Biochemical studies on lysin, a cell wall degrading enzyme released during fertilization in Chlamydomonas

New methods have been developed for the purification and characterization of the cell wall degrading enzyme, lysin, which is released into the medium during the mating reaction of the biflagellated alga Chlamydomonas reinhardtii. A quantitative spectrophotometric assay that detects the number of cells losing walls was used to devise a procedure for the 60-fold purification of lysin from the medium of mating gametes with a 30% yield of activity. Molecular sieve and ion exchange chromatography in combination with SDS-PAGE showed that lysin was a single polypeptide with an Mr of 60,000. High-performance liquid chromatography and sucrose density gradient centrifugation of lysin activity were used to obtain an estimate of 66,000 D for the nondenatured molecular weight of lysin, indicating that lysin behaves as a monomer.