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 M-r of 39 k Da whereas CDP II appears as a doubler of 43 and 40 kDa. Both enzymes requi re free thiol, the same concentration of Ca2+ for half maximal activation, and are inactivated by thiol protease inhibitors. Our results show that des pite these similarities the two enzymes are different because affinity-puri fied CDP II antibodies do nor. cross-react with CDP I antigen in Western bl ots. In contrast, there is a strong cross-reaction between the two 43 and 4 0 kDa CDP II peptides and their respective antibodies. Both enzymes cleave preferentially the carboxy terminus of Arg and to a limited extent Lys on t he 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 phospho rylable serine residue(s). (C) 1999 Societe francaise de biochimie et biolo gie moleculaire / Editions scientifiques et medicales Elsevier SAS.