Nucleotide sequence of the 18S ribosomal ribonucleic acid gene from two teleosts and two sharks and their molecular phylogeny.

The 18S rRNA sequence was determined for two teleostean fish species, Fundulus heteroclitus and Sebastolobus altivelis, and two sharks, Squalus acanthias and Echinorhinus cookei. To study the molecular phylogeny of these taxa, the sequences were compared with 18S rRNA sequences of the Coelacanth Latimeria chalumnae, the frog Xenopus laevis, and humans. Maximum parsimony analysis of the sequences resulted in a single most parsimonious tree that is in agreement with the expected phylogeny. The correct phylogenetic tree was also found when using S. altivelis alone as the teleost representative. In contrast, the most parsimonious tree found by using F. heteroclitus as the teleost representative presented anomalous groupings (the teleost branch being grouped with humans), matching results previously obtained. However, a bootstrap analysis showed that some branches containing anomalous relationships were not significantly supported. An explanation for this peculiarity, the differences between our tree and previously identified ones, and their phylogenetic implications are discussed.     

Hydrostatic pressure alters the time course of GTP

The effects of hydrostatic pressure on the receptor-stimulated exchange of guanosine triphosphate (GTP) for guanosine diphosphate (GDP) on the a subunit of G proteins were studied in two congeneric marine teleost fishes that differ in their depths of distribution. The poorly hydrolyzable GTP analog [35S]guanosine 5'-[gamma-thio]triphosphate ([35S]GTP[S]) was used to monitor the modulation of signal transduction by the A1 adenosine receptor agonist N6-R-(phenylisopropyl)adenosine (R-PIA) in brain membranes of the scorpaenids Sebastolobus alascanus and S. altivelis. The maximal binding (Bmax) and dissociation constant (K(d)) values, determined from equilibrium binding isotherms at atmospheric pressure (5 degrees C), were similar in the two species. The Bmax values for these species are much lower than literature values for mammalian brain tissue (25 degrees C); however, the K(d) values of the teleost and mammalian G proteins are similar. The EC50 values for the A1 adenosine receptor agonist R-PIA were similar in the two species. Hydrostatic pressure of 204 atm altered the binding of [35S]GTP[S]; basal [35S]GTP[S] binding decreased 25%. The A1 adenosine receptor agonist R-PIA and the muscarinic cholinergic receptor agonist carbamyl choline stimulated [35S]GTP[S] binding at 1 and 204 atm. At atmospheric pressure the half-time (t1/2) of [35S]GTP[S] binding differed between the two species. The GTP[S] on rate (k(on)) is larger in the shallower-living S. alascanus. Increased hydrostatic pressure altered the time course, decreasing the t1/2 in both species. The pressures that elicit this change in the time course differ between the species. However, interpolating over the range of in situ pressures the species experience, the values are similar in the two species. The guanyl nucleotide binding properties of the G protein a subunits appear to be conserved at the environmental temperatures and pressures the species experience.     

Comparison of LDH-B4 allozymes of macrourids (Teleostei: Gadiformes) by electrophoresis and peptide mapping

The heart-type lactate dehydrogenase (LDH-_B4) homologs of two species of Nezumia and eight species of Coryphaenoides are isomobile on two commonly used electrophoretic buffer systems. To test the hypothesis that the homologs possess the same primary structures, the allozymes from N. bairdii and four species of Coryphaenoides were purified by affinity chromatography on an oxamate aminohexyl Sepharose column and digested with trypsin. The resulting peptide mixtures were then mapped using reversed-phase high-performance liquid chromatography. The peptide maps of the enzyme homologs indicate that the overall similarity of the homologs is high, but unique peptides in each species indicate that the allozymes are not identical in primary structure.     

