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H Jörnvall

Publications and source records attributed to H Jörnvall.

At least 19 recordsLinked to original sources
PubMed1997

Guinea pig and bovine zeta-crystallins have distinct functional characteristics highlighting replacements in otherwise similar structures.

zeta-Crystallin, a major cytosolic protein of guinea pig lens, has been characterized as an NADPH:quinone oxidoreductase (EC 1.6.5.5) [Rao et al. (1992) J. Biol. Chem. 267, 97-103]. A bovine lens homolog with 83% sequence identity was found to have very different functional characteristics. While the bovine lens zeta-crystallin exhibits similar physicochemical properties, such as molecular weight, hydropathy profile, and predicted secondary structure, and exhibits strong immunological cross-reactivity with the guinea pig and human lens zeta-crystallins, it shows minimal quinone oxidoreductase activity. On the other hand, bovine lens zeta-crystallin, but not guinea pig zeta-crystallin, showed a strong binding affinity to single-stranded DNA (ssDNA) that could be competed with NADPH, the specific cofactor of zeta-crystallin. NADH and dextran sulfate did not affect this characteristic of bovine zeta-crystallin and the enzyme showed no binding affinity for the heparin-Ultragel A4R. Two-dimensional electrophoresis of bovine lens zeta-crystallin showed a distinct pattern of posttranslational charge modification as compared to the guinea pig protein. Alignment of eight zeta-crystallin sequences, and computer modelling of the bovine and human forms based on the crystallographically analyzed Escherichia coli form, suggest that if loss of a functional residue accounts for the lowered catalytic activity of the bovine protein, Tyr 52 of the E. coli enzyme, and the equivalent Tyr present in all known mammalian forms except the bovine, is the likely candidate. In the bovine form this tyrosine is replaced by histidine.

Amino Acid Sequence↗
PubMed1997

Characterization of a marsupial glutathione transferase, a class Alpha enzyme from Brown Antechinus (Antechinus stuartii).

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.

Amino Acid Sequence↗
PubMed1997

Active site directed mutagenesis of 3 beta/17 beta-hydroxysteroid dehydrogenase establishes differential effects on short-chain dehydrogenase/reductase reactions.

Mutagenetic replacements of conserved residues within the active site of the short-chain dehydrogenase/reductase (SDR) superfamily were studied using prokaryotic 3 beta/17 beta-hydroxysteroid dehydrogenase (3 beta/17 beta-HSD) from Comamonas testosteroni as a model system. The results provide novel data to establish Ser 138 as a member of a catalytically important "triad" of residues also involving Tyr151 and Lys155. A Ser-->Ala exchange at position 138 results in an almost complete (> 99.9%) loss of enzymatic activity, which is not observed with a Ser-->Thr replacement. This indicates that an essential factor for catalysis is the ability of side chain 138 to form hydrogen bond interactions. Mutations in the NAD(H) binding region, in strands beta A, beta D, and adjacent turns, reveal two additional residues, Thr12 and Asn87, which are important for correct binding of the coenzyme and with a differential effect on the reactions catalyzed. Thus, mutation of Thr12 to Ala results in a complete loss of the 3 beta-dehydrogenase activity, whereas the 3-oxoreductase activity remains unchanged. On the other hand, a T12S substitution yields a protein with unaltered catalytic constants for both reactions, revealing that a specific hydrogen bond is critical for the dehydrogenase activity. Our interpretation of the available crystal structure of 3 alpha/20 beta-HSD from Streptomyces hydrogenans suggests a hydrogen bond in that enzyme between the Thr12 side chain and the backbone NH of Asn87 rather than the coenzyme, indicating that this hydrogen bond to the beta D strand might determine a crucial difference between the reductive and the oxidative reaction types. Similarly, mutation of Asn87 to Ala results in an 80% reduction of kcat/Km in the dehydrogenase direction but also unchanged 3-oxoreductase properties. It appears that the binding of NAD+ to the protein is influenced by local structural changes involving strand beta D and turn beta A to alpha B.

17-Hydroxysteroid Dehydrogenases↗
PubMed1996

A novel type I receptor serine-threonine kinase predominantly expressed in the adult central nervous system.

