Studies of the cysteine proteinases of the cellular slime mold Dictyostelium discoideum have been aided by a simple acid treatment step that was incorporated into the standard one-dimensional gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis assay procedure. The step involved immersing the separating gel in 10% (v/v) glacial acetic acid for 30-60 s immediately after electrophoresis. This modified approach revealed the presence of acid-activatable forms of some enzymes with noticeable increases in their ability to hydrolyze gelatin, a substrate present in the sodium dodecyl sulfate-polyacrylamide gels, and peptidyl amidomethylcoumarins. The activation has been analyzed using extracts of dormant spores from which cysteine proteinase activity had previously appeared low or virtually absent. The major acid-activatable proteinase had an apparent molecular mass of 48 kDa. Its activation was not due to autocatalysis as it was not prevented by mercuric chloride, an inhibitor of the enzyme, and was not accompanied by a significant change in electrophoretic mobility. It was most likely due to a conformational change and/or the removal of a low molecular weight inhibitor. The acid treatment has also revealed the presence of acid-activatable cysteine proteinases in vegetative cells, in which cysteine proteinase activity is present at high levels, as well as among enzymes from the developmental cells which have much lower cysteine proteinase activity. Indeed novel developmental forms were detected at some stages. These results provide additional insight concerning cysteine proteinase expression at various stages during development in the slime molds. A developmental model is presented which suggests that the crypticity of the cysteine proteinases in dormant spores may be governed by proton pumps and endogenous lysosomotropic agents.

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1. Ovomucoids were purified from Muscovy duck, domestic duck and domestic goose. 2. Peptide maps of cyanogen bromide-cleaved ovomucoids from Muscovy duck and domestic duck were very similar to one another, but differed from that of goose. 3. Muscovy duck ovomucoid showed the same protease inhibitory pattern as ovomucoid from domestic duck, inhibiting trypsin in the molar ratio of 1:2 and chymotrypsin 1:1. 4. Inhibitory complexes could be detected between chymotrypsin and ovomucoid from both Muscovy and domestic duck, but not from goose, by using non-denaturing gels. 5. No complexes could be detected between DFP-inactivated chymotrypsin and any of the ovomucoids. 6. The results show that of ovomucoid from Muscovy duck more closely resembles that from domestic duck than goose.