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria. III) The primary structure of thermophilic lactate dehydrogenase from Bacillus stearothermophilus. Hydroxylamine-, o-iodosobenzoic acid- and tryptic-fragments. The complete amino-acid sequence

Based on the partial sequence of the cyanogen bromide fragments [Tratschin, J.D., Wirz, B., Frank, G. and Zuber, H. (1983) Hoppe-Seyler's Z. Physiol. Chem. 364, 879-892], the amino-acid sequence of thermophilic lactate dehydrogenase from B. stearothermophilus was completed by the preparation and sequencing (sequenator, carboxypeptidase A and Y) of further overlapping fragments. Suitable peptide fragments were obtained by lactate dehydrogenase cleavage with hydroxylamine, o-iodosobenzoic acid and trypsin. The polypeptide chain of thermophilic lactate dehydrogenase from B. stearothermophilus consists of 317 amino-acid residues. While sequence homology with mesophilic lactate dehydrogenase of higher organisms reaches 35%, it is substantially higher with this mesophilic enzyme of bacillae (greater than 60%, B. megaterium, B. subtilis). The secondary structure elements and amino-acid residues of the active site of thermophilic lactate dehydrogenase deducted from primary structure data were compared with those from the mesophilic enzyme, the same was done for the internal sequence homology at the nucleotide-binding units. A comparative structure analysis (matrix system) based on the primary structure data of thermophilic enzyme should provide insight into the characteristic structure differences between thermophilic and mesophilic lactate dehydrogenase.     

The alpha-mannosidase from Canavalia ensiformis seeds: chemical and kinetic properties and action on animal lymphocytes

The alpha-mannosidase from Canavalia ensiformis was characterized with respect to molecular mass, glycoprotein nature, amino-acid composition, enzymic properties and action on animal cells. The enzyme is composed of two pairs of subunits (molecular mass 44 and 66 kDa) which form a tetramer (220 kDa). The larger subunit is glycosylated, the smaller one is not. Both subunits have similar amino-acid compositions. The larger subunit contains a surplus of alanine, aspartic acid/asparagine, histidine, phenylalanine and tyrosine, the smaller one a surplus of glutamic acid/glutamine, serine and threonine. The enzyme is subject to product inhibition by mannose. It stimulates the proliferation of B-lymphocytes from nude mice.     

Proteinchemical and kinetic features of gramicidin S synthetase

The amino-acid compositions of both enzymes of gramicidin S synthetase were determined. These proteins contain a high number of acidic amino-acid residues. Phenylalanine racemase, the light enzyme, was sequenced from the N-terminus until position 10. The kinetics of the thioester formation reactions were studied. The half-life times of these processes under substrate saturation conditions were found in the range between seconds and a few minutes. The valine activation at the heavy enzyme was detected as one of the rate-limiting steps of the biosynthesis of gramicidin S.     

Purification and properties of porcine testis acylphosphatase

Acylphosphatase has been purified from porcine testis and its properties were compared with those of porcine skeletal muscle acylphosphatase. The molecular weight of the testis enzyme was found to be 10,600, similar to that of porcine skeletal muscle acylphosphatase, on sedimentation equilibrium analysis. The specific activity of the testis enzyme was 10,800 mumol/min/mg at 25 degrees C with benzoyl phosphate as substrate, i.e., higher than that of the muscle enzyme, 7,200 mumol/min/mg, under the same conditions. The pI of the testis enzyme was 8.3, i.e., lower than that of the muscle enzyme, 10.6. There were marked differences in the amino acid compositions of the two enzymes. In particular two histidine residues were present in the testis enzyme but none were present in the muscle enzyme, and no cysteine residue was present in the testis enzyme but one was present in the muscle enzyme. The carboxyl terminal amino acid of the testis enzyme seemed to be lysine, while that of the muscle enzyme is tyrosine. The peptide maps of the testis and muscle enzymes indicated considerable differences in the amino acid sequences of the two enzymes. Differences in the antigenic structures of the two enzymes were demonstrated on enzyme linked immunoassaying and double immunodiffusion. These results indicate that the porcine testis acylphosphatase is an isozyme different from the porcine skeletal muscle acylphosphatase.     

