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A Patthy

Publications and source records attributed to A Patthy.

At least 37 records · Page 2Linked to original sources
PubMed1994

A novel alpha-amylase inhibitor from amaranth (Amaranthus hypocondriacus) seeds.

The major alpha-amylase inhibitor (AAI) present in the seeds of Amaranthus hypocondriacus, a variety of the Mexican crop plant amaranth, is a 32-residue-long polypeptide with three disulfide bridges. Purified AAI strongly inhibits the alpha-amylase activity of insect larvae (Tribolium castaneum and Prostephanus truncatus) and does not inhibit proteases and mammalian alpha-amylases. AAI was sequenced with the automated Edman method, and the disulfide bridges were localized using enzymatic and chemical fragmentation methods combined with N-terminal sequencing. AAI is the shortest alpha-amylase inhibitor described so far which has no known close homologs in the sequence data bases. Its residue conservation patterns and disulfide connectivity are related to the squash family of proteinase inhibitors, to the cellulose binding domain of cellobiohydrolase, and to omega-conotoxin, i.e. a group of small proteins termed "knottins" by Nguyen, D. L., Heitz, A., Chiche, L., Castro, B., Boigegrain, R., Favel, A., and Coletti-Previero, M. ((1990) (Biochimie 72, 431-435) The three-dimensional model of AAI was built according to the common structural features of this group of proteins using side-chain replacement and molecular dynamics refinement techniques.

Amino Acid Sequence↗
PubMed1994

Staphylococcal protein A is a novel heterologous substrate for the HIV-1 protease.

Upon in vitro processing of the recombinant HIV-1/gag p24 protein, expressed in Escherichia coli as a fusion protein, by HIV-1 protease, a cleavage site within the staphylococcal protein A fusion partner was found. N-terminal sequencing of the protein A fragments showed that HIV-1 protease cleavage occurred between phenylalanine-235 and tyrosine-236 within the sequence Gln-Asn-Ala-Phe/Tyr-Glu-Ile-Leu (QNAF/YEIL) in the IgG-binding domain C of the protein A encoded by the pRIT2T fusion gene vector (Pharmacia). Results presented here have proven that the protease-sensitive site is viable in vitro on the protein A alone and other chimeric protein, protein A/beta-galactosidase. A possible significance of this phenomenon in biotechnology work is discussed.

Amino Acid Sequence↗
PubMed1994

Self-methylation of BspRI DNA-methyltransferase.

The DNA (cytosine-5)-methyltransferase (m5C-MTase) M.BspRI is able to accept the methyl group from the methyl donor S-adenosyl-L-methionine (AdoMet) in the absence of DNA. Transfer of the methyl group to the enzyme is a slow reaction relative to DNA methylation. Self-methylation is dependent on the native conformation of the enzyme and is inhibited by S-adenosyl-L-homocysteine, DNA and sulfhydryl reagents. Amino acid sequencing of proteolytic peptides obtained from M.BspRI, which had been methylated with [methyl-3H]AdoMet, and thin layer chromatography of the modified amino acid identified two cysteines, Cys156 and Cys181 that bind the methyl group in form of S-methylcysteine. One of the acceptor residues, Cys156 is the highly conserved cysteine which plays the role of the catalytic nucleophile of m5C-MTases.

Amino Acid Sequence↗
PubMed1994

Alteration of the specificity of ecotin, an E. coli serine proteinase inhibitor, by site directed mutagenesis.

The gene of ecotin, an E. coli proteinase inhibitor, was cloned, and by site-directed mutagenesis the active site residue of the protein, Met84, was mutated to Lys, Arg and Leu. The recombinant wild-type and mutant inhibitors were overexpressed in E. coli, purified to homogeneity and their inhibitory effects on trypsin, chymotrypsin and elastase were compared. Of these serine proteinases trypsin is the most strongly inhibited by wild type ecotin and its mutants. According to our results the character of residue 84 of ecotin significantly but not dramatically modifies the specificity of the inhibitor.

