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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.
Highlights
Chlamydomonaslytic enzyme of the cell wall,which is released during agglutination of gametes of opposite mating types, has been characterized as a metalloprotease
Effects of Microbial Protease Inhibitors on LyticActiuityPrevious study has shown that metal ion chelators such as EDTA,EGTA,and1,lO-phenanthrolineandSH-blocking agents such as p-chloromercuribenzoic acid, HgC12, and iodoacetate are effective in inactivating the lytic enzyme (10)
Leupeptin, chymostatin, and E-64, which show activity against serineandthiolproteases (24-28), hadno effect onthe activity of lytic enzyme
Summary
Preparation of Cell Walls-The nonfusing mt+ mutant imp-1 (17) was used forthe preparationof cell walls. Our imp-l stock (originally obtained as CC-462 from the Chlamydomonas Genetics Center, Department of Botany, Duke University)casts off and accumulates abundant cell walls in the medium. This property is similar to that of the strain used for the cell wall preparation by Hills et al (12). After 30 minof incubation at room temperature, cell walls were washed three timesin the buffer solution by centrifugations a t 10,000 X g for 10 min and resuspended at 20 mg/ml. Treatment with Lytic Enzyme-Cell walls and their components were resuspended in 10 mM Tris acetate, pH 7.5, at a concentration of 1mg of protein/ml, anda 100-plportion was treated with100 units of lytic enzyme.
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