Differential expression pattern of Rab-GDI isoforms during the parotid gland secretion cycle.

Rab GDP dissociation inhibitor (GDI) plays an important role in regulating the GDP/GTP cycle of small GTP binding proteins of the Rab family. It also regulates their association to membranes. The small family of Rab-GDI consists of several closely related isoforms, the functional differences between which are still unknown. Here we show that multiple GDI isoforms are expressed in rat parotid gland and that the individual GDI isoforms have a characteristic expression both at the RNA and at the protein level, during the parotid secretory cycle. GDIalpha, the major isoform in brain, is expressed throughout the secretory process and is equally distributed between cytoplasmic and membranous fractions. In contrast, an isoform related to, but different from GDIbeta is found predominantly in the cytoplasmic fraction and its expression is detected only after beta-adrenergic stimulation of the gland, at the end of the secretion phase, when exocytosis is already completed. The induction of such a GDI isoform at the beginning of the recovery stage correlates with the expression pattern of Rab1 and Rab5, but not Rab2 and Rab4. Our results suggest different functional roles for multiple GDI isoforms along the secretion and recovery phases in rat parotid gland.

The possible involvement of trypsin-like enzymes in germination of spores of Bacillus cereus T and Bacillus subtilis 168.

Germination of spores of Bacillus cereus T and Bacillus subtilis 168 was inhibited by the trypsin inhibitors leupeptin and tosyllysine chloromethyl ketone (TLCK) and by the substrates tosylarginine methyl ester (TAME), benzoyl-L-arginine-p-nitroanilide (L-BAPNA) and D-BAPNA. Potencies of these inhibitory compounds were estimated by finding the concentration which inhibited 50% germination (ID50), 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 B. cereus T, all the compounds inhibited early and late events with the same ID50. In B. subtilis, TAME inhibited early and late events at the same ID50, but all other inhibitors had a lower ID50 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 B. subtilis 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 B. cereus T and in germinated spores of B. subtilis by means of three chromogenic substrates: benzoyl-L-phenylalanyl-L-valyl-L-arginine-p-nitroanilide (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 Ki values for the above inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Trypsinlike enzymes from dormant and germinated spores of Bacillus cereus T and their possible involvement in germination.

Trypsin-like enzymes were studied in dormant, activated, and germinated spores of Bacillus cereus T. Dormant spores contained two heat-labile enzyme activities. One was extractable with 2 M KCl and hydrolyzed azo-albumin. The second, a trypsinlike activity, was not extractable with 2 M KCl and hydrolyzed benzoyl-L-arginine-p-nitroanilide. Because of their heat instability, these two enzyme activities are probably not involved in the germination of heat-activated spores. Upon germination of heat-treated spores, a trypsinlike protease which was not detected in intact dormant spores was activated or exposed. This enzyme, when measured in intact germinated spores, hydrolyzed benzoyl-DL-arginine-p-nitroanilide but not azo-albumin and was inhibited in situ by sulfhydryl-blocking reagents such as p-chloromercuribenzoic acid and Hg2+. There was a correlation between the inhibition of germination and enzymatic activity by sulfhydryl-blocking reagents. The enzyme was also inhibited by leupeptin, tosyl-L-lysine chromoethyl ketone, and tosyl-L-arginine methyl ester. Good correlation existed between the inhibition of germination and enzymatic activity by these agents. Electron micrographs showed that in the presence of trypsin inhibitors, the spores did not lose their cortex. The protein extracts of the inhibited spores formed a somewhat different electrophoretic pattern in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the protein extracts of dormant or germinated spores.

The pattern of protein synthesis in spoIVC mutants of Bacillus subtilis resuspended in sporulation medium.

Two spoIVC mutants of Bacillus subtilis were labelled with [35S]methionine either at the time of resuspension in sporulation medium or 1, 2 or 3 h later, and radioactive proteins were detected after cell extracts had been subjected to two-dimensional gel electrophoresis. The mutants completed almost all of the changes in protein synthesis that occur in the wild-type in these conditions. A heavily labelled protein was found in the mutants that has also been observed in a spoO mutant but does not occur in the wild-type.

Effect of inhibitors of trypsin-like proteolytic enzymes Bacillus cereus T spore germination.

The germination of Bacillus cereus T spore suspensions is partially prevented by several inhibitors of trypsin-like enzymes. Leupeptin, antipain, and tosyl-lysine-chloromethyl ketone are effective inhibitors, whereas chymostatin, elastatinal, and pepstatin are inactive. A synthetic substrate of trypsin, tosyl-arginine-methyl ester, also inhibits germination. Its inhibitory effect decreases as a function of incubation time in the presence of spores and is abolished by previous hydrolysis with trypsin. Germinating, but not dormant, spore suspensions hydrolyze tosyl-arginine-methyl ester; its hydrolysis is insensitive to chloramphenicol, sulfhydryl reagents, and EDTA. A crude extract of germinated B. cereus spores contains a trypsin-like enzyme whose activity, as measured by hydrolysis of benzoyl-arginine p-nitroanilide, is sensitive to germination-inhibitory compounds such as leupeptin, tosyl-arginine-methyl ester, and tosyl-lysine-chloromethyl ketone. Spore suspensions exposed to the above inhibitors under germination conditions lose only part of their heat resistance and some 10 to 30% of their dipicolinic acid content. Part of the germinating spore population becomes "phase grey" under phase optics. Based on a study of the inhibition of germination by protease inhibitors and the activity of a protease in germination spores and spore extracts, it is suggested that the activity of a trypsin-like enzyme may be involved in the mechanism of the breaking of dormancy in spores of B. cereus T.

In vitro translation of messenger ribonucleic acid from sporulating and nonsporulating strains of Bacillus subtilis.

An Escherichia coli translation system supplemented with ribonucleic acid from sporulating Bacillus subtilis produces unique polypeptides which are missing among translation products of ribonucleic acid from six early sporulation mutants.