Amino Acid Sequence↗
PubMed1993

Interactions of USF and Ku antigen with a human DNA region containing a replication origin.

By means of a combination of ion-exchange and sequence-specific affinity chromatography techniques, we have purified to homogeneity two protein complexes binding in a human DNA region (B48) previously recognized to contain a DNA replication origin. The DNA sequence used for the protein purification (B48 binding site) contains a binding site for basic-helix-loop-helix DNA binding proteins. The first complex is composed of two polypeptides of 42- and 44-kDa; its size, heat stability, and target DNA sequence suggest that it corresponds to transcription factor USF; furthermore, the 42-kDa polypeptide is recognized by antibodies raised against 43-kDa-USF. The second complex is represented by equimolar amounts of two proteins of 72 and 87 kDa; microsequencing of the two species indicated that they correspond to the human Ku antigen. In analogy with Ku, they produce a regular pattern of footprints without an apparent sequence-specificity, and their binding can be competed by unspecific DNA provided that it contains free ends. The potential role of B48 binding site and of these cognate proteins in origin activation is discussed.

Amino Acid Sequence↗
PubMed1992

Cleavage of the Lys196-Ser197 bond of prolyl oligopeptidase: enhanced catalytic activity for one of the two active enzyme forms.

Prolyl oligopeptidase, a representative of a new family of serine proteases, is remarkably sensitive to ionic strength and has two catalytically active forms, which interconvert with changing pH [Polgár, L. (1991) Eur. J. Biochem. 197, 441-447]. To reveal whether conformational changes are associated with these effects, prolyl oligopeptidase was digested with trypsin. SDS gel electrophoresis studies demonstrated that tryptic digestion of the 75-kDa native protein generated two fragments, one having a molecular mass of 51 kDa and the other of 26 kDa. The digestion was markedly dependent on the ionic strength. Specifically, the digestion proceeded more rapidly in 0.05 M Hepes buffer than in 0.05 M Hepes buffer containing 0.5 M NaCl. Moreover, the nicked enzyme formed at low ionic strength was not stable but degraded and inactivated during an extended incubation. The digestion experiments suggested that alteration in the ionic strength elicits conformational changes in native prolyl oligopeptidase, and this may account for the enhanced catalytic activity observed at higher ionic strength. The two fragments of the nicked prolyl oligopeptidase did not separate during size-exclusion chromatography under nondenaturing conditions on a Superose 12 column and eluted in place of the native enzyme, indicating that they were strongly associated. The reactive serine residues of the nicked enzyme was labeled with tritiated diisopropyl phosphofluoridate, and the fragments were separated by size-exclusion chromatography in urea.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

The isolation and partial characterization of trypsinogen, pancreatic secretory trypsin inhibitor and multiple forms of chymotrypsinogen and trypsin from the pancreas of the ostrich (Struthio camelus).

1. PSTI, two chymotrypsinogens and two trypsins were purified to homogeneity by acid extraction, salt fractionation, SP-Sephadex C-50 chromatography and RP-HPLC. 2. A third chymotrypsinogen, a trypsinogen and another trypsin were purified using an alkaline extraction procedure, followed by Trasylol- and Benzamidine-Sepharose affinity chromatography and hydroxylapatite chromatography. 3. The enzymes differed in amino acid composition as well as in specific activities towards synthetic amidase and esterase substrates. 4. N-terminal amino acid sequences were determined for one chymotrypsinogen and one trypsin.

Amino Acid Sequence↗
PubMed1989

Comparative studies of the elastin content of normal and emphysematous human lungs with special regard to antitrypsin deficiency.

