Properties of the interaction of fluoride- and guanylyl-5'-imidodiphosphate-regulatory proteins with adenylate cyclase.

Abstract

The mechanism of activation of adenylate cyclase by guanylyl-5'-imidodiphosphate [Gpp(NH)p] and NaF has been investigated by studying the reconstitution of Gpp(NH)p and NaF sensitivity of an enzyme rendered insensitive to these agents by differential detergent extraction of a particulate brain enzyme. Such reconstitution can be achieved by the addition of macromolecular regulatory factors from membranes of various tissues. Trypsin digestion and thermal inactivation provide evidence for the existence of two distinct regulatory functions, one capable of restoring the Gpp(NH)p response and another the NaF response. The regulatory protein(s) seem to interact with their respective activators in an easily reversible, divalent cation-independent reaction. This appears to be followed by a high-affinity interaction between the catalytic and regulatory components of adenylate cyclase in a slow, temperature-dependent, divalent cation-dependent process that produces the persistently activated state of the enzyme. The enzyme activation can be reversed by methods that separate catalytic from regulatory components and the resulting enzyme activity can be restimulated by the reconstitution technique.The molecular mechanisms of activation of adenylate cyclase by NaF and guanylyl-5'-imidodiphosphate [Gpp(NH)p], and the relevance of such mechanisms to the hormonal modulation of this enzyme, have been difficult to investigate due to the lack of appropriate techniques for the resolution and reconstitution of the various molecular components of the adenylate cyclase system. We have reported (1) the development of such a technique which involves the preparation of a Gpp(NH)p-and NaF-insensitive brain particulate adenylate cyclase by differential detergent extraction. The enzyme regained its characteristic Gpp(NH)p and NaF responses upon the addition of one or more proteins solubilized from membrane preparations of various tissues (1). These regulatory proteins can be partially separated from solubilized adenylate cyclase by gel filtration on Ultrogel AcA 34 columns. We report here the utilization of this reconstitution technique to help elucidate the mode of interaction of the various components of this multifactorial system. Thermal and trypsin inactivation of the Gpp(NH)p-and NaF-reconstituting activities suggests that the two activities are functionally separable. Gpp(NH)p and NaF appear to interact reversibly with the corresponding regulatory proteins in a divalent cation-independent step, followed by a divalent cation-dependent step that leads to the persistent state of enzyme activation. Techniques that separate regulatory from catalytic components can also reverse the enzyme activation.The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 3693 The Gpp(NH)p-and NaF-insensitive adenylate cyclase was prepared by a modification of a published procedure (1, ...

Molecular complexes involved in the regulation of adenylate cyclase.

“…The largest molecular form derived from Triton shells may include one or more cytoskeletal elements. The intermediate molecular entity appears to be similar in size to that reported earlier (21) and purified subsequently (15 (17,28). The recent determination of the subunit structure of the N-protein (15) should facilitate further elucidation of these molecular changes.…”

“…In addition, we also demonstrated a reversible interaction between this protein and p[NH]ppG, leading to a conformational change in the N-protein which altered its sensitivity to proteolysis and thermal inactivation (17). This activated N-protein formed a highaffinity complex with a catalytic component, resulting in the activation of adenylate cyclase (17). Affinity chromatography (18) and somatic cell variants (19) have also been used to isolate and characterize the N-protein.…”

“…The G-N' complex dissociates readily after the removal of free G (17). However, G-N' binds tightly to the catalytic component of adenylate cyclase (E) to produce the active state of the enzyme (17).…”

“…We had previously detected both a particulate and a soluble guanyl nucleotide-binding protein (N-protein) which mediated the activation of adenylate cyclase by guanyl nucleotides in several rat tissues (16,17). In addition, we also demonstrated a reversible interaction between this protein and p[NH]ppG, leading to a conformational change in the N-protein which altered its sensitivity to proteolysis and thermal inactivation (17). This activated N-protein formed a highaffinity complex with a catalytic component, resulting in the activation of adenylate cyclase (17).…”

“…The development ofspecific reconstitution (16)(17)(18)(19) and labeling (8,18,20) techniques has resulted in a better understanding of the adenylate cyclase system. We had previously detected both a particulate and a soluble guanyl nucleotide-binding protein (N-protein) which mediated the activation of adenylate cyclase by guanyl nucleotides in several rat tissues (16,17).…”