Calcium-dependent Adenylate Cyclase from Rat Cerebral Cortex REVERSIBLE ACTIVATION BY SODIUM FLUORIDE*

The adenylate cyclase activity of a particulate preparation of rat cerebral cortex is comprised of two contributing components, only one of which requires a Ca’+-dependent regulator protein (CDR) for activity. The CDR-dependent component was stabilized by CDR. responded to increasing free Ca’+ concentrations biphasically (activation then inhibi-tion), and was inhibited by high ratios of Mg’+ to Ca’+ and by chlorpromazine (0.1 to 0.5 m&. This component, which represented approximately 80% of the basal activity of a cortex homogenate, was completely deactivated by the removal of CDR during the preparation of the particulate fraction. Adenylate cyclase activity which was not dependent on CDR was inhibited with increasing free Ca’+ concentrations, had elevated activity at high ratios of Mg’+ to Ca”, and was not affected by chlorpromazine (0.1 to 0.5 mx). The CDR-dependent component was activated 50 to 100% by 5 rnM NaF. Activation required the presence of Ca”, was facilitated by warming to 37”, and was immediately and completely reversed by assaying with or washing with ethylene glycol bis(P-aminoethyl

The adenylate cyclase activity of a particulate preparation of rat cerebral cortex is comprised of two contributing components, only one of which requires a Ca'+-dependent regulator protein (CDR) for activity. The CDR-dependent component was stabilized by CDR. responded to increasing free Ca'+ concentrations biphasically (activation then inhibition), and was inhibited by high ratios of Mg'+ to Ca'+ and by chlorpromazine (0.1 to 0.5 m&. This component, which represented approximately 80% of the basal activity of a cortex homogenate, was completely deactivated by the removal of CDR during the preparation of the particulate fraction. Adenylate cyclase activity which was not dependent on CDR was inhibited with increasing free Ca'+ concentrations, had elevated activity at high ratios of Mg'+ to Ca", and was not affected by chlorpromazine (0.1 to 0.5 mx). The CDR-dependent component was activated 50 to 100% by 5 rnM NaF. Activation required the presence of Ca", was facilitated by warming to 37", and was immediately and completely reversed by assaying with or washing with ethylene glycol bis(P-aminoethyl ether)N,N'-tetraacetic acid (EGTA). Alternately, the component which did not respond to CDR was activated 4-to &fold by F-. This activation was not influenced by Ca'+ or CDR and was not reversed by EGTA.
Total adenylate cyclase activity of the particulate preparation was activated approximately S-fold by F-when Ca'+ and CDR were present. Each component when fully activated by F-comprised about half of the overall activity. Pretreatment of the preparation with Ca'+ and CDR for 1 h at 37" resulted in the selective inactivation of enzyme which was not dependent on CDR. The remaining adenylate cyclase activity was lo-to 30-fold dependent on CUR and retained the characteristics of the CDR-dependent component of the original preparation.

RESULTS
Properties of Particulate Adenylate Cyclase from Rat Cerebral Cortex -Homogenates of freshly prepared cerebral cortex in EGTA-containing buffer were found in initial experiments to respond to low concentrations of Ca"+ with a 4-fold activation of adenylate cyclase activity ( Table I). The addition of up to 1 pg of homogeneous Ca"+-dependent regulator (CDR), however, added only slightly to this response. Previous work with detergent-dispersed preparations of brain adenylate cyclase had established that the Cay+ dependence of this enzyme was conferred by CDR (6). Therefore various washing proce- dures with chelating agents were tested in efforts to remove the large amount of endogenous CDR from the cortex preparations. The series of washes detailed under "Experimental Procedures" involving EDTA-containing imidazole buffers effectively eliminated CDR from the particulate enzyme (Table I). Activation of the adenylate cyclase resulting from the addition of Ca"+ alone was completely eliminated from this preparation. Addition of both Ca'+ and CDR, however, provided a 4fold activation. Recovery of total enzyme activity as assayed with the addition of Ca'+ and CDR was approximately 50%. These investigations were extended to ascertain the Mg2' concentration dependence of the adenylate cyclase preparation at various concentrations of Ca"+ with and without added CDR ( Table II). The enzyme activity as assayed without CDR (CDR-independent activity) increased at least 4-fold as Mg'+ was elevated from 0.5 to 10 mM. Adenylate cyclase activity was highest under conditions with EGTA in excess of added Ca'+ concentrations.
Concentrations of Ca2+ in excess of EGTA were inhibitory.
Addition of CDR to the assay elicited increased adenylate cyclase activity at each MgY' and Ca"+ concentration tested except for no added Ca'+. The increment in activity due to CDR (CDR-dependent activity) increased with increasing Mg" concentrations provided that CaY' was optimized. For example, at low Ca"+ (125 PM) a biphasic effect, activation followed by inhibition, was observed as Mg'+ was raised from 0.5 to 10 mM. At higher CaYi concentrations only the activation phase was seen. Concentrations of Ca"+ in excess of EGTA were inhibitory.
The highest ratio (4:l) of CDRdependent to CDR-independent activity occurred at 1 to 3 mM Mgy' and 200 PM Ca"+.
The Ca'+ concentration dependence of the adenylate cyclase activity was examined in greater detail at 3 mM Mg'+ ( Fig. 1). An inhibitory response of the activity as assayed without CDR was clearly evidenced as added Ca2+ approached the concentration of EGTA in the assay. Addition of CDR to the enzyme assay provided a biphasic response to Ca'+, with a 4-fold maximal activation occurring at 150 to 200 PM added Ca'+. Determinations by atomic absorption spectrophotometry of the Ca'+ concentrations contaminating the enzyme and assay reagents were in the range of 20 to 40 PM. Enzyme preincubated with Ca'+ and CDR for 20 min at 0" before assay at 37" required less Ca'+ for the both the activation and the inhibitory phases than did the non-pretreated enzyme. The large decline observed for the activity of adenylate cyclase pretreated at 37" did not occur during enzyme assays at 37" with Ca'+ and CDR or with Ca2+ alone (Fig. 4) Ca'+ CDR, however, and assayed at optimal Ca2+ was found to retain much higher degrees of total activity than enzyme pretreated with Ca*+ alone.
In the experiment illustrated in Fig. 6, the enzyme activity retained in the preparation after l-h pretreatment with CaZ+. CDR was more than 30fold dependent on Ca'+ while the response of the enzyme to F-(at inhibitory EGTA) was reduced lo-fold. It is noteworthy that pretreatment of the enzyme with EGTA alone or with Ca'+ alone provided identical results for each time point examined (data not shown).
The data for activities determined by assay either with EGTA and F-or with optimal Ca"+ and CDR were corrected for the basal activities observed at inhibitory concentrations of EGTA to calculate the increment in enzyme activity due to For to Ca'+. CDR.
The values of these increments were expressed as percentages of maximal activity which was defined as the activity of samples not exposed to 37", and were plotted as a function of time of pretreatment at 37" (Fig. 6,  with NaF and assayed with (a--A) or without (A-A) CDR, and (r) pretreated with NaF and CDR and assayed with CDR (0-O).
NaF concentration after transfer to the assay from the pretreatment was 0.8 mM.
On the other hand, the CDR-dependent increment of activity fell more rapidly for the Ca'+-pretreated preparation than for the CaZ+-and CDR-pretreated sample.