzymatically, and little is known about their species or class variabilities.

P 1 - N 6 -(4-azidophenylethyl)adenosine- P 2 -4-(3-azidopyridinio)butyl diphosphate was synthesized with an [8- 14 C]adenine label. This bifunctional photoaffinity labelling reagent inactivates lactate dehydrogenase from pig heart upon irradiation with light of wavelength 300–380 nm. Stoichiometry of binding and enzymatic parameters suggest that the analogue is bound to the coenzyme binding site and that adjacent residues are modified. Four radioactive peptides were isolated by reverse-phase HPLC after tryptic digestion of the labelled protein. Amino-acid sequence analysis identified the peptides and correlation with the three-dimensional structure of dogfish lactate dehydrogenase reveals that the peptides correspond to positions affecting the coenzyme binding site, consistent with proper affinity labelling. Two of the peptides, Ile-77 → Lys-81 and Asp-82 → Asn-88, are located close to the adenine binding site. Low recovery of Thr-86 in combination with the detection of additional products in the sequence analysis indicates that this residue is modified by the photoaffinity label. The two other peptides (positions 119–124 and 318–328) are located next to the substrate binding site; their label is lost upon treatment with pyrophosphatase, showing that they are linked to the pyridinio moiety of the coenzyme analogue.

The nature of the isozyme differences in the class-I alcohol dehydrogenase structure from the lizard, Uromastix hardwickii, was determined and related to those in the human and horse enzymes, for which isozyme structures have also been established. The Uromastix isozymes differ much (at a total of 72 positions, 19%) but, in spite of this, have similar properties and were not obtained resolved. Their structures were analyzed in mixture, and the two sub-sets of peptides obtained could be distinguished by evaluation of the recovery ratios within the peptide pairs. The isozymes have class-I activities, with an ethanol dehydrogenase activity of 0.6 U/mg and no formaldehyde dehydrogenase activity, have typical class-I structures, and are composed of N-terminally acetylated 375-residue subunits (a and b). Importantly, variability patterns between the isozymes are reminiscent of those both in the other two lines with isozymes (primates and horse) and in the class distinctions of the enzyme. Hence, the variability pattern since the distant stage of class-I emergence is also visible within the more recent isozyme divergence, illustrating a continuity in the evolution of isozymes to classes (and then to enzymes). The pattern also links the different levels of multiplicity and may suggest an acceptability in common to duplications and mutations, compatible with the emergence of novel functions.

We have studied many alcohol dehydrogenases and related enzymes with the aim of defining functional properties, structural patterns, and evolutionary relationships. From this, four major conclusions have been drawn: The enzymes are clearly multiple and represent different protein families. Within the MDR family (medium-chain dehydrogenases/reductases), repeated duplications at different levels have produced the enzymes, classes, and isozymes that are now visible in human, mammalian, and other lines (Jornvall et al., 1987; Hjelmqvist et al., 1995a). Of the alcohol dehydrogenases, class III, with its glutathione-dependent formaldehyde dehydrogenase activity (Koivusalo et al., 1989), appears to be the parent form, locking much of the alcohol dehydrogenase family to cellular detoxication reactions (Danielsson and Jornvall, 1992). Separate, internal molecular architectures are present (Danielsson et al., 1994a). Class III has a protein-classical pattern, with a low variability overall like functionally constant enzymes in general, and with variable regions in non-functional segmens. The other classes are more variable, both overall and in their functional segments, in a protein-atypical manner, indicating evolution of new functions, or “enzymogenesis” (Danielsson and Jornvall, 1992). Functional convergence toward ethanol activity has occurred in many lines. Thus, the ethanol-active enzymes in yeast, prokaryotes, plants, and animals all appear to have separate origins (Jornvall, 1994).

Ten different alcohol dehydrogenases, representing several classes of the enzyme and a wide spread of organisms, were analyzed for patterns of N-terminal structures utilizing a combination of conventional and mass spectrometric peptide analysis. Results show all forms to be N-terminally acetylated and allow comparisons of now 40 such alcohol dehydrogenases covering a large span of forms and origins. Patterns illustrate roles of acetylation in proteins in general, define special importance of the class I N-terminal acetylation, and distinguish separate acetylated structures for all classes, as well as a common alcohol dehydrogenase motif.

