| Abstract: | Chlamydomonas lytic enzyme of the cell wall, which is released during agglutination of gametes of opposite mating types, has been characterized as a metalloprotease. The purified enzyme contains zinc. Removal of zinc with EDTA results in an inactive, metal-free apoenzyme, and Co2+ restores the activity most effectively. Among various protease inhibitors of microbial origin, pepstatin A, chymostatin, antipain, leupeptin, and E-64 do not inactivate the enzyme, whereas phosphoramidon causes a complete loss of lytic activity. Cysteine, histidine, aspartic acid, and glutamic acid also inhibit the activity. The lytic enzyme splits casein and RNase A into several polypeptides of lower molecular masses. To determine which polypeptides of the cell wall are sensitive to the lytic enzyme, we first separated the intact cell walls into sodium perchlorate-soluble and -insoluble components, treated them with enzyme, and then analyzed them by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. We conclude that only 2 of 16 polypeptides are digested by exposure to the enzyme and that the sensitive polypeptides belong to the salt-insoluble component of the cell wall. The mechanism of cell wall digestion with the lytic enzyme is discussed. |
