Purification and Characterization of a Vegetative Lytic Enzyme Responsible for Liberation of Daughter Cells During the Proliferation of Chlamydomonas Reinhardtii

A vegetative lytic enzyme (VLE) of Chlamydomonas reinhardtii mediates digestion of the cell walls of mother cells (sporangia) to allow release of daughter cells after miotic cell division in the vegetative cell cycle. This enzyme is secreted into the culture medium concurrently with the appearance of daughter cells in synchronized cultures. Using an assay that monitors digestion of the mother cell wall, we purified VLE by ion-exchange and gel-filtration chromatography from the medium of synchronized cultures. The purified enzyme was a basic glycoprotein with an apparent molecular mass of 120 kDa on gel filtration and 130 kDa on SDS-PAGE. Thus, VLE appeared to behave as a monomer. The enzyme acted specifically on the mother cell wall and was unable to digest the cell walls derived from single vegetative cells. The enzymatic activity was inhibited by PMSF, p-APMSF, TLCK, HgCl2, iodoacetate, EGTA, EDTA and 1, 10-phenanthroline. VLE cleaved several synthetic model peptides on the carboxyl side of a Lys or Arg residue, indicating that it is a protease that acts on protein in the mother cell wall in vivo to release the daughter cells.