Cell, Vol. 23, 543-549. February 1981, Copyright 0 1981 by MIT Calmodulin Activates NAD Kinase of Sea Urchin Eggs: an Early Event of Fertilization David Epel,* Chris Patton,* Robert W. Wallace+ and Wai Yiu Cheungt * Hopkins Marine Station Department of Biological Sciences Stanford University Pacific Grove, California 93950 t Department of Biochemistry St. Jude Children’s Research Hospital Memphis, Tennessee 38101 Summary NAD kinase, one of the first enzymes activated after fertilization of sea urchin eggs, is regulated by Ca*+ and calmodulin in vitro. The evidence is the require- ment for low amounts of Ca*+ (Kd for Ca*+ of 4 x lo-’ M) and the dissociation of a heat-stable acti- vator from the enzyme which is similar to calmodulin on the basis of radioimmunoassay, activation of bovine brain phosphodiesterase and coelectropho- resis of a major protein of the activator fraction with bovine calmodulin. Also, the calcium stimulation of the enzyme is prevented by trifluoperarine, an in- hibitor of calmodulin-associated reactions. In vivo studies show that the enzyme is activated by artifi- cial parthenogenesis regimes that increase cyto- solic Ca*+, but not by ammonia activation which only partially activates eggs and bypasses the Ca*+- rise step. These in vitro and in vivo studies indicate that calmodulin is part of the linkage between the rise in Ca*+ at fertilization and the turning on of egg metabolism. Introduction The current view of egg activation is that a transient rise in free calcium occurring shortly after sperm-egg fusion triggers embryonic development (reviews by Epel, 1978, 1980; Steinhardt et al., 1980). The mech- anism by which the rise in Ca*+ initiates the series of events leading to development is not clear. Recent progress on other cell types indicates that the actions of Ca2+ are mediated through a class of homologous Ca*+-binding proteins, of which calmodulin appears to be the most versatile (reviewed by Cheung, 1980; Means and Dedman, 1980). In the presence of Cap+, calmodulin undergoes a conformational change to a more helical configuration and interacts with its recep- tor protein, whose activity is thus modified. Calmodulin complexes have been found to alter the activity of enzymes such as phosphodiesterase and cellular pro- cesses, such as the depolymerization of microtubules (see Cheung, 1980; Means and Dedman, 1980). During fertilization of the sea urchin egg, events that are turned on at the time of the calcium rise include the cortical exocytosis and the activation of NAD kinase (Epel, 1978). This enzyme, which cata- lyzes the phosphorylation of NAD to NADP NAD + ATP -+ NADP + ADP is transiently activated, causing the conversion of about one-fourth of the cellular NAD into NADP (Epel, 1964). The time of increased NAD kinase activity in vivo (Epel, 1964) corresponds to that of elevated intracellular calcium (Steinhardt et al., 1977) and it seemed reasonable that this enzyme might be regu- lated by calmodulin. Indeed, Anderson and Cormier (1979) recently demonstrated that calmodulin stimu- lated an NAD kinase of higher plants and calmodulin has been identified in sea urchin gametes (Jones et al., 1978; Head et al., 1979; Nishida and Kumagai, 1980). In this communication we present evidence that demonstrates that sea urchin NAD kinase is regulated by calmodulin. The major criteria are: NAD kinase is activated by less than micromolar concentrations of calcium and this augmentation of activity is reversed by EGTA; Ca2+ sensitivity is lost when an endogenous activator is resolved from the enzyme; the activator stimulates bovine brain phosphodiesterase, cross- reacts with an antibody to calmodulin and on electro- phoresis comigrates with calmodulin; enzyme activity can be restored by bovine brain calmodulin; and the stimulation of NAD kinase activity is inhibited by tri- fluoperazine, a known inhibitor of calmodulin. In ad- dition, we show that NAD kinase activity in vivo is regulated by calcium. Results Calcium Sensitivity of NAD Kinase Activity To determine whether the enzyme was sensitive to Ca’+. a homogenate (5% v/v) of unfertilized eggs was prepared, centrifuged at 100,000 x g for 60 min and the supernatant fluid dialyzed for 48 hr against six changes of 0.02 M MES containing 0.05 M DTT (pH 6.5). Enzyme activity was then assayed in various EGTA concentrations in the standard assay mix, ex- cept that the pH was buffered to pH 7.0 with 0.1 M HEPES, final concentration. (This pH corresponds to the cytoplasmic pH during the period when the en- zyme is active in vivo [Shen and Steinhardt, 19781.) Figure 1 shows that maximal inhibition (96%) required 150 PM EGTA and activity was 50% inhibited by 50 /.LM EGTA. To determine more exactly the affinity for Ca’+, a Ca*+-EGTA buffer system was prepared using a com- puter program (Perrin and Sayce, 1967) provided by R. Steinhardt (University of California at Berkeley) and using a stability constant of 10.7. We used 10 mM EGTA and added Ca”+ ion to achieve the final free Ca2+ levels indicated in Figure 2. A basal level of