The presence of essential histidine residues in manganese(III)-containing acid phosphatase from sweet potato

Chemical modification studies of manganese(III)-containing acid phosphatase [EC 3.1.3.2] were carried out to investigate the contributions of specific amino-acid side-chains to the catalytic activity. Incubation of the enzyme with N-ethylmaleimide at pH 7.0 caused a significant loss of the enzyme activity. The inactivation followed pseudo-first-order kinetics. Double log plots of pseudo-first-order rate constant vs. concentration gave a straight line with a slope of 1.02, suggesting that the reaction of one molecule of reagent per active site is associated with activity loss. The enzyme was protected from inactivation by the presence of molybdate or phosphate ions. Amino acid analyses of the N-ethylmaleimide-modified enzyme showed that the 96%-inactivated enzyme had lost about one histidine and one-half lysine residue per enzyme subunit without any significant decrease in other amino acids, and also demonstrated that loss of catalytic activity occurred in parallel with the loss of histidine residue rather than that of lysine residue. Molybdate ions also protected the enzyme against modification of the histidine residue. The enzyme was inactivated by photooxidation mediated by methylene blue according to pseudo-first-order kinetics. The pH profile of the inactivation rates of the enzyme showed that an amino acid residue having a pKa value of approximately 7.2 was involved in the inactivation. These studies indicate that at least one histidine residue per enzyme subunit participates in the catalytic function of Mn(III)-acid phosphatase.     

Chemical modification of dopamine beta-hydroxylase

Dopamine beta-hydroxylase (3,4- dihydroxyphenylethylamine ,ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1) is the terminal enzyme in the biosynthetic pathway of norepinephrine. Chemical modification studies of this enzyme were executed to investigate contributions of specific amino-acid side-chains to catalytic activity. Sulfhydryl reagents were precluded, since no free cysteine residue was detected upon titration of the denatured or native protein with 2-chloromercuri-4-nitrophenol. Incubation of enzyme with diazonium tetrazole caused inactivation of the protein coupled with extensive reaction of lysine and tyrosine residues. Reaction with iodoacetamide resulted in complete loss of enzymatic activity with reaction of approximately three histidine residues; methionine reaction was also observed. Modification of the enzyme using diethylpyrocarbonate resulted in complete inactivation of the enzyme, and analysis of the reacted protein indicated a loss of approx. 1.7 histidine residues per protein monomer with no tyrosine or lysine modification observed. The correlation of activity loss with histidine modification supports the view that this residue participates in the catalytic function of dopamine beta-hydroxylase.     

Pyruvoyl-dependent histidine decarboxylases. Comparative sequences of cysteinyl peptides of the enzymes from Lactobacillus 30a, Lactobacillus buchneri, and Clostridium perfringens

The two cysteinyl residues present in histidine decarboxylase from Lactobacillus 30a differ greatly in reactivity. One (class 1) reacts readily in the native state with dithiobis-(2-nitrobenzoate) with complete loss of enzyme activity; the other (class 2) reacts only after denaturation of the enzyme (Lane, R. S., and Snell, E. E. (1976) Biochemistry 15, 4175-4179). These differences in reactivity permitted use of covalent (disulfide) chromatography to isolate separate peptides that contain these two residues. Sequence analysis showed that the class 1 cysteinyl residue is at position 147 in a hydrophilic portion of the alpha chain (Huynh, Q. K., Recsei, P. A., Vaaler, G. L., and Snell, E. E. (1984) J. Biol. Chem. 259, 2833-2839), while the class 2 cysteinyl residue is present at position 71, adjacent to a hydrophobic portion of the same chain. Cysteinyl peptides identical with or homologous to the class 2 cysteinyl peptide of the Lactobacillus 30a enzyme were isolated from the alpha subunits of histidine decarboxylases from Lactobacillus buchneri and Clostridium perfringens, respectively. The L. buchneri enzyme also contained a peptide homologous to the class 1 cysteinyl peptide from Lactobacillus 30a. However, no corresponding peptide was present in the enzyme from C. perfringens, in which the second cysteinyl residue of the alpha chain occupies position 3, very near the essential pyruvoyl residue. This enzyme, unlike those from Lactobacillus 30a or L. buchneri, also contains one cysteinyl residue in its beta chain. Although Cys 147 is an active site residue in histidine decarboxylase from Lactobacillus 30a, the absence of a corresponding residue in the C. perfringens enzyme confirms previous indications (Recsei, P. A., and Snell, E. E. (1982) J. Biol. Chem. 257, 7196-7202) that this SH group is not essential for decarboxylase action.     

