CALCIUM AS A REGULATOR OF BACILLUS-MEGATERIUM CYTOPLASMIC PROTEOLYTICACTIVITY IN-VITRO

We studied the effect of calcium cation on protease activity in the cr ude cytoplasm of Bacillus megaterium cells treated with netropsin, an antibiotic which suppresses sporulation. Only negligible proteolytic a ctivity was found at the currently used 1 mM CaCl2 in spite of the pre sence of a high concentration of Ca2+-dependent serine protease ISP1. The Ca2+-mediated activation of this protease in the cytoplasmic prepa ration was perceptible only at [Ca2+] > 5 mM and reached a maximum at [Ca2+] > 20 mM. This 'delay' in activation (in terms of Ca2+ concentra tion) was reduced by diluting the cytoplasmic preparation or purifying ISP1. Kinetic studies of the proteolytic reaction in the crude cytopl asm suggested that the activation was caused by Ca2+-dependent modific ation of the native protease molecule. Immunological detection reveale d Ca2+-induced sequential processing of the ISP1 molecule to shorter a ctive form(s). The suppression of this processing at lower Ca2+ concen trations was probably caused by the presence of Ca2+-binding compounds that chelated calcium. Their M-r in the cytoplasmic preparation was l ower than 3000. Possible involvement of these compounds in regulation of ISP1 activity in vivo is discussed. (C) 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.