Purification, properties and developmental regulation of a Ca(2+)-independent serine-cysteine protease from Allomyces arbuscula.
Reproductive differentiation in Allomyces takes place against the background of substrate limitation, a sharp increase in intracellular proteolysine and the induction of at least one specific protease. The aim of this report is to describe the purification, properties and developmental regulation of this enzyme. The enzyme has been partially purified by a combination of ion exchange chromatography, ultrogel filtration and affinity chromatography. The purified enzyme in SDS-PAGE appeared as a doublet of M(r) 40-43 kDa. Two bands corresponding to a relative molecular mass of 40-43 kDa were also apparent in activity gels. The protein has an apparent molecular mass in the region of 43 kDa as estimated by gel filtration. The enzyme therefore, seems to be a monomer of 43 kDa. The second band in SDS-PAGE and activity gels is probably the proteolyzed form of the enzyme. The protease recognized alanine and to a lesser extent phenylalanine in the P1 position when assayed with a range of synthetic peptides. The active site of the enzyme contains a reactive serine residue, as shown by its inhibition with PMSF and soya bean trypsin inhibitor. There is probably a reactive cysteine residue as well since the enzyme activity is strongly inhibited by HgCl2, a thiol group binding reagent. The enzyme is present in zoospores but disappears progressively during germination and hyphal growth. It reappears when actively growing cultures are transferred to dilute salt solution. In conclusion, we have purified a serine-cysteine protease of M(r) 43 kDa. This enzyme has a very restricted substrate specificity and appears to be developmentally regulated.
Ojha
M
M
Laboratory of General Microbiology, University of Geneva, Switzerland.
D000595:Amino Acid Sequence; D002118:Calcium / Q000494:pharmacology*; D008411:Chytridiomycota / Q000201:enzymology* / Q000254:growth & development; D003546:Cysteine Endopeptidases / Q000302:isolation & purification*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013172:Spores, Fungal / Q000201:enzymology; D013379:Substrate Specificity
D015853:Cysteine Proteinase Inhibitors; D003546:Cysteine Endopeptidases; D002118:Calcium