Elastase-cathepsin G inhibitors eglin b and eglin c differ by a single Tyr----His substitution. A micro-method for the identification of amino-acid substitution

The structures of eglin b and eglin c, both potent inhibitors of human neutral granulocytic proteinase elastase and cathepsin G, were compared by micro amino-acid analysis and peptide mapping techniques. Eglin b and eglin c differ by one amino-acid substitution in the middle of the polypeptide chain. Tyrosine residue at position 35 of eglin c was substituted by histidine in eglin b. This amino-acid substitution requires one base exchange (U----C) at the DNA level and apparently does not affect the reactive site of eglins. Though without disulfide linkages, eglins are very rigid molecules and can be effectively digested by trypsin only after rigorous acid incubation.     

Aromatic boronic acids as probes of the catalytic site of human plasma lecithin-cholesterol acyltransferase

We have recently proposed a catalytic mechanism for human plasma lecithin-cholesterol acyltransferase (EC 2.3.1.43) (J. Biol. Chem. (1986) 261, 7032-7043), implicating single serine and histidine residues in phosphatidylcholine cleavage and two cysteine residues in cholesterol esterification. We now confirm the involvement of serine and histidine in catalysing the phospholipase A2 action of lecithin-cholesterol acyltransferase by demonstrating the inhibition of this activity by phenylboronic acid (Ki = 1.23 mM) and m-aminophenylboronic acid (Ki = 2.32 mM), inhibitors of known serine/histidine hydrolases. The specificity of the interaction of aromatic boronic acids with catalytic serine and histidine residues and the putative formation of a tetrahedral adduct between boron and the lecithin-cholesterol acyltransferase serine hydroxyl group which is similar to the transition-state intermediate formed between phosphatidylcholine and the catalytic serine residue was suggested by: substrate protection against inhibition by phenylboronic acids; a much reduced incorporation of phenylmethane[35S]sulphonyl fluoride into the enzyme in the presence of phenylboronic acid; the lack of interaction of histidine- or serine-modified enzyme with immobilized phenylboronic acid in the presence of glycerol (Ve/Vo = 2.7 and 2.3 respectively) when compared to the native enzyme (Ve/Vo = 5.25). Fatty acyl-lecithin-cholesterol acyltransferase, produced by incubation of the enzyme with a lecithin-apolipoprotein A-I proteoliposome substrate, was not retarded upon the sorbent column (Ve/Vo = 1.5). Modification of the enzyme's two free cysteine residues with 5,5'-dithiobis(2-nitrobenzoic acid) or potassium ferricyanide reduced (Ve/Vo = 3.5) but did not abolish retardation on the sorbent column, indicating that these modifications resulted in steric hinderance of the interaction of the boron atom with the lecithin-cholesterol acyltransferase serine hydroxyl group. These data suggest that the serine and histidine residues are proximal within the enzyme catalytic site and that both cysteine thiol groups are close to the serine hydroxyl group. The presence of significant amino-acid sequence homologies between lecithin-cholesterol acyltransferase, triacylglycerol lipases and the transacylases of fatty acid synthase is also reported.     

