Two novel endopeptidases released into the medium during mating of gametes of Chlamydomonas reinhardtii.
During the mating reaction between mt+ and mt- gametes of Chlamydomonas reinhardtii, two novel endopeptidases, each of which was able to digest the B chain of insulin, were released into the culture medium, together with a gamete lytic enzyme (GLE) which is responsible for digestion of the gametic cell walls. Both endopeptidases and GLE were copurified from the mating medium by column chromatography on DEAE-cellulose and concanavalin A. Gel filtration separated the peptidases, which were unable to digest gametic cell walls, into two fractions, endopeptidase-1 and endopeptidase-2. These enzymes were also separated from GLE, which was unable to digest the B chain of insulin. Endopeptidase-1, with a molecular mass of about 200 kDa, cleaved the B chain of insulin at the Ala14-Leu15 peptide bond, and this activity was inhibited by EDTA. Endopeptidase-2, with a molecular mass of about 110 kDa, digested the peptide at the Leu15-Tyr16 peptide bond and was sensitive to iodoacetate and chymostatin. When the cell walls of gametes of either mating-type were digested prior to mating with exogenously added GLE, the two endopeptidases were released into the medium, a result that suggests that they are stored, like GLE, outside the plasmalemma.
Matsuda
Y
Y
Department of Biology, Faculty of Science, Kobe University, Japan.
Saito
T
T
Taketoshi
T
T
D000595:Amino Acid Sequence; D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000502:physiology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism; D008969:Molecular Sequence Data
D007328:Insulin; D010450:Endopeptidases