Receptor serine-threonine kinases (RSTK) mediate inhibitory as well as stimulatory signals for growth and differentiation by binding to members of the transforming growth factor-beta (TGF-beta) superfamily. Over 12 different RSTKs have been isolated so far, displaying wide expression in peripheral tissues and in the nervous system. Here we report the isolation and characterization of a novel type I RSTK termed activin receptor-like kinase-7 (ALK-7) that, unlike other members of this receptor family, is predominantly expressed in the adult central nervous system. The ALK-7 gene encodes a 55-kDa cell-surface protein that exhibits up to 78% amino acid sequence identity in the kinase domain to previously isolated type I receptors for TGF-beta and activin. In the extracellular domain, however, ALK-7 is more divergent, displaying comparable similarities with all members of the ALK subfamily. RNase protection and in situ hybridization studies demonstrated a highly specific mRNA distribution restricted to neurons in several regions of the adult rat central nervous system, including cerebellum, hippocampus, and nuclei of the brainstem. Receptor reconstitution and cross-linking experiments indicated that ALK-7 can form complexes with type II RSTKs for TGF-beta and activin in a ligand-dependent manner, although direct binding of ALK-7 to ligand in these complexes could not be demonstrated. The specific expression pattern of ALK-7, restricted to the postnatal central nervous system, indicates that this receptor may play an important role in the maturation and maintenance of several neuronal subpopulations.

Activin Receptors↗
PubMed1996

Isozyme multiplicity with anomalous dimer patterns in a class III alcohol dehydrogenase. Effects on the activity and quaternary structure of residue exchanges at "nonfunctional" sites in a native protein.

The isozymes of class III alcohol dehydrogenase/glutathione-dependent formaldehyde dehydrogenase from cod were characterized. They exhibited three unexpected properties of general interest. First, these dimeric isozymes, derived from two types of subunit (h and l, for high- and low-activity forms), were recovered from liver preparations in only the homodimeric ll and heterodimeric hl combinations. Dissociation and reassociation of the isolated hl form in vitro also resulted in lower yields of the hh than the ll homodimer, although class III subunits are usually freely associable over wide borders of divergence (human and Drosophila). The h and l primary structures show that both chain types are characteristic of class III enzymes, without large amino acid replacements at positions of known subunit interactions. Hence, the hh dimer partial restriction indicates nontraditional alterations at h-subunit interfaces. The structure provides a possible explanation, in the form of h-chain modifications that may influence the anchoring of a loop at positions of two potentially deamidative beta-aspartyl shifts at distant Asn-Gly structures. Second the ll and hl forms differ in enzymatic properties, having 5-fold different K(m) values for NAD+ at pH 8, different K(m) values for S-(hydroxymethyl)glutathione (10 versus 150 microM), and different specific activities (4.5 versus 41 units/mg), with ll resembling and hl deviating from human and other class III alcohol dehydrogenases. However, functional residues lining substrate and coenzyme pockets in the known conformations of homologous forms are largely identical in the two isozymes [only minor conservative exchanges of Val/Leu116, Val/Leu203, Ile/Val224, and Ile/Val269 (numbering system of the human class I enzyme)], again indicating effects from distantly positioned h-chain replacements. Third, the two isozymes differ a surprising amount in amino acid sequence (18%, the same as the piscine/ human difference), reflecting a remarkably old isozyme duplication or, more probably, discordant accumulation of residue exchanges with greater speed of evolution for one of the subunits (h chain) than is typical for the slowly evolving class III alcohol dehydrogenase.

Aldehyde Oxidoreductases↗
PubMed1996

Arabidopsis formaldehyde dehydrogenase. Molecular properties of plant class III alcohol dehydrogenase provide further insights into the origins, structure and function of plant class p and liver class I alcohol dehydrogenases.

A glutathione-dependent formaldehyde dehydrogenase (class III alcohol dehydrogenase) has been characterized from Arabidopsis thaliana. This plant enzyme exhibits kinetic and molecular properties in common with the class III forms from mammals, with a K(m) for S-hydroxymethylglutathione of 1.4 microM, an anodic electrophoretic mobility (pI: 5.3-5.6) and a cross-reaction with anti-(rat class III alcohol dehydrogenase) antibodies. The enzyme structure, deduced from the cDNA sequence, fits into the complex system of alcohol dehydrogenases and shows that all life forms share the class III protein type. The corresponding mRNA is 1.4 kb and present in all plant organs; a single copy of the gene is found in the genome. The class III structural variability is different from that of the ethanol-active enzyme types in both vertebrates (class I) and plants (class P), although class P conserves more of the class III properties than class I does. Also the enzymatic properties differ between the two ethanol-active classes. Active-site variability and exchanges at essential residues (Leu/Gly57, Asp/Arg115) may explain the distinct kinetics. These patterns are consistent with two different metabolic roles for the ethanol-active enzymes, a more constant function, reduction of acetaldehyde during hypoxia, for class P, and a more variable function, the detoxication of alcohols and participation in metabolic conversions, for class I. A sequence motif, Pro-Xaa-Ile/Val-Xaa-Gly-His-Glu-Xaa-Xaa-Gly, common to all medium-chain alcohol dehydrogenases is defined.

Alcohol Dehydrogenase↗
PubMed1996

Molecular modelling of human gastric alcohol dehydrogenase (class IV) and substrate docking: differences towards the classical liver enzyme (class I).