Alcohol dehydrogenase class I from kiwi liver has been purified, analyzed, and compared with that of other alcohol dehydrogenases. The results show that several avian and mammalian forms of the enzyme exhibit parallel evolutionary patterns in two independent lineages of a single protein, establishing a pattern in common. Furthermore, the data correlate the enzyme evolutionary pattern with that of 12S rRNA. Biologically, the patterns complement those on ratite and other avian relationships. Functionally, the enzyme has a low Km with ethanol and a branched-chain residue at position 141, like the mammalian enzymes but in contrast to the other characterized ratite enzyme (with Ala-141 and a higher Km). This pattern of natural variability suggests a frequent but not fully complete correlation between a large residue size at position 141 and tight ethanol binding.

Alcohol dehydrogenases of different types are common enzymes in nature. Two of these families, the medium-chain dehydrogenase/reductase family, MDR, and the shortchain dehydrogenase/reductase family, SDR, are well studied and known since long, but have experienced a recent “explosion” of new knowledge, extension and importance. The MDR family includes the classical zinc-containing liver alcohol dehydrogenases encompassing the classes of human liver alcohol dehydrogenase, while the SDR family includes the Drosophila alcohol dehydrogenase, which has shorter subunits, no similar metal requirements, other sub-domain arrangements with different structural relationships, and other subunit interactions.

Mammalian alcohol dehydrogenases (ADH) constitute a well-studied enzyme system composed of sub-forms at different levels of multiplicity. The family has diverged into a number of different enzymes. At the next level (Fig. 1), fairly different forms (“classes”) of alcohol dehydrogenase, with distinct structural and enzymatic properties, occur. The subsequent level constitutes still more similar forms (“isozymes”) with gradual differences in properties and fewer residue exchanges.

Mammalian alcohol dehydrogenase (ADH, EC 1.1.1.1) is a complex enzymatic system composed of multiple molecular forms, which have been grouped into classes according to their enzymatic and structural characteristics. In addition to the three classes (I-III) early recognized (Vallee and Bazzone, 1983), more recent studies have led to the identification of three more classes: class IV (Pares et al., 1990, Pares et al., 1992), class V (Yasunami et al., 1991), and class VI (Zheng et al., 1993).

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.

Alcohol dehydrogenases and related enzymes constitute a complex system of proteins derived from gene duplications at minimally four different levels. The system includes proteins of different type regarding family relationships and overall organiza tion. It also includes different enzymes within each family, as well as different classes of the enzymes, and different isozymes within the classes, apart from allelic variants. We have studied these relationships, starting with the horse liver alcohol dehydrogenase (Jornvall, 1970, Eklund et al., 1976), distinguishing the parallel evolution of separate enzyme types (Jornvall et al., 1981) and successively characterizing both the “medium-chain” (Jornvall et al., 1987) and “short-chain” (Persson et al., 1991) alcohol dehydrogenase families. Recently, we have characterized several novel forms, including both mammalian enzymes and those from other vertebrate lines, as well as from further, distantly related sources. Together, this has made it possible to deduce relationships of the functional and structural organization of the enzyme system, tracing gene duplications, original forms, and functional properties.

