Su-Chen Tsai

Hydroxylamine-stable covalent linkage of myristic acid in Goα, a guanine nucleotide-binding protein of bovine brain

G0 alpha, a guanine nucleotide-binding protein with a strong homology to the G1 alpha and Gs alpha regulatory proteins of adenylate cyclase, is shown to contain myristic acid. The attachment of myristate to the protein is stable to hydroxylamine treatment, and since the amino-terminal sequence of G0 alpha is typical of proteins with amino-terminal myristate, the inference is strong that G0 alpha is also myristylated at its amino-terminal glycine.

Reversible inactivation of soluble liver guanylate cyclase by disulfides

Summary Guanylate cyclase activity was reduced in soluble fractions from rat liver slices incubated with diamide, N-ethylmaleimide, or 5′, 5′-dithiobis-2-nitrobenzoate; addition of GSH or dithiothreitol to assays restored activity. Inactivation of purified rat liver guanylate cyclase by p -hydroxymercuribenzoate at 0°C was completely reversed by dithiothreitol. p -Hydroxymercuribenzoate and GSSG caused reversible inactivation of calf liver guanylate cyclase. Dithiobisnitrobenzoate and cystamine were more potent than GSSG; all were more effective at 30°C than at 0°C. It appears that guanylate cyclase activity in intact cells could be modulated by reversible modification of critical sulfhydryl groups, e.g., by thiol:disulfide exchange involving glutathione.

Effects of nitroprusside and nitroglycerin on cGMP content and PGI2 formation in aorta and vena cava

Nitroprusside (NP) and nitroglycerin (NG) are potent vasodilators that are used clinically on the basis of their abilities to cause relaxation of smooth muscle. In vitro, both agents cause activation of guanylate cyclase, resulting in increased intracellular cGMP. They also have effects on arachidonate metabolism. Despite apparent similarities in their mechanisms of action, the two drugs have different therapeutic applications based in part on differences in their effectiveness on the arterial and venous systems in vivo. To understand better their target tissue preference, slices of aorta and vena cava were incubated with the agents; cGMP and the vasodilatory prostanoid, prostacyclin, were quantified. NP was more effective in increasing the cGMP content of aorta than of vena cava; it was more active than NG in both tissues. Prostaglandin formation by vascular tissue was influenced by the preliminary equilibration period. Under optimal conditions, it appeared that NG enhanced prostacyclin formation in aorta more than did NP. This in vitro model for NP and NG action may be useful in studying the mechanisms of action of these and other vasoactive agents.

Participation of a guanine nucleotide-binding protein cascade in cholera toxin activation of adenylate cyclase

Guanine nucleotide-binding (G) proteins are involved in several transmembrane signaling systems. Choleragen (cholera toxin) activates adenylate cyclase by catalyzing the ADP-ribosylation of Gs alpha, the stimulatory G protein of the cyclase system. This reaction is enhanced by another guanine nucleotide-binding protein termed ADP-ribosylation factor or ARF that was purified from bovine brain membranes [R. A. Kahn and A. G. Gilman, Journal of Biological Chemistry (1986) 261, 7906-7911]. It was recently found that this ARF also increases the NAD:agmatine and NAD:protein ADP-ribosyltransferase, NAD glycohydrolase and auto-ADP-ribosylation activities of the toxin. We have purified and characterized two soluble proteins from bovine brain that act in a similar fashion to enhance choleragen activity in each of these reactions. The membrane and soluble factors are all proteins of approximately 19 kDa that require GTP or GTP analogues for activity and are ADP-ribosylated by the toxin. The ARF proteins apparently interact directly with choleragen in a GTP-dependent fashion to increase its catalytic activity and thus are part of a G protein cascade through which the toxin activates adenylate cyclase. The physiological function of the ARF proteins, as well as their possible relationships to the ras oncogene products and/or the family of G proteins that includes Gs alpha, remains to be determined.

Separation of the 24 kDa substrate for botulinum C3 ADP-ribosyltransferase and the cholera toxin ADP-ribosylation factor

Botulinum C3 ADP-ribosyltransferase modifies a approximately 24 kDa membrane protein believed to bind guanine nucleotides. Cholera toxin ADP-ribosylation factors are approximately 19 kDa GTP-binding proteins that directly activate the toxin. To evaluate a possible relationship between C3 ADP-ribosyltransferase substrate and ADP-ribosylation factor, they were partially purified from bovine brain. ADP-ribosylation factor, but not C3 ADP-ribosyltransferase substrate, stimulated auto-ADP-ribosylation of the choleragen A1 subunit whereas C3 ADP-ribosyltransferase substrate, but not ADP-ribosylation factor, was ADP-ribosylated by C3 ADP-ribosyltransferase. Thus, although both may be GTP-binding proteins, no functional similarity between ADP-ribosylation factor and C3 ADP-ribosyltransferase substrate was found.