The elastin content and the amino acid composition of elastin in emphysematous and healthy control lung tissues were comparatively studied. Decreased lung elastin content was found in the emphysematous patients of both normal and reduced trypsin inhibitor activity compared to normal control subjects. In the emphysematous patients with reduced inhibitor level a more significantly decreased elastin level was found than in those emphysematous patients with normal inhibitor level. There was no difference between the two groups in either the amino acid composition of elastin or the ratio of nonpolar to polar amino acid components. There was no significant correlation demonstrable between the age and the elastin content of the lung. This applied to the control group as well as to the emphysematous patients with normal trypsin inhibitor activity. The results indicate that no genetically abnormal pulmonary elastin structure is a predisposing risk factor but the reduced elastin content induced by elastase enzymes leading to changed mechanical properties is responsible for the development of pulmonary emphysema.

Adult↗
PubMed1988

[Biocytin13]dynorphin A 1-13 amide: a potential probe for the kappa-opioid receptor.

A dynorphin A 1-13 amide (DYN) derivative biotinylated in position lysine 13 (B-DYN) has been prepared by automated solid-phase peptide synthesis. The derivative retained its ability to bind to avidin, and B-DYN-avidin complex showed a dissociated half-life of 10 hr at 37 degrees C. Opioid receptor binding was measured in membrane preparations of rat brain (mu), NG-108-15 neuroblastoma-glioma hybrid cells (delta), and guinea pig cerebellum (kappa). Biotinyl substitution of DYN either did not effect receptor binding (delta) or slightly reduced binding affinity (mu and kappa). Binding of B-DYN to the kappa receptor was very tight, with an IC50 value in the low picomolar range, while binding to mu and delta sites was over two orders of magnitude lower. Preassociation of B-DYN with avidin resulted in a reduction of the affinities to the investigated opioid receptors by 100- to 1000-fold. However, the apparent affinity of B-DYN-avidin for the kappa-opioid receptor is sufficient to suggest that B-DYN may be a useful tool for kappa-opioid receptor assay, localization, and purification.

Animals↗
PubMed1987

Selective alteration of substrate specificity by replacement of aspartic acid-189 with lysine in the binding pocket of trypsin.

To test the role of Asp-189 which is located at the base of the substrate binding pocket in determining the specificity of trypsin toward basic substrates, this residue was replaced with a lysine residue by site-directed mutagenesis. Both rat trypsinogen and Lys-189 trypsinogen were expressed and secreted into the periplasmic space of Escherichia coli. The proteins were purified to homogeneity and activated by porcine enterokinase, and their catalytic activities were determined on natural and synthetic substrates. Lys-189 trypsin displayed no catalytic activity toward arginyl and lysyl substrates. Further, there was no compensatory change in specificity toward acidic substrates; no cleavage of aspartyl or glutamyl bonds was detected. Additional studies of substrate specificity involving gas-phase sequence analyses of digested natural substrates revealed an inherent but low chymotrypsin-like activity of trypsin. This activity was retained but modified by the Asp to Lys change at position 189. In addition to hydrolyzing phenylalanyl and tyrosyl peptide bonds, the mutant enzyme has the unique property of cleaving leucyl bonds. On the basis of computer graphic modeling studies of the Lys-189 side chain, it appears that the positively charged NH2 group is directed outside the substrate binding pocket. The resulting hydrophobic cavity may explain the altered substrate specificity of the mutant enzyme. The relatively low chymotrypsin-like activity of both recombinant enzymes may be due to distorted positioning of the scissile bond with respect to the catalytic triad rather than to the lack of sufficient interaction between the hydrophobic side chains and the substrate binding pocket of the enzyme.

Amino Acid Sequence↗
PubMed1986

Distribution and stress-induced increase of glutamate and aspartate levels in discrete brain nuclei of rats.

Concentrations of glutamate and aspartate have been measured in 45 microdissected brain areas and nuclei in rat. Both amino acids are ubiquitously present and distributed unevenly in the central nervous system. Very high glutamate levels were found in the cerebellum and the insular cortex, high levels in neocortical and limbic cortical areas, and in the nuclei of the medial hypothalamus. Aspartate is distributed rather uniformly with the highest concentration in the hypothalamic arcuate nucleus and the lowest in the midbrain central gray matter and the cerebellum. Acute formalin (pain) stress elevated glutamate and aspartate levels in the cortical areas and substantia nigra significantly, but had minor or no effects on other brain nuclei. Increased locomotor and behavioral activities due to a high dose of amphetamine resulted in a 2-5-fold increase of glutamate and aspartate concentrations, particularly in the biogenic amine-containing brain nuclei.

