Proteinases of silkmoth moulting fluid: Physical and catalytic properties

Proteolytic activity of silkmoth moulting fluid is attributable to two catalytically similar enzymes that appear during the last week of adult development in the pharate adult stage. These proteinases have been partially purified and separated by sucrose density-gradient centrifugation and DEAE-cellulose column chromatography. The two enzymes are present in approximately equal amounts. Both are weakly acidic, with a mol. wt. of 30,000 and 34,000, respectively. They show maximal stability and optimal enzymatic activity near the mildly alkaline pH characteristic of moulting fluid. Inhibitory effects of diisopropyl fluorophosphate and of high concentrations of Hg²⁺ suggest the presence of serine and histidine in the active centres, and inactivation by soybean trypsin inhibitor and by benzamidine further suggests some similarities to mammalian trypsin. Tyrosinase rapidly and irreversibly inactivates the proteinases. The enzymes hydrolyse several protein substrates but have a strict esterase specificity, restricted to esters of arginine and lysine, with a marked preference for arginine. The physical and catalytic properties of the enzymes are compared with those of mammalian trypsin and of invertebrate serine proteinases having trypsin-like specificity.