Characterization of the Family of Mammalian Genes Encoding ADP- ribosylation Factors

ADP-ribosylation factors (ARFs) are ~20-kDa guanine nucleotide-binding proteins that stimulate the in vitro cholera toxin-catalyzed ADP-ribosylation of the α subunit of Gs (the stimulatory GTP-binding protein of the adenylyl cyclase system) (Kahn and Gilman, 1984, 1986; Bobak et al., 1990). Membrane (mARF) and soluble (sARF I and sARF II) ADP-ribosylation factors that enhance cholera toxin-catalyzed ADP-ribosylation of Gsα, and simple guanidino compounds were identified in bovine brain (Tsai et al., 1987, 1988). Subsequent molecular cloning of the cDNAs that encode the ARFs revealed the existence of a larger family of related guanine nucleotide-binding proteins. To date, six mammalian ARF cDNAs (ARFs 1 to 6) have been isolated from bovine and/or human libraries (Sewell and Kahn, 1988; Price et al., 1988; Bobak et al., 1989; Monaco et al., 1990; Tsuchiya et al., 1991). Based on the deduced amino acid sequences, mammalian ARFs represent a family of 20kDa guanine nucleotide-binding proteins clearly different from members of the ras and ras-like (20-25 kDa) superfamily. In particular, the signature sequences of the regions believed to be involved in guanine nucleotide binding and GTP hydrolysis in the ARF proteins more closely resemble those in the heterotrimeric G protein a subunits than those found in the ras, rho, rac,rap, ral, and rab families (Price et al., 1990). The GTP-hydrolysis domain GXXXXGK is completely conserved across the mammalian ARFs as GLDAAGK. The sequence DVGG which forms the DXXG consensus sequence that has been proposed to coordinate binding to Mg2+ and the β phosphate of GDP and the sequence NKQD (the NKXD consensus sequence) which is thought to contribute to the specificity of interaction with the purine ring of GTP, are found in all the deduced ARF sequences (Price et al., 1990).

Isolation and Immunological Properties of Adenosine Kinase

Bovine liver adenosine kinase is a 43 kDa protein that catalyzes the transfer of phosphate from GTP or ATP to adenosine. Its immunological properties were compared to other GTP-binding proteins of approximately 40 kDa, in particular those involved in signal transduction, such as Gs and Gi, the stimulatory and inhibitory regulatory proteins of adenylyl cyclase, Gt, from the visual excitation system, and Go, a similar protein of unknown function. Antibodies elicited in rabbits against adenosine kinase did not significantly cross-react with other guanyl nucleotide-binding proteins. Antibodies against the other GTP-binding proteins did not react with adenosine kinase. Thus these GTP-binding proteins do not exhibit immunological cross-reactivity.

Activation of kidney guanylate cyclase by cobalt

Abstract Guanylate cyclase activity and cyclic nucleotide content were studied in individual slices from guinea pig kidneys. Basal guanylate cyclase activity, assayed in homogenates or in particulate fractions (100,000 g × 1 h ), and the tissue content of cGMP and cAMP were greater in the inner than in the outer (entirely cortical) slices. The fraction of guanylate cyclase activity recovered in the supernatant was greater in the cortex. Taurodeoxycholate increased activity of the particulate cyclase but decreased that of the supernatant enzyme. Activity of the particulate was increased ca. 200% and that of the supernatant >500% by 1 m m NaN 3 . Supernatant activity was markedly increased by 0.1 m m Co 2+ , which had no effect on the particulate enzyme. (Incubation of kidney slices with 2 m m Co 2+ did not alter their cGMP content, but caused a small increase in the cAMP content of slices containing medullary tissue.) Basal guanylate cyclase activity in fresh supernatants increased linearly with pH from 5.9 to 9, whereas in the presence of Co 2+ there was a clear maximum at pH 7.3 to 7.5. Incubation of fresh supernatant fractions at 37 °C for 3 h increased guanylate cyclase activity and abolished Co 2+ activation. The relationship between Co 2+ activation and that resulting from incubation remains to be defined. It seems probable, however, that these phenomena reflect regulatory properties of the supernatant guanylate cyclases of kidney and other tissues.

Activation of the NAD Glycohydrolase, NAD:Agmatine and NAD:Gsα ADP-Ribosyltransferase and Auto-ADP-Ribosylation Activities of Choleragen by Guanyl Nucleotide and Soluble Proteins Purified from Bovine Brain

Choleragen (cholera toxin) exerts its effects on animal cells by activating adenylate cyclase, thereby increasing intracellular cAMP content (1). The A1 protein of choleragen, released from the holotoxin by reduction of a single disulfide bond linking the A1 and A2 proteins, catalyzes the mono- ADP-ribosylation of Gsα, a regulatory component of the adenylate cyclase system that is responsible for the GTP-dependent activation of the cyclase catalytic unit. ADP-ribosylation of Gsα apparently increases its sensitivity to GTP and its dissociation from the inhibitory Gβγ complex (1).

Stimulation of Choleragen Enzymatic Activities by GTP and a Membrane Protein from Bovine Brain

Activation of adenylate cyclase by choleragen results from the toxincatalyzed ADP-ribosylation of a regulatory component of the cyclase system, Gsα, a guanine nucleotide-binding protein involved in stimulation of the cyclase catalytic unit (1). The ADP-ribosylation reaction and cyclase activation are enhanced by soluble and membrane components (2–9). One membrane protein, known as ADP-ribosylation factor or ARF, was extensively purified by Kahn and Gilman and shown to be a GTP-binding protein (8, 9).