Claus J. Schmitges

Molecular resolution and reconstitution of the GPP(NH) and NAF sensitive adenylate cyclase system

Abstract When a special detergent-extraction procedure is applied to rat brain particulate fractions, the latters adenylate cyclase activity becomes virtually unresponsive to NaF or Gpp (NH)p (guanylyl-5′-imidodiphosphate) despite the fact that under these conditions the enzyme does not appear to be removed (i.e., solubilized) from the membranes. Addition of exogenous fractions of detergent-solubilized membranes or of water-soluble samples of homogenates, obtained from various tissues, restores the stimulation of the enzyme by both Gpp(NH)p and NaF. These findings indicate that the stimulation caused by these agents is mediated by one or more regulatory component(s), and that these are molecular components physically distinct from the enzyme itself. The regulatory component(s) appear to be proteinaceous in nature and sensitive to SH-reactive reagents. The properties of the reconstituted system resemble those of the original particulate adenylate cyclase. This system may serve as a convenient tool for the study of the molecular properties of adenylate cyclase and of the basis of its regulatory control.

Demonstration of choleragen-dependent ADP-ribosylation in whole cells and correlation with the activation of adenylate cyclase

Abstract 3T3C 2 mouse fibroblasts rendered permeable to ( α −32 P)NAD + show cholera toxin-dependent labeling of a 45,000 m.w. protein and of a doublet of polypeptides around 52,000 m.w. These same bands are ADP-ribosylated in broken cells. Membranes prepared from pigeon erythrocytes pretreated with choleragen show a decrease in subsequent cholera toxin-specific ADP-ribosylation of a 43,000 m.w. polypeptide. Both whole cell and broken cell adenylate cyclase activation and toxin-specific ADP-ribosylation are reversed specifically by low pH and high concentrations of toxin and nicotinamide in all systems. Thus ADP-ribosylation appears to be relevant to the molecular action of choleragen in whole cells as well as in broken cells.

Role of Specific Membrane Lipids in Modulating the Activity of Adenylate Cyclase

Male Sprague-Dawley rats were decapitated and their brains removed; subsequent operations were performed at 0°C. The brain was homogenized in 8 vol (vol/wt) of 3 mM MgCl2, 3 mM DTT, 50 mM Tris-HCl pH 8.4 and centrifuged for 10 min at 40,000 g,,..,. This procedure was repeated once and the pellet was homogenized in 8 vol (vol/wt) of 1 mM MgCl2, I mM DTT, 10%o glycerol (vol/vol), 0.5% sodium deoxycholate (DOC), 50 mM Tris-HCI, pH 8.4 (Buffer A). The homogenate was centrifuged for 45 min at 300,000 g,..., and the supernatant, containing solubilized enzyme, retained. Enzyme in the supernatant was separated from solubilized lipid by chromatography on Ultrogel AcA34 equilibrated with buffer A. When required, phospholipid, dissolved in chloroform:methanol (2:1 vol/vol) and dried under a stream of N2, was dispersed in the solubilized enzyme preparation. Enzyme activity was measured at 300C (10).

Concerning the nonspecificity of methylation inhibitors

Most of the evidence for the essential participation of S-adenosylmethionine (AdoMet)-mediated methylation reactions in eukaryotic cell functions derives from the use of chemical probes that have been found to be inhibitory to these various cell functions and that are also known to be inhibitory, in a direct or an indirect manner, to some cellular methylation reaction(s). Compounds most often employed experimentally as methylation reaction probes include adenosine (Ado), 3-deazaadenosine (c3Ado), 5’-deoxy-5’-S-isobutylthioadenosine (SIBA) and 5’-deoxy-5’-S-isobutylthio-3-deazaadenosine (c3SIBA). The use of all these compounds as methylation reaction probes is based upon the findings of numerous investigators that S-adenosylhomocysteine (AdoHcy) is a potent inhibitor of many AdoMet-utilizing methyltransferases (see review by Borchardt, 1977). In many of the studies employing Ado and c3Ado, L-homocysteine (Hcy) has been observed to potentiate the physiological effects of these nucleosides (e.g., Ishizaka et al., 1980; Morita et al., 1981; Pike et al., 1978; Rabe et al., 1980; Zimmerman et al., 1978). This potentiation by Hcy has been interpreted as additional evidence that Ado and c3Ado are affecting cell function as the result of an intracellular buildup of AdoHcy or S-3-deazaadenosylhomocysteine (c3AdoHcy), respectively, and consequent inhibition of one or more methyltransferases.

Some Perspectives on the Hormone-stimulated Adenylate Cyclase System

A central question in biology concerns how regulation of cellular processes is achieved. A major aspect of this relates to the mechanism by which extracellular factors, e.g., growth factors, neurotransmitters, and hormones, exert their effects. These agents modify the intracellular status of the cell, affecting either a broad range of cell types (e.g. growth hormones) or a more restricted population (e.g. ACTH). A further characteristic is the time span within which the effects become apparent which may be long (e.g. NGF), short (e.g. cAMP generation) or both long and short (e.g. insulin effect on growth and glucose transport). An even more rapid effect is detected with those agents which seem to modulate ion-fluxes (e.g. acetyl- choline). The cell can thus show a very broad time-spectrum of responses. Long term action is believed to reflect a pleiotropic action at the level of the genome (e.g. transcription, translation, and cell division), whereas the short term effects seem to be specific alterations designed to meet particular environmental needs (e.g. cAMP production leading to glycogenolysis). This represents coarse and fine tuning of cellular metabolism, allowing the cell to efficiently co-ordinate adaptations to changing environmental conditions.