Publications (4)5.36 Total impact
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Article: Structure, expression and function of Allomyces arbuscula CDP II (metacaspase) gene.
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ABSTRACT: Allomyces arbuscula, a primitive chytridiomycete fungus, has two Ca(2+)-dependent cysteine proteases, the CDP I and CDP II. We have cloned and analyzed the nucleotide sequence of CDP II gene and domain structure of the protein. Blast analysis of the sequence has shown that the protein belongs to a newly described member of caspase superfamily protein, the metacaspase, a CD clan of C14 family cysteine protease, we hence-forth name it as AMca 2 (Allomyces metacaspase 2). Southern hybridization studies have shown that the gene exists in a single copy per genome. The transcriptional analysis by Northern hybridization has confirmed our previous results that the protein is developmentally regulated, i.e. present in active growth phase but disappears during nutritional stress which also induces reproductive differentiation, indicating that the protein promotes cell growth, not death. The recombinant gene product expressed in Escherichiacoli has all the catalytic properties of native enzyme, i.e. sensitivity to protease inhibitors and substrate specificity. There is an absolute requirement of Ca(2+) for the activation of catalytic activity and the presence of R residue at the cleavage site (P1 position) in the substrate. The presence of a second basic residue, either R or K, in the P2 position strongly inhibits the catalytic activity which is stimulated by the presence of P and to a lesser extent G at this site. Peptide substrates with D at the cleavage site are not recognised and therefore not cleaved. The enzyme activity is inhibited by EDTA-EGTA, cysteine protease inhibitors and a specific peptide inhibitor Ac GVRCHCL TFA, but not by E64, although a potent inhibitor of cysteine proteases.Gene 03/2010; 457(1-2):25-34. · 2.34 Impact Factor -
Article: A novel N(alpha)-acetyl alanine aminopeptidase from Allomyces arbuscula.
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ABSTRACT: An N(alpha)-acetyl alanine aminopeptidase has been purified from the aquatic fungus Allomyces arbuscula. The apparent molecular mass of the enzyme was estimated to be 280 kDa by gel filtration through calibrated Sephacryl S300 column. In SDS-PAGE, the purified enzyme appeared as a single band of M(r) 80 kDa. Catalytic activity of the enzyme was inhibited by specific serine protease inhibitors, 3,4-DCI and APMSF, as well as SH reacting compounds, HgCl(2) and iodoacetate, indicating that the enzyme is a serine protease with some functional SH group(s) involved in the catalytic reaction. 3H-DFP was used to label the reactive serine of the enzyme. When the labeled protein was analyzed in SDS-PAGE, most of the label appeared in the M(r) 80 kDa band, however, a few additional faster migrating minor bands were also seen, probably representing a minor degradation product of the enzyme. The enzyme cleaved mainly N(alpha)-acetlylated alanine, although a small but negligible activity was also obtained with acetylated leucine and phenylalanine. The role of the enzyme in N-end rule proteolysis is discussed.Biochimie 05/2002; 84(4):309-19. · 3.02 Impact Factor -
Article: Comparative studies of Ca2+-dependent proteases (CDP I and CDP II) from Allomyces arbuscula
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ABSTRACT: Allomyces arbuscula, an aquatic fungus, contains two Ca2+-dependent neutral cysteine proteases (CDP I and CDP II), eluting respectively, at 0.07 and 0.2 M NaCl from DEAE cellulose columns. The purified CDP I has a Mr of 39 kDa whereas CDP II appears as a doublet of 43 and 40 kDa. Both enzymes require free thiol, the same concentration of Ca2+ for half maximal activation, and are inactivated by thiol protease inhibitors. Our results show that despite these similarities the two enzymes are different because affinity-purified CDP II antibodies do not cross-react with CDP I antigen in Western blots. In contrast, there is a strong cross-reaction between the two 43 and 40 kDa CDP II peptides and their respective antibodies. Both enzymes cleave preferentially the carboxy terminus of Arg and to a limited extent Lys on the cleavage site. This primary specificity is governed by the nature of the amino acids in the P2 and P3 positions. In general either Pro or Gly in P2 is required, with preference for Pro and in P3 position, Gly over Val. CDP II has higher catalytic activity than CDP I. The sulfhydryl reagent NEM is a more potent inhibitor of CDP I than CDP II. Although the function of the phosphorylable site(s) is not clear, both CDP I and CDP II contain phosphorylable serine residue(s).Biochimie. -
Article: Structure and Properties of Casein Kinase IIs from Allomyces arbuscula Phosphorylating Serine Residues
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ABSTRACT: Structure and properties of casein kinase Its from Allomyces arbuscula phosphorylating serine residues. Experimental Mycology 18, 349–362. Two casein kinase (CK) activities have been purified from the aquatic fungus Allomyces arbuscula and have been referred to as CK IIA and CK IIB. Both enzymes have the properties characteristic of animal and yeast casein kinase II, i.e., tetrameric holoenzyme, ability to use ATP as well as GTP as donor of phosphate group in phosphotransferase reactions, inhibition of kinase activity by heparin, and inability to use basic protein like histone H1 as substrate. The two enzymes differ from each other by their affinity to DE-52-cellulose, CK IIA does not bind to DE-52 while CK IIB does. The kinetic parameters of the two enzymes also differ. The Km of CK IIA has been found to be 7.7 μM, 20.83 μM, and 0.96 mM and that of CK IIB 22.2 νM, 41.7 ~LM, and 1.86 mM, for casein, ATP, and Mgt2+ , respectively. Both α and β subunits of CK IIA and CK IIB undergo autophosphorylation. Polylysine is inhibitory to autophosphorylation of a subunit whereas spermine, protamine, and histone H1 are stimulatory. Phosphoamino acid analysis showed that serine was the phospho-accepting amino acid. The phosphopeptide analysis showed that the trypsin recognition sites of the α and β subunits in CK IIA and CK IIB are different.Experimental Mycology.