Chemical phosphorylation of histidine-containing peptides based on the sequence of histone H4 and their dephosphorylation by protein histidine phosphatase

Using peptides based on the amino acid sequences surrounding the two histidine residues in histone H4, we have investigated the kinetics of the phosphorylation and dephosphorylation reactions of their histidine residues, when reacted with potassium phosphoramidate, by ¹H NMR. We have been able to estimate rate constants for the reactions and have shown that there are differences in the kinetics between the two peptides. The kinetics of hydrolysis of phosphoramidate was measured by ³¹P NMR and protein histidine phosphatase (PHP) was shown to catalyse the reaction. We have shown that the dephosphorylation of the phosphohistidine of the phosphopeptides is catalysed by PHP. In terms of substrate specificity, there is a small preference for 1-phosphohistidine compared to 3-phosphohistidine, although the rate accelerations for hydrolysis induced by the enzyme were 1100- and 33,333-fold, respectively. The kinetics of both the phosphorylation and dephosphorylation reactions depend on the amino acid sequence surrounding the histidine. PHP shows greater substrate specificity for the peptide whose sequence is similar to that around histidine 18 of histone H4. PHP was unable to catalyse the dephosphorylation of histone H4 that had been phosphorylated with a histone H4 histidine kinase.     

A missense mutation found in human lactate dehydrogenase-B (H) variant gene

A human lactate dehydrogenase-B mutant gene was isolated from a genomic DNA library constructed from a patient with unstable LDH-B (heart) subunit. The nucleotide sequences of seven protein-coding exons were determined and a single nucleotide substitution of G by A at Arg codon CGC in exon 4 was found. This mutation results in an amino-acid replacement of a conserved arginine by histidine at the residue "173," which is involved in an anion-binding site at the P-axis of LDH subunits.     

The amino-acid sequence of rabbit Cu-Zn superoxide dismutase

The primary structure of Cu-Zn superoxide dismutase from rabbit liver was investigated. The reduced and S-carboxymethylated enzyme was treated with cyanogen bromide, trypsin or Staphylococcus aureus proteinase V8. The resulting peptides were separated by high-performance liquid chromatography and sequenced by automated Edman degradation. With the exception of the N- and C-terminus the complete sequence was established by means of overlapping peptides. The N-terminus is blocked and thus not susceptible to Edman degradation. The amino-acid composition of the tryptic N-terminal peptide corresponds to that of the cytoplasmatic Cu-Zn superoxide dismutases of other mammals investigated. The chromatographic behaviour of these N-terminal peptides on a reversed phase C18 column is also identical, thus suggesting also for the rabbit Cu-Zn superoxide dismutase the N-terminal sequence Ac-Ala-Thr-Lys. The C-terminus was demonstrated to have the sequence -Ile-Ala-Pro by enzymatic degradation with carboxypeptidase Y. The complete amino-acid sequence of the rabbit Cu-Zn superoxide dismutase consists of 152 amino-acids and shows the expected homology to other Cu-Zn enzymes published so far. The aspartate and six histidine residues known to complex the metal ions are conserved at homologous positions. This also applies for the arginine residue near the C-terminus which is supposed to direct the anionic superoxide radical towards the active centre of the enzyme. The amino acid sequence of the rabbit Cu-Zn superoxide dismutase corresponds to those of other mammals in more than 80% of its amino-acid residues. From a total of 152 amino-acid residues the rabbit shares with rat 128, with mouse 130, with horse 127, with pig 126/127, with cattle 130 and with man 131 amino acids in homologous positions. However the Cu-Zn superoxide dismutases of closely related mammals like rats and mice differ in only five amino acid residues of their sequence. A phylogenetic closer relatedness between lagomorphs and rodents than between other orders of mammals, could not be derived from the sequence data given. Rather rodents and lagomorphs are to be considered as two evolutionary independent orders of mammals.     

