Characterization of guanylyl cyclase in purified myelin

Abstract

This study was undertaken to characterize the enzymatic properties of the particulate guanylyl cyclase previously shown to be present at a high level of activity in purified rat brain myelin. Significant activation was achieved by both Lubrol-PX and Triton X-100, the latter being somewhat more effective. A pH optimum of 7.8 was observed, compared to 7.4 for microsomes. Employing 1.2 mM GTP with 1% Triton X-100, linearity of response was observed up to 60 min and approximately 1.2 mg of myelin protein. Kinetic analysis revealed Km values of 0.258mM and 0.486mM for myelin and microsomes, respectively, similar values being obtained by Lineweaver-Burke analysis or Direct Linear Plot. Vmax values were 20 and 266 pmol/mg protein/min for myelin and microsomes, respectively. Washing of the myelin with 0.5 M NaCl or 0.1% Na taurocholate did not remove a significant amount of guanylyl cyclase activity, indicating the enzyme to be intrinsic to the myelin sheath.

Membrane association of nitric oxide-sensitive guanylyl cyclase in cardiomyocytes

“…pH-dependence of GC NO was identical for the two locations and similar to that described previously [17,31]. Membrane GC NO had a significantly lower EC 50 value for GTP than the cytosolic, but the difference was small and its physiological relevance is doubtful.…”

Cyclic GMP-Dependent Pathways Protect Differentiated Oligodendrocytes from Multiple Types of Injury

“…ANP, an activator of particulate gaunylate cyclases, elevated cGMP in astroglia and microglia, but not OLs, suggesting that soluble guanylate cyclases were responsible for the NO-mediated increases in cGMP in OLs. However, with more quantitative biochemical methods, Grabow et al [36] found that particulate guanylate cyclase is enriched in the myelin membrane. Thus particulate guanylate cyclase is likely to be expressed in OLs, as substantiated by our finding that ANP is protective in OLs, presumably by elevating cGMP levels via particulate guanylate cyclase.…”

Myelin contains neutral sphingomyelinase activity that is stimulated by tumor necrosis factor-?

“…Detection of these activities broadens the repertoire of signaling processes previously demonstrated to occur in myelin. These have included reactions mediated by (1) ␤-adrenergic and muscarinic cholinergic receptors leading to activation and inhibition, respectively, of adenylyl cyclase (Larocca et al, 1987a), (2) another muscarinic receptor subtