Germination of spores of T and 168 was inhibited by the trypsin inhibitors leupeptin and tosyllysine chloromethyl ketone (TLCK) and by the substrates tosylarginine methyl ester (TAME), benzoyl-L-arginine--nitroanilide (L-BAPNA) and D-BAPNA. Potencies of these inhibitory compounds were estimated by finding the concentration which inhibited 50 % germination (ID), as measured by events occurring early (loss of heat resistance), at an intermediate stage [dipicolinic acid (DPA) release], and late in germination (decrease in optical density). In T, all the compounds inhibited early and late events with the same ID. In , TAME inhibited early and late events at the same ID, but all other inhibitors had a lower ID for late events than for early events. This suggests that a trypsin-like enzyme activity is involved at two sequential stages in the germination of spores, one occurring at or before the loss of heat resistance and one at or before the decrease in optical density. Different trypsin-like activities were detected in broken dormant spores and germinated spores of T and in germinated spores of by means of three chromogenic substrates: benzoyI-L-phenylalanyl-L-valyl-L-arginine--nitroaniIid (L-PheVA), L-BAPNA and D-BAPNA. Separation of extracts of germinated spores on non-denaturing polyacrylamide gels showed that in both species the substrates were hydrolysed by three distinct enzymes with different electrophoretic mobilities. The three enzymes had different values for the above inhibitors. The possibility that L-BAPNase from T might be involved in the initial germination steps was suggested by the similarity of ID values for germination and values for inhibition of the enzyme by TAME and TLCK, and by the fact that both germination and the L-BAPNase were reversibly inhibited by TLCK.


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