A three-dimensional model of the human class IV alcohol dehydrogenase has been calculated based upon the X-ray structure of the class I enzyme. As judged from the model, the substrate-binding site is wider than in class I, compatible with the differences in substrate specificities and the large difference in Km value for ethanol. Substrate docking performed for the class I structure and the class IV model show all-trans-retinol and 11-cis-retinol to bind better to the class IV enzyme. The calculations also indicate that 16-hydroxyhexadecanoic acid binds in a different manner for the two enzyme classes. A simulation of coenzyme-binding indicates that the adenine ring of the coenzyme might be differently bound in class IV than in class I, decreasing the interactions with Asp-223 which is compatible with the higher k(cat) values for class IV.

Alcohol Dehydrogenase↗
PubMed1996

Molecular characterization of a tissue-polypeptide-specific-antigen epitope and its relationship to human cytokeratin 18.

Tissue-polypeptide-specific antigen (TPS) from the human colon adenocarcinoma cell line WiDr was purified using the monoclonal antibody M3 as a probe. Upon SDS/polyacrylamide gradient gel electrophoresis, several TPS-positive bands were detected (corresponding to 13 kDa, 22 kDa and a doublet at 42 kDa). The 13-kDa moiety was purified about 30,000-fold by a 5-step protocol. The electro-phoretically homogeneous component was obtained in a 7% yield of the total TPS activity of the crude extract. N-terminal sequence analysis showed the presence of an N-terminally truncated molecule and identified the 13-kDa TPS component as a fragment of human cytokeratin 18, with a major from starting at position 284 of the parent molecule. Laser-desorption mass spectrometry showed the presence of one major component with a molecular mass corresponding to a C-terminal end close to position 396 (which gives 12776 Da for the form with non-truncated N-terminus). The M3 antibody was also used to screen a human prostate cDNA lambda gt11 library. Four identical phage clones were detected, each producing a fusion protein with beta-galactosidase and the M3-positive component. PCR amplification showed the presence of an approximately 1200-bp insert, and sequence analysis revealed it to contain a 996-nucleotide fragment corresponding to residues 103-429 of human cytokeratin 18 (plus a non-coding human desmin artifact fragment). Smaller fragments, engineered by PCR and expressed as fusion proteins using the pET3xc vector in Escherichia coli, showed that the M3 epitope is localized to cytokeratin 18, residues 322-340. Two other TPS-active monoclonal antibodies were localized to cytokeratin 18 with similar techniques, ascribing an epitope (to M21) to residues 414-429 and another (to M24) to residues 139-297. Combined, the results demonstrate that TPS reactivity is derived from specific epitopes of human cytokeratin 18.

Antibodies, Monoclonal↗
PubMed1996

Alcoholytic deblocking of N-terminally acetylated peptides and proteins for sequence analysis.

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.

Acetylation↗
PubMed1996

Fructose-1,6-bisphosphatase. Primary structure of the rabbit liver enzyme. 'Intermediate' variability of an oligomeric protein.

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.

Adenosine Monophosphate↗
PubMed1996

Leukotriene A4 hydrolase: protection from mechanism-based inactivation by mutation of tyrosine-378.

Leukotriene A4 (LTA4) hydrolase [(7E,9E,11Z,14Z)-(5S,6S)-5,6-epoxyicosa-7, 9,11,14-tetraenoate hydrolase; EC 3.3.2.6] is a bifunctional zinc metalloenzyme that catalyzes the final step in the biosynthesis of the potent chemotactic agent leukotriene B4 (LTB4). LTA4 hydrolase/aminopeptidase is suicide inactivated during catalysis via an apparently mechanism-based irreversible binding of LTA4 to the protein in a 1:1 stoichiometry. Previously, we have identified a henicosapeptide, encompassing residues Leu-365 to Lys-385 in human LTA4 hydrolase, which contains a site involved in the covalent binding of LTA4 to the native enzyme. To investigate the role of Tyr-378, a potential candidate for this binding site, we exchanged Tyr for Phe or Gln in two separate mutants. In addition, each of two adjacent and potentially reactive residues, Ser-379 and Ser-380, were exchanged for Ala. The mutated enzymes were expressed as (His)6-tagged fusion proteins in Escherichia coli, purified to apparent homogeneity, and characterized. Enzyme activity determinations and differential peptide mapping, before and after repeated exposure to LTA4, revealed that wild-type enzyme and the mutants [S379A] and [S380A]LTA4hydrolase were equally susceptible to suicide inactivation whereas the mutants in position 378 were no longer inactivated or covalently modified by LTA4. Furthermore, in [Y378F]LTA4 hydrolase, the value of kcat for epoxide hydrolysis was increased 2.5-fold over that of the wild-type enzyme. Thus, by a single-point mutation in LTA4 hydrolase, catalysis and covalent modification/inactivation have been dissociated, yielding an enzyme with increased turnover and resistance to mechanism-based inactivation.

