Transient and steady state kinetics of the interaction of guanyl nucleotides with the adenylyl cyclase system from rat liver plasma membranes. Interpretation in terms of a simple two-state model.

Abstract

Transient and steady state kinetics of the interaction of liver plasma membrane adenylyl cyclase with GTP and the GTP analogues guanyl-5'-yl imidodiphosphate (GMP-P(NH)P), guanyl-5'-yl diphosphonate (GMP-P(CH2)P), and guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) was studied before and after treatment of membranes with preactivated cholera toxin. (a) In control experiments, GTP stimulated the enzyme partially and without a noticeable lag, while GTP analogues stimulated to varying degrees (GTP gamma S approximately GMP-P(NH)P greater than GMP-P(CH2)P greater than or equal to GTP) and with lag periods that varied with the nucleotide (GMP-P(NH)P approximately GMP-P(CH2)P greater than GTP gamma S). (b) In toxin-treated membranes, transient kinetics and degree of activation by GTP analogues were unaltered, while activation by GTP remained rapid and became as effective as the most effective of the analogues. (c) Toxin treatment had no effect on the concentration-effect curves for GTP analogues but resulted in a 5- to 9-fold lowering of the apparent K alpha with which GTP stimulates the system. These results indicate that the degree of stimulation of the liver adenylyl cyclase by GTP and its analogues is independent of the susceptibility of the beta-gamma bond of the guanosine triphosphate to be hydrolyzed and that lags in progress curves are not due to slow dissociation of "resident" GDP molecules. The time transients of the interaction of the rat liver adenylyl cyclase with combinations of GTP and GMP-P(NH)P were studied and found to be of the competitive type regardless of the time and sequence of addition of the two nucleotides. The results are consistent with GMP-P(NH)P interacting with and dissociating from the system very slowly and not leading to the formation of an irreversibly activated state of the enzyme. The data obtained on regulation of the basic (no hormone added) liver adenylyl cyclase by guanyl nucleotides are interpreted in terms of a two-state enzyme model (Birnbaumer, L., Bearer, C.F., and Iyengar, R. (1980) J. Biol. Chem. 255, 3552-3557) and the results are discussed in the light of current views of regulation of adenylyl cyclases and involvement of a GTPase. Our experiments are consistent with, but not proof for, presence of a cholera toxin-inhibited GTPase in intimate association with the liver adenylyl cyclase.