Gametic flagellar enzyme, but not the vegetative enzyme, is regulated by phosphorylation and dephosphorylation. Both the regulation by cell–cell interactions and the differences between the vegetative and gametic forms of the enzyme are matter of interest. Cyclic adenosine monophosphate (cAMP) is important for the control of flagellar motility; gametes must carefully regulate low, basal levels of this molecule when they are not interacting with gametes of the opposite type. But, on flagellar adhesion with a gamete of the opposite mating type, cAMP levels raise several folds as the cells prepare for fusion. While this adhesion-dependent regulation is critical for gametes, evidence is lacking that vegetative cells, which do not undergo flagellar adhesion, exhibit any dramatic regulation of the enzyme. This chapter summarizes methods for studying Chlamydomonas adenylyl cyclase in both vegetative and gametic flagella. The methods detail the chemicals, reagents, and solutions, the processes of flagellar isolation and assaying adenylyl cyclase. The chapter also describes the evaluating parameters of the assay conditions—divalent cations and detergents. Both gametic and vegetative adenylyl cyclases have requirement for Mg2+, the optimal concentration of Mg2+ for both enzymes being about 2.5 mM. Both the gametic and vegetative enzymes are inhibited by free Ca2+ in the millimolar range. The gametic adenylyl cyclase is more sensitive to Ca2+ than the vegetative enzyme and is inhibited by micromolar concentrations of free Ca2+. The vegetative enzyme seems to be less sensitive to detergents than the gametic enzyme. There is discussion on the regulation of gametic and vegetative adenylyl cyclase—ATP-dependent inhibition and heat-induced activation of gametic adenylyl cyclase; and adhesion-induced activation of adenylyl cyclase in gametic flagella. During gametogenesis the appearance of the gametic form of the enzyme is coincident with the acquisition of the ability of the cells to undergo sexual signaling and cell fusion.
ASJC Scopus subject areas
- Cell Biology