PURIFICATION AND CHARACTERIZATION OF CARNOSINE SYNTHETASE FROM MOUSE OLFACTORY BULBS

Carnosine synthetase was purified about 500-fold from mouse olfactory bulb to a specific activity of approx 25 nmol/min/mg. This is an increase of 800-fold over that previously reported for this enzyme from rat brain and 11 times higher than the most highly purified enzyme from chicken pectoral muscle. ATP was essential for activity and could not be replaced by ADP. NAD had no effect on the synthesis of carnosine. Of the β-alanine analogues tested, the purified mouse enzyme incorporated only γ-aminobutyric acid and β-amino-n-butyric acid into peptide linkage with histidine. Synthesis of carnosine by the mouse olfactory bulb enzyme was competitively inhibited by the histidine analogues, 1-methyl histidine and 3-methyl histidine, with K_i values which were at least 40 times the K_m value for histidine (16 μM). Ornithine and lysine were more efficient β-alanine acceptors than 1-methyl histidine for the mouse enzyme. Enzyme from olfactory epithelium and leg skeletal muscle of mice also showed higher K_i values for 1–methyl histidine than the K_m value for histidine. In contrast, carnosine-anserine synthetase from chicken pectoral muscle gave K_m values for histidine, 1-methyl histidine and 3-methyl histidine, which were all in the range of 4–12 μM. The differences in substrate specificity between the enzyme from mouse and chicken implies alternate routes of anserine synthesis in these species and predicts the occurrence of certain novel peptides in mouse brain.     

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria, X. Analysis of structural elements responsible for the differences in thermostability and activation by fructose 1,6-bisphosphate in the lactate dehydrogenases from B. stearothermophilus and B. caldolyticus by protein engineering

The amino-acid sequences of the lactate dehydrogenases (LDH) from B. stearothermophilus and B. caldolyticus differ at only 10 positions. The properties of these enzymes however show substantial differences. The LDH from B. stearothermophilus is activated by Fru-P2 and has a higher thermostability (10 degrees C) than the enzyme from B. caldolyticus which cannot be activated by Fru-P2. To correlate these functional differences to the structural properties, we have constructed a set of hybrid- and point-mutants of the two LDHs. The amino acids at positions 207, 209B, and 209C could be identified to confer the property of activation by Fru-P2 to the enzymes. This part of the enzyme is to a large extent also responsible for the different thermostabilities of these two proteins.     

An active site homology model of phenylalanine ammonia-lyase from Petroselinum crispum.

The plant enzyme phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) shows homology to histidine ammonia-lyase (HAL) whose structure has been solved by X-ray crystallography. Based on amino-acid sequence alignment of the two enzymes, mutagenesis was performed on amino-acid residues that were identical or similar to the active site residues in HAL to gain insight into the importance of this residues in PAL for substrate binding or catalysis. We mutated the following amino-acid residues: S203, R354, Y110, Y351, N260, Q348, F400, Q488 and L138. Determination of the kinetic constants of the overexpressed and purified enzymes revealed that mutagenesis led in each case to diminished activity. Mutants S203A, R354A and Y351F showed a decrease in kcat by factors of 435, 130 and 235, respectively. Mutants F400A, Q488A and L138H showed a 345-, 615- and 14-fold lower kcat, respectively. The greatest loss of activity occurred in the PAL mutants N260A, Q348A and Y110F, which were 2700, 2370 and 75 000 times less active than wild-type PAL. To elucidate the possible function of the mutated amino-acid residues in PAL we built a homology model of PAL based on structural data of HAL and mutagenesis experiments with PAL. The homology model of PAL showed that the active site of PAL resembles the active site of HAL. This allowed us to propose possible roles for the corresponding residues in PAL catalysis.     

Histidine for Treatment of Uraemic Anaemia

A group of 28 uraemic patients on dialysis treatment were given daily supplements of histidine by mouth. Plasma amino-acid concentration, plasma iron, serum transferrin, packed cell volume, and reticulocyte count were all measured before and after two months of histidine supplementation. The treatment raised the plasma histidine concentration and at the same time there was a rise in transferrin and iron levels and packed cell volume. Reticulocyte counts fell after two months of histidine supplementation.