Animals↗
PubMed1986

Concentrations of GABA and glycine in discrete brain nuclei. Stress-induced changes in the levels of inhibitory amino acids.

Concentrations of gamma-aminobutyric acid (GABA), glycine and serine have been measured in 44 microdissected areas of the brain of the rat. All three amino acids were ubiquitously present and distributed unevenly in the brain. Very high levels of GABA were found in the anterior hypothalamic and medial preoptic nuclei and the substantia nigra; high levels were found in the interpeduncular and red nuclei in the mesencephalon and in several hypothalamic nuclei. Glycine was distributed fairly uniformly with large concentrations in certain lower brainstem nuclei. In these areas, the concentrations of glycine exceeded those of serine, while the serine-glycine ratio was 4.5:1 in the caudate nucleus, 4:1 in the cerebellum and 2.5:1 in the cerebral cortical areas. Acute stress induced with formalin (pain) resulted in a significant depletion of levels of GABA in the hypothalamus and the lower brainstem but not in the cortical areas. In the same animals, concentrations of glycine doubled in the cerebral cortex and remained unchanged elsewhere in the brain. Increased motor and behavioral activity after the acute administration of a large dose of amphetamine were associated with a 2-5-fold increase in the levels of glycine in brain, and markedly elevated the concentrations of GABA in the major biogenic amine-containing cell groups only (substantia nigra, locus coeruleus and dorsal raphe).

Animals↗
PubMed1986

Taurine levels in discrete brain nuclei of rats.

Concentrations of taurine have been measured in 44 microdissected rat brain nuclei or areas. Taurine is ubiquitously present and distributed unevenly in the rat brain: the ratio of the highest (pyriform cortex) to lowest (midbrain reticular formation) concentrations is 4.7:1. High taurine levels were found in cerebral cortical areas, caudate-putamen, cerebellum, median eminence, and supraoptic nucleus. Acute pain stress reduced taurine levels in the hypothalamus and the lower brainstem nuclei but not in cortical areas. Increased locomotor and behavioral activities following a high dose of amphetamine elevated taurine concentrations significantly in the substantia nigra and locus ceruleus.

Animals↗
PubMed1985

Action of neutral metalloendopeptidase ("enkephalinase") on beta-endorphin.

Human beta-endorphin was digested by neutral metalloendopeptidase from rabbit kidney and the products were isolated and identified. Based on the structure and yield of the fragments, the major cleavage sites were identified with the Leu17-Phe18, Gly3-Phe4, Pro13-Leu14 and Ile22-Ile23 peptide bonds of the beta-endorphin structure. The cleavage of the Leu17-Phe18 bond appears to be the rate-limiting step of the enzymic conversion similarly to the previously proposed pathways of beta-endorphin degradation by brain homogenates and synaptic membranes.

Animals↗
PubMed1982

The primary structure of human beta-lipotropin. Further peptide sequencing resolves the controversy and suggests the existence of only one human beta-lipotropin.

Human beta-lipotropin isolated in Hungary from frozen pituitary glands was purified by high-performance liquid chromatography in Canada. The amino acid sequence of the first 30 residues was determined. Trypsin, trypsin/papain, and trypsin/thermolysin fragments were obtained for the disputed region containing residues 9-25 of beta-lipotropin. Their amino acid composition and sequence established beyond doubt that only one human beta-lipotropin sequence is present. These results suggest the presence of only one gene coding for human pro-opiomelanocortin, the precursor of adrenocorticotropin and beta-endorphin and resolve the controversy over the sequence of human beta-lipotropin.

Amino Acid Sequence↗