Background: Annotations of completely sequenced genomes reveal that nearly half of the genes identified are of unknown function, and that some belong to uncharacterized gene families. To help resolve such issues, information can be obtained from the comparative analysis of homologous genes in model organisms. Results: While characterizing genes from the retinitis pigmentosa locus RP26 at 2q31-q33, we have identified a new gene, ORMDL1, that belongs to a novel gene family comprising three genes in humans (ORMDL1, ORMDL2 and ORMDL3), and homologs in yeast, microsporidia, plants, Drosophila, urochordates and vertebrates. The human genes are expressed ubiquitously in adult and fetal tissues. The Drosophila ORMDL homolog is also expressed throughout embryonic and larval stages, particularly in ectodermally derived tissues. The ORMDL genes encode transmembrane proteins anchored in the endoplasmic reticulum (ER). Double knockout of the two Saccharomyces cerevisiae homologs leads to decreased growth rate and greater sensitivity to tunicamycin and dithiothreitol. Yeast mutants can be rescued by human ORMDL homologs. Conclusions: From protein sequence comparisons we have defined a novel gene family, not previously recognized because of the absence of a characterized functional signature. The sequence conservation of this family from yeast to vertebrates, the maintenance of duplicate copies in different lineages, the ubiquitous pattern of expression in human and Drosophila, the partial functional redundancy of the yeast homologs and phenotypic rescue by the human homologs, strongly support functional conservation. Subcellular localization and the response of yeast mutants to specific agents point to the involvement of ORMDL in protein folding in the ER.

Penicillium purpurogenum produces at least two acetyl xylan esterases (AXE I and II). The AXE II cDNA, genomic DNA and mature protein sequences were determined and show that the axe 2 gene contains two introns, that the primary translation product has a signal peptide of 27 residues, and that the mature protein has 207 residues. The sequence is similar to the catalytic domain of AXE I from Trichoderma reesei (67% residue identity) and putative active site residues are conserved, but the Penicillium enzyme lacks the linker and cellulose binding domain, thus explaining why it does not bind cellulose in contrast to the Trichoderma enzyme. These results point to a possible common ancestor gene for the active site domain, while the linker and the binding domain may have been added to the Trichoderma esterase by gene fusion.

Class III alcohol dehydrogenase from the lizard Uromastix hardwickii has been characterized. This non-mammalian, gnathostomatous vertebrate class III form allows correlations of structures and functions of this class, the traditional class I alcohol dehydrogenase, and other well-studied proteins. Catalytically, results show similar recoveries and activities of all vertebrate class III forms independent of source, similar activities also in invertebrates but in lower amounts, and considerably higher specific activities in microorganisms. Structurally, variability patterns are consistent throughout the vertebrate system with a ratio in accepted point mutations versus class I of 0.4. This ratio between different classes of a zinc enzyme is comparable to that between different heme proteins (cytochrome c and myoglobin), suggesting defined but non-identical functions also for the alcohol dehydrogenase classes.

Liver alcohol dehydrogenase of the ethanol-active type (‘class I enzyme’) from the lizard, Uromastix hardwickii, was purified and screened for relationships with other vertebrate forms of the enzyme. Two differernt acetylated N-termini (acetyl-Gly and acetyl-Ser) and further positional differences already in the N-terminal segments establish the presence of two types of protein chain. The multiplicity is different from that hitherto detected within vertebrate class I alcohol dehydrogenase isozymes but typical of that which would be expected for subunits of different classes. In particular, relationships to class II or to class II-related forms appear likely. This may indicate yet further vertebrate alcohol dehydrogenase multiplicity or discovery of a class II non-mammalian enzyme. The results give prospects of defining gene duplications corresponding to more than one alcohol dehydrogenase class split to at an early vertebrate stage.

Two different insulin-like growth-factor (IGF)-binding proteins have been found in human blood, one of high molecular mass and dependent on growth hormone for synthesis, the other of low molecular mass and independent of growth hormone. The small IGF-binding protein is abundant in human amniotic fluid. Its amino acid sequence has now been determined by direct analysis of the protein and its proteolytic fragments. Also, by immunoscreening a partial cDNA clone was isolated from a human hepatoma cell line. The mature protein consists of 234 amino acids and is coded for by an mRNA of approximately 1700 nucleotides in length. The primary structure of the protein reveals 18 Cys residues in N-terminal and C-terminal clusters and an Arg-Gly-Asp peptide sequence, common to extracellular proteins binding to receptors of the integrin family. A protein-sequence polymorphism was detected at position Ile/Met-228, indicating possible allelic variation. The 3'-untranslated mRNA sequence has a high A + T content and shows five copies of an ATTTA sequence, which has been shown to be involved in the regulation of the stability of certain mRNAs coding for growth-regulating proteins.