Comparative studies of Ca2+-dependent proteases (CDP I and CDP II) from Allomyces arbuscula.
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).
Ojha
M
M
Départment de Botanique et Biologie végétale, Université de Genève, Sciences III, Geneva, Switzerland.
Cattaneo
A
A
Schwendimann
B
B
D002118:Calcium / Q000378:metabolism*; D047390:Casein Kinase II; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005658:Fungi / Q000201:enzymology*; D008970:Molecular Weight; D010766:Phosphorylation; D011480:Protease Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D013379:Substrate Specificity
D011480:Protease Inhibitors; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D010450:Endopeptidases; D002118:Calcium