Purification of the regulatory component of adenylate cyclase.

Abstract

The regulatory component (G/F) of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from rabbit liver plasma membranes has been purified essentially to homogeneity. The purification was accomplished by three chromatographic procedures in sodium cholate-containing solutions, followed by three steps in Lubrol-containing solutions. The specific activity of G/F was enriched 2000-fold from extracts of membranes to 3-4 mumol x min-1 x mg-1 (reconstituted adenylate cyclase activity). Purified G/F reconstitutes guanine nucleotide-, fluoride-, and hormone-stimulated adenylate cyclase activity in the adenylate cyclase-deficient variant of S49 murine lymphoma cells. G/F also recouples hormonal stimulation of the enzyme in the uncoupled variant of S49. Preparations of pure G/F contain three polypeptides with approximate molecular weights of 52,000, 45,000, and 35,000. The active G/F protein behaves as a multisubunit complex of these polypeptides. Treatment of G/F with [32P]NAD+ and cholera toxin covalently labels the molecular weight 52,000 and 45,000 polypeptides with 32P.

The differential detergent solubilization of adenylate cyclase and polypeptides ADP‐ribosylated with cholera toxin suggests an excess of G/F protein relative to adenylate cyclase in rat pancreatic plasma membranes

“…The Mr-values of 42 000 and 50 000, estimated for the 2 major pancreatic bands, was comparable to that of ADP-ribosylated polypeptides from other G/F oligoproteins [7,12,14] and, further, the mobilities of the 42 kM r pancreatic component ( fig.2A(2)) and of the single 42 kM r component from human erythrocyte ghosts [8,9], labelled for comparison ( fig.2A(4)) were the same.…”

“…The same conclusion has been attained for polypeptides 45 kM r and 52-53 kM r in membranes from $49 lymphoma and HTCA cells [7], and polypeptides 42 kM r and 47 kM r from fibroblasts [23]. Similarly, polypeptides 45 kM r and 55 kM r are the only one to be ADP-ribosylated by cholera toxin in G/F from rat liver membranes, a 130 kM r oligoprotein made of 3 polypeptides: 30 kMr, 45 kM r and 55 kM r [14]. In contrast, there is only one ADP-ribosylated polypeptide in pigeon [5,6] and human [8,9] erythrocyte ghosts and in thymus [12] …”

Fluoroaluminates activate transducin‐GDP by mimicking the γ‐phosphate of GTP in its binding site

“…its decay to opsin. Fluorides in the millimolar range confer upon the GDP-bound protein structural and functional properties of the GTP-bound one [4,8]. The requirement of a trace amount of aluminum was recently demonstrated for fluoride activation of hormonal systems and it was suggested that AlF: was indeed the active species [9].…”

Streptococcal glycoprotein-induced tumour cell growth inhibition involves the modulation of a pertussis toxin-sensitive G protein

“…IAP catalyses ADP ribosylation of a cysteine residue in the asubunit of inhibitory GTP-binding (Gi) protein, and thereby blocks interactions between Gi protein and receptors (Katada and Ui, 1982;Murayama and Ui, 1983). On the other hand, CTX activates stimulatory GTP-binding (Gs) protein by ADP-ribosylating an arginine residue of the a-subunit in Gs protein (Northup et al, 1980;Hepler and Gilman, 1992). The finding that the activity of SAGP was diminished by IAP suggested the involvement of Gi protein in the expression of SAGP activity.…”