Worm Breeder's Gazette 7(2): 70
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
We are interested in the biochemical pathways of intracellular protein turnover and in the possible roles of proteolytic processes in development. We have been concentrating on the enzyme Cathepsin D because preliminary experiments with crude extracts of C. elegans showed that proteolysis at acid pH was >95% inhibited by pepstatin, a known inhibitor of carboxyl proteases. We have purified the wild-type enzyme in a single step by affinity chromatography on pepstatin-agarose. We can obtain about 5mg of 'pure' enzyme from 15gm of N2 in one afternoon. The purified enzyme shows 6-7 bands (MW = 32-42 Kdal) on SDS gels. We believe, on the basis of digestion studies with exo- and endo-glycosidases, that Cathepsin D is a complex (sialylated) glycoprotein and that this multiplicity of bands represents heterogeneity of the oligosaccharide sidechains. It is not yet clear whether this heterogeneity exists in vivo or represents partial degradation during purification. Gel filtration of crude extracts indicates that the enzyme is a monomer. The proteolytic specificity of the enzyme is similar, but not identical, to that of Cathepsin D from bovine spleen. We have isolated one mutant deficient in Cathepsin D, and have ( unofficially) named the gene cad-1. Mutant homozygotes have 10-20% of wildtype enzyme levels. The cad-1 gene has been assigned to the right extremity of LG II, 33% from sqt-2, 22% from dpy-10 and 23% from unc-4. Other markers on the right extremity of LG II have been useless for reasons of shared phenotypes, so more precise mapping remains to be done. The cad-1 gene appears to be a structural gene on two grounds: 1) The enzyme level in cad-1/+ heterozygotes is precisely the mean of the levels in mutant and wild-type homozygotes. 2) The pure enzyme from the cad-1 mutant has one-third the activity per molecule of the wild- type enzyme. A search for electrophoretic differences is in progress. Our cad-1 mutant has an 'odd' appearance of the intestine, looking less dark than wild-type under the dissecting microscope, and having highly vacuolated intestinal cells in the EM. It has a reasonably high tendency to egg retention and internal hatching (frequency similar to a lin-7; nzyme level) and lays only unfertiIized eggs at 25 C. Reproduction is not rescued by N2 maIes at 25 C. Conversely, cad-1 males preincubated for 24 hr. at 25 C remain fertile. Growth of the mutant at 25 C is quite poor, even though the in vitro activity of its Cathepsin D shows a temperature coefficient (Q10 = 1.9 in the range 16 C to 37 C) no different from that of wild-type. It is not yet totally clear that the ts properties are attributable directly to the cad-1 mutation.