Purification of a soluble isoform of guanylyl cyclase-activating-factor synthase.

Abstract

The soluble form of guanylyl cyclase-activating-factor (GAF) synthase from rat cerebellum was purified to homogeneity by sequential affinity chromatographic steps on adenosine 2',5'-bisphosphate (2',5'-ADP)-Sepharose and calmodulin-agarose. Enzyme activity during purification was bioassayed by the L-arginine-, NADPH-, and with a molecular mass ofabout 155 ± 3 kDa. These data suggest that soluble GAF synthase purified from rat cerebellum is a homodimer of 155-kDa subunits and that enzyme activity is dependent upon the presence of calmodulin.In various mammalian cell types [e.g., macrophages (1), endothelial cells (2), polymorphonuclear neutrophils (3, 4), brain (5, 6), and neuronal cell lines (7, 8)], L-arginine is oxidized to yield the endothelium-derived relaxing factor with physicochemical and pharmacological properties similar to nitric oxide (NO) or a NO-containing compound (9, 10). Both within the same cell and in specific target cells, this NO-like factor (or group offactors) activates soluble guanylyl cyclase, thereby acting as an intra-and intercellular signal molecule to regulate the intracellular concentration of cGMP (11)(12)(13)(14). Earlier studies have demonstrated that L-arginine could activate guanylyl cyclase in brain and neuronal cells (15, 16); however, the mechanisms were not elucidated. This laboratory has proposed (17, 18) that hormonal regulation of cGMP accumulation could be mediated through such a signal transduction pathway. Since the exact chemical structure of this ubiquitous factor (or group of factors) remains to be clarified (19,20), we suggest the preliminary name of guanylyl cyclase-activating factor (GAF). The formation of GAF from L-arginine is catalyzed by an NADPH-dependent GAF synthase (6,(21)(22)(23)(24)(25). GAF synthase and its target enzyme, soluble guanylyl cyclase, represent the protein components of this signal-transduction pathway (13,14,17,18,26,27). In rat cerebellum, GAF synthase activity is due to a cytosolic Ca2+/calmodulin-regulated isoform of the enzyme. GAF synthase was found to catalyze the conversion of L-arginine to both L-citrulline and a labile NO-like activator of soluble guanylyl cyclase (i.e., GAF). The enzyme was purified by the sequential use of two affinity chromatography columns, ADP-Sepharose and calmodulin-agarose, from the crude supernatant fraction to homogeneity (8925-to 10,076-fold, assayed by citrulline and soluble guanylyl cyclase stimulation, respectively). Purified GAF synthase is a (homo)dimer of protein subunits of 155 ± 3 kDa. Some of these observations have been presented in abstract form (28-30).MATERIALS AND METHODS Materials. Calmodulin-agarose and Sepharose 4B were obtained from Sigma. All other column materials and standard marker proteins were purchased from Pharmacia. Glycine and Tris (Bio-Rad) and SDS (Boehringer Mannheim) were of electrophoresis grade. All other reagents were of analytical grade. Biotinylated calmodulin, biotinylated alkaline phosphatase, and avidin were kindly provided

Barium Activates Rat Cerebellar Nitric Oxide Synthase

“…2b). The EC50 value for Ca 2+ was 200 nM, similar to those obtained in previous studies (1,2). The EC50 for Ba2+ was 50 pM, which is 250 times higher than that for Ca 2+.…”

“…Nitric oxide (NO) is thought to be a messenger mol ecule in cerebella, and the characteristics of cerebellar nitric oxide synthase (NOS) have been well-defined; activation of NOS is absolutely dependent on Ca 2' and calmodulin (1,2), and this underlies the mechanism of the physiological response to N-methyl-D-arpartate (NMDA) in cerebella. Glutamate and NMDA release endothelium derived relaxing factor (EDRF)-like messengers from cere bellar cells (3) increase cGMP levels (4-6) and enhance the conversion of L-arginine to L-citrulline (4, 6) through NMDA-receptor activation in cerebellar cells.…”

Dose dependent effects of reactive oxygen and nitrogen species on the function of neuronal nitric oxide synthase

“…These include protein-protein interactions [22][23][24][25][26], protein phosphorylation [27][28][29], endogenous inhibitory N-methylated L-arginines [30,31], subcellular localization [32], subunit dimerization [33], as well as product feedback inhibition, by which excess NO down-regulates the amount of subsequent NO synthesis. NOS activity can also be altered by oxidative stress.…”

Purified retinal nitric oxide synthase enhances ADP‐ribosylation of rod outer segment proteins

“…RetNOS activity was determined by the formation of [3H]L-citrulline from [3H]e-arginine as previously described [24,25]. Briefly, samples (50 /ll) were incubated for 5 min at 37°C in the presence of 25 mM Tris-HCl (pH 7. applied to 1-ml columns of Dowex 50X8-200 (Sigma).…”

“…Enzymatic properties indicate a relationship between the retinal enzyme and the neuronal NOS type I from mammalian brain [24,25]. So far the retinal enzyme has not been purified or cloned and therefore knowledge of its molecular and enzymatic properties is limited.…”