Olle Danielsson

The Alcohol Dehydrogenase System

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.

Peptide repertoire of human cerebrospinal fluid: novel proteolytic fragments of neuroendocrine proteins

Polypeptides in human cerebrospinal fluid (CSF), isolated by phase separation in chloroform-methanol-water and reversed-phase HPLC, were characterised by sequence analysis and mass spectrometry. This identified the presence of peptide fragments of testican, neuroendocrine specific protein VGF, neuroendocrine protein 7B2, chromogranin B/secretogranin I, chromogranin A, osteopontin, IGF-II E-peptide and proenkephalin. The majority of these fragments were generated by proteolysis at dibasic sites, suggesting that they are derived by activities related to prohormone convertase(s). Several of the fragments have previously not been detected, and their functions in CSF or elsewhere are unknown. A characteristic feature of all these fragments is a very high content of acidic residues, in particular glutamic acid. In addition to the fragments of neuroendocrine proteins, endothelin-binding receptor-like protein 2, ribonuclease 1, IGF-binding protein 6, albumin, alpha1-acid glycoprotein 1, prostaglandin-H2 D-isomerase, apolipoprotein A1, transthyretin, beta2-microglobulin, ubiquitin, fibrinopeptide A, and C4A anaphylatoxin were found.

Enzyme and Isozyme Developments within the Medium-Chain Alcohol Dehydrogenase Family

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.

Multiplicity of N-terminal structures of medium-chain alcohol dehydrogenases Mass-spectrometric analysis of plant, lower vertebrate and higher vertebrate class I, II, and III forms of the enzyme

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.

Fast atom bombardment mass spectrometry and chemical analysis in determinations of acyl-blocked protein structures

Peptide generation and fast atom bombardment mass spectrometry in combination with conventional chemical analysis was used to identify the blocking group and establish the N‐terminal structure of six different proteins at the nanomole level. In this manner, the first terminal structures of three non‐mammalian alcohol dehydrogenases were determined, demonstrating the presence of N‐terminal acetylation in these piscine, amphibian, and avian enzymes. Similarly, two different yeast glucose‐6‐phosphate dehydrogenases and a minor variant of a human alcohol dehydrogenase were found to be acetylated. The exact end location of C‐terminal structures was also established. Together, the analyses permit the definition of terminal regions and blocking groups, thus facilitating the delineation of remaining structures.

Selective insulin-like growth factor-I antagonist inhibits mouse embryo development in a dose-dependent manner

OBJECTIVE To study the role of a synthetic insulin-like growth factor-I receptor (IGF-IR) antagonist, picropodophyllin, for mouse preimplantation embryo development in vivo and in vitro. DESIGN In vitro and in vivo study. SETTING Hospital-based research unit. ANIMALS FVB/N mice and mouse embryos. INTERVENTION(S) The effect of picropodophyllin in mouse embryo development in vivo and in vitro, immunohistochemistry, ELISA, polymerase chain reaction. MAIN OUTCOME MEASURE(S) Embryo development, presence of IGF-IR, messenger RNA expression, IGF-I synthesis. RESULT(S) The effect of picropodophyllin on embryo development in vitro and in vivo was not reversible. Mice treated with picropodophyllin 1 to 3 days after mating had a reduced number of blastocysts, 40.5% versus 78.8%, and a higher number of embryos with delayed development, 48.6% versus 11.5%. Insulin-like growth factor-IR protein is present in both phosphorylated and nonphosphorylated form at all stages of embryo development. The relative IGF-IR messenger RNA expression was highest in the oocyte and reduced during development to blastocyst stage. Insulin-like growth factor-I in culture media was reduced after picropodophyllin treatment. CONCLUSION(S) We conclude that IGF-I has an important role in normal mouse embryo development and that its receptor plays an essential role in the embryonic genome activation process.

Alcohol Dehydrogenase Variability

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).

Crystallisation and crystallographic investigations of cod alcohol dehydrogenase class I and class III enzymes.

Cod liver alcohol dehydrogenase of class‐hybrid properties has been crystallized as an NAD+—pyrazole complex in the monoclinic space group P21 with cell dimensions a = 103.3 Å, b = 47.4 Å, c = 80.7 Å, β = 104.6°, and with one dimer in the asymmetric unit. The position of the dimer molecule in the crystal was determined by molecular replacement methods at 3.0 Å resolution. The successful search model was the poly‐alanine structure of the horse enzyme. Side chains were then replaced according to the amino acid sequence of the cod enzyme, and the structure has been refined at 2.8 Å to an R‐factor of 0.26. Cod liver class III alcohol dehydrogenase crystallizes in the monoclinic space group C2 with cell dimensions a = 127.5 Å, b = 76.6 Å, c = 93.4 Å, β = 99.4° and with probably one dimer in the asymmetric unit.

Site-directed mutagenesis and enzyme properties of mammalian alcohol dehydrogenases correlated with their tissue distribution

Site-directed mutagenesis of mammalian alcohol dehydrogenases has helped to explain functional differences between enzymes within the protein family and traced these characteristics to specific amino acid residues. A threonine/serine exchange at position 48 in the human beta/gamma subunits can explain sensitivity to testosterone inhibition, as well as steroid dehydrogenase activity. It is possible to correlate the glutathione-dependent formaldehyde dehydrogenase activity of class III alcohol dehydrogenase with an arginine at position 115. Tissue distribution analysis of the three initially established classes of mammalian alcohol dehydrogenase show pronouncedly different patterns. Class I alcohol dehydrogenase is widespread but varies between the tissues, and exists in small amounts in the brain. The occurrence of class II is limited in contrast to the class III enzyme which is abundant in all tissues examined. The latter probably reflects the need for scavenging of formaldehyde in cytoprotection. Additional enzyme forms of mammalian alcohol dehydrogenase have been detected and have to be investigated further, together with the enzymes characterized earlier, regarding their physiological role in alcohol metabolism.

Tissue Distribution of Alcohol and Sorbitol Dehydrogenase mRNAs

Alcohol dehydrogenase (ADH) of class I is the principal enzyme in liver ethanol oxidation, and has been the subject of much research. It has been studied in many species and is a part of the enzyme system now constituting ADHs at large. The different mammalian ADHs can be divided into at least five classes according to structural properties (Pares et al., 1992). Class I is the classical liver ADH (Vallee and Bazzone, 1983), and class III ADH is the glutathione-dependent formaldehyde dehydrogenase (Koivusalo et al., 1989). ADH of class II shows a higher Km for ethanol than class I, and exhibits activity toward norepinephrine metabolites (Mardh et al., 1986), but is less studied than class I and class III ADH. Class IV is a stomach ADH characterized in rat and man (Pares et al., 1990; 1992; Moreno and Pares, 1991), and class V is a DNA-derived human structure recently reported (Yasunami et al., 1991).