Neuroblastoma adenylate cyclase. Role of 2-chloroadenosine, prostaglandin E1, and guanine nucleotides in regulation of activity.

Neuroblastoma adenylate cyclase is activated by 2-chloroadenosine, prostaglandin E1, and 5'-guanylylimidodiphosphate [GMP-P(NH)P]. However, the process of activation by the first two compounds is different from that induced by the third. Prostaglandin E1 and 2-chloroadenosine activation is rapid, producing elevated activities which are constant throughout a 20-min assay. In contrast, GMP-P(NH)P activation is slow and although the activity is elevated within 1 min, it continues to increase for up to 12 min before attaining a maximal constant value. Activation is more rapid when either prostaglandin E1 or 2-chloroadenosine is present with GMP-P(NH)P. Activation of the enzyme by GMP-P(NH)P appears to be retarded by endogenous nucleotides as suggested by the following observations: (a) if the enzyme is incubated at 30 degrees with 5 mM MgCl2 for 5 to 7 min, GMP-P(NH)P then produces maximal activation without a detect able lag; (b) if, during this incubation, nucleotides, a nucleotide regenerating system, or EDTA (instead of MgCl2) are present, subsequent GMP-P(NH)P activation is slow; and (c) in the assays which contain a nucleotide regenerating systm and MgATP as substrate, the Km for GMP-P(NH)P is 6 +/- 2 muM. However, in the assays using MgAMP-P(NH)P as substrate but no nucleotide regenerating system, the Km is 0.5 +/- 0.2 muM. GPD and GTP do not replace GMP-P(NH)P as an enzyme activator in any of our assays systems, and in fact, are potent inhibitors of GMP-P(NH)P enzyme activation. Prostaglandin E1 and 2-chloradensine do not alter significantly the Km for GMP-P(NH)P but do decrease the ensyme's sensitivity of GDP. Proposed is a hysteretic model of neuroblastoma adenylate cyclase, which shows the enzyme responding slowly to rapid changes in GMP-P(NH)P concentration due to the slow displacement of the tightly bound endogenous guanine nucleotides by GMP-P(NH)P. Additionally, prostaglandin E1 and 2-chloroadenosine increase the rate of GMP-P(NH)P activation by decreasing the enzyme's affinity for these endogenous guanine nucleotides.