Base Sequence↗
PubMed1996

Photoaffinity labelling of lactate dehydrogenase from pig heart with a bifunctional NAD(+)-analogue.

P1-N6-(4-azidophenylethyl)adenosine-P2-4-(3-azidopyridinio)b utyl diphosphate was synthesized with an [8-14C]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.

Affinity Labels↗
PubMed1996

Biochemical and antibacterial analysis of human wound and blister fluid.

Fluid from a post-operative wound, six leg ulcers and a large blister were collected and analysed by biochemical, microbiological and immunological techniques. The results were compared with those from sera. All samples were lyophilized and extracted twice with 60% aqueous acetonitrile containing 1% trifluoroacetic acid. The pooled supernatants were lyophilized, redissolved, and the fluid extracts were characterized by six techniques (the blister exudate only with three): reverse-phase HPLC, Edman degradation, mass spectrometry, Western blot analysis, inhibition zone assay on plates with Bacillus megaterium (anti-Bm activity) and zone clearing on plates with cell walls from Micrococcus luteus (a lysozyme assay). The material corresponding to HPLC peaks of the wound fluid extract was identified as: histone H2B fragments 1-11,1-15 and 1-16, intact thymosin beta-4, defensins HNP1, 2 and 3, lysozyme and the peptide antibiotic FALL-39 and its precursor(s). The HPLC-separated blister fluid was extremely rich in anti-Bm activity (mainly defensins) and lysozyme. It may also contain factors not identified before. The plate assays scored 50-fold differences in anti-Bm activities and more than 10-fold differences in lysozyme, factors which together with thymosin could be active in wound healing. It is concluded that analysis of wound fluid yields peptide and activity patterns with novel fragments of important peptides, and quantitative differences, that can be useful to understand molecular mechanisms of wound healing further.

Adult↗
PubMed1996

Glutaredoxin-3 from Escherichia coli. Amino acid sequence, 1H AND 15N NMR assignments, and structural analysis.

The primary and secondary structure of glutaredoxin-3 (Grx3), a glutathione-disulfide oxidoreductase from Escherichia coli, has been determined. The amino acid sequence of Grx3 consists of 82 residues and contains a redox-active motif, Cys-Pro-Tyr-Cys, typical of the glutaredoxin family. Sequence comparison reveals a homology (33% identity) to that of glutaredoxin-1 (Grx1) from E. coli as well as to other members of the thioredoxin superfamily. In addition to the active site cysteine residues, Grx3 contains one additional cysteine (Cys65) corresponding to one of the two non-active site (or structural) cysteine residues present in mammalian glutaredoxins. The sequence-specific 1H and 15N nuclear magnetic resonance assignments of reduced Grx3 have been obtained. From a combined analysis of chemical shifts, 3JHNalpha coupling constants, sequential and medium range NOEs, and amide proton exchange rates, the secondary structure of reduced Grx3 was determined and found to be very similar to that inferred from amino acid sequence comparison to homologous proteins. The consequences of the proposed structural similarity to Grx1 are that Grx3, while possessing a largely intact GSH binding cleft, would have a very different spatial distribution of charged residues, most notably surrounding the active site cysteine residues and occurring in the proposed hydrophobic protein-protein interaction area. These differences may contribute to the observed very low Kcat of Grx3 as a reductant of insulin disulfides or as a hydrogen donor for ribonucleotide reductase. Thus, despite an identical active site disulfide motif and a similar secondary structure and tertiary fold, Grx3 and Grx1 display large functional differences in in vitro protein disulfide oxido-reduction reactions.

Amino Acid Sequence↗
PubMed1996

An essential tyrosine residue of Aspergillus polygalacturonase.

Based on strict conservation of a tyrosine residue in 24 polygalacturonases, tyrosine modification was assessed in two different forms of the Aspergillus enzyme. The second subform was unknown in structure but submitted to sequence analysis and was found also to have the conserved tyrosine residue. Results of chemical modifications are consistent in showing inactivation of the proteins with all tyrosine-reactive agents tested, acetic anhydride, N-acetyl imidazole, and tetranitromethane. Furthermore, after acetylation, regeneration of enzyme activity was possible with hydroxylamine. Spectrophotometric pH titration showed that one accessible tyrosine residue is ionized at pH 9.3-9.5, whereas the remaining, masked residues are all ionized at pH 10.5. It is concluded that one tyrosine residue is catalytically important, in agreement with the inactivation and reactivation data, that this residue is accessible, and that it is likely to correspond to the strictly conserved residue observed in all forms.

Acetic Anhydrides↗
PubMed1996

Linking of isozyme and class variability patterns in the emergence of novel alcohol dehydrogenase functions. Characterization of isozymes in Uromastix hardwickii.

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.

Alcohol Dehydrogenase↗