NO and cGMP constitute an autocrine, paracrine, and possible endocrine signal transduction system. Cytosolic NO-responsive guanylyl cyclase can be stimulated by NO derived from its own cell, from similar or distinct neighboring cell types within a tissue, from a circulating pool of NO (as NO+ equivalents coupled to plasma protein thiol groups), or from pharmacologic agents, the nitrovasodilators. NO and cGMP together comprise an especially wide-ranging signal transduction system when one considers (i) the many roles of cGMP in physiological regulation, including smooth muscle relaxation, visual transduction, intestinal ion transport, and platelet function; (ii) the many sources, biochemical interactions, and functions of NO; and (iii) the interactions of cGMP and its affected pathways with other signaling systems such as phosphoinositides, eicosanoids, cAMP and Ca2+.

Nitric oxide-stimulated modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by (5)(6)(7)(8). After purification and microsequencing of the radiolabeled protein from human erythrocytes or platelets or rat brain, it was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) (9-12). Incorporation of radiolabel into isolated GAPDH was also stimulated by NO, meaning that an ADPThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.ribosyltransferase was not needed and suggesting that the protein was automodified (9-13). Label was released from GAPDH by treatment with mercuric ion, consistent with the presence of an ADP-ribosylcysteine linkage (9-13). The active-site cysteine of GAPDH was suggested to be the site of modification, so that ADP-ribosylation could account for the observed loss of GAPDH activity, even though the extent of modification was very low (=14%) (9-13).We have now further characterized the GAPDH modification. On the basis of radiolabeling, both the adenine and nicotinamide moieties of NAD were incorporated into the enzyme and released by treatment with Hg2+, indicating that GAPDH was modified covalently with NAD, not with ADPribose.

No abstract

Brefeldin A (BFA) is a fungal metabolite that exerts profound and general Inhibitor actions on membrane transport. At least some of the BFA effects are due to inhibition of the GDP-GTP exchange on the ADP-ribosylation factor (ARF) catalyzed by membrane protein(s). ARF activation is likely to be a key event The fungal toxin brefeldin A (BFA) has been widely used to analyze the mechanisms of membrane transport. The effects of BFA include the disappearance of non-clathrin-coated buds and transport vesicles, the inhibition of constitutive secretion, and a series of changes in'shape, location, and function of the organelles of the exocytic and endocytic pathways (1-4). These changes are preceded and probably, at least in part, caused by the release ofa set ofproteins from the Golgi complex including two major non-clathrin coat proteins, the ADP-ribosylation factor (ARF, a small Ras-like GTPase) and 8COP, a component of the cytosolic protein complex "coatomer" (5-7). Moreover, BFA inhibits the GDP-GTP exchage on ARF catalyzed by a Gojgi protein and the binding of ARF to Golgi membranes, suggesting that the components involved in ARF association to transport organelles may be the primary targets of BFA and a key site of regulation of vesicular transport pathways (8, 9).ARF, in addition to being involved in membrane transport, has long been known as a cofactor in the ADP-ribosylation of the a subunit of the GTP-binding protein Gs by cholera toxin and to be able to interact directly with cholera toxin (10-12). ADP-ribosylation is a posttranslational modification of proteins produced by the transfer of ADP-ribose from NAD to specific amino acid residues, which can be catalyzed by both bacterial toxins and eukaryotic enzymes (13). Analogous to its role in the activation of the exogenous ADP-ribosyltransferase cholera toxin, one of the physiological functions of ARF may be to activate an endogenous cellular mono(ADPribosyl)transferase (11). Indeed, a family of brain mono(ADP-ribosyl)transferases has been reported to be sensitive to ARF (14). These considerations prompted us to determine whether BFA, possibly by perturbing ARF binding, might affect cellular ADP-ribosylations. Here we report that BFA markedly stimulates the ADP-ribosylation of two cytosolic proteins of38 and 50 kDa (p38 and p50). p38 appears to be identical with an isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme and a multifunctional protein that has been implicated in several cellular processes (15)(16)(17)(18)(19)(20)(21)(22) 1114The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.