title: Novel bifunctional alkaline protease inhibitor: protease inhibitory activity as the biochemical basis of antifungal activity.
abstract: An alkaline protease inhibitor (API) from a Streptomyces sp. NCIM 5127 was shown to possess antifungal activity against several phytopathogenic fungi besides its antiproteolytic (anti-feedent) activity [J. V. Vernekar et al. (1999) Biochem. Biophys. Res. Commun. 262, 702-707]. Based on the correlation between antiproteolytic and antifungal activities in several tests such as copurification, heat inactivation, chemical modification, and its binding interaction with the fungal protease, we demonstrate, for the first time, that the dual function of API is a consequence of its ability to inhibit the essential alkaline protease. The parallel enrichment of both the functions during purification together with the heat inactivation of API leading to the concomitant loss of the two activities suggested their presence on a single molecule. Chemical modification of API with NBS resulted in the complete loss of antiproteolytic and antifungal activities, with no gross change in conformation implying the involvement of a Trp residue in the active site of the inhibitor and the presence of a single active site for the two activities. Treatment of API with DTT abolished both the activities although the native structure of API remained virtually unaffected, indicating the catalytic role of the disulfide bonds. Inactivation of API either by active site modification or by conformational changes leads to the concurrent loss of both the antiproteolytic and antifungal activities. Experimental evidences presented here serve to implicate that the antifungal activity of API is a consequence of its protease inhibitory activity.
authors.affiliation: Division of Biochemical Sciences, National Chemical Laboratory, Pune, 411008, India.
authors.forename: J V
authors.initials: JV
authors.lastname: Vernekar
authors.forename: A M
authors.initials: AM
authors.lastname: Tanksale
authors.forename: M S
authors.initials: MS
authors.lastname: Ghatge
authors.forename: V V
authors.initials: VV
authors.lastname: Deshpande
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000935:Antifungal Agents / Q000302:isolation & purification / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D001665:Binding Sites; D020092:Conidiobolus / Q000187:drug effects*; D004220:Disulfides; D006358:Hot Temperature; D008826:Microbial Sensitivity Tests; D011480:Protease Inhibitors / Q000302:isolation & purification / Q000494:pharmacology*; D011485:Protein Binding; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D014364:Tryptophan
chemical_list: C433242:API protein, Streptomyces; D000596:Amino Acids; D000935:Antifungal Agents; D001426:Bacterial Proteins; D004220:Disulfides; D011480:Protease Inhibitors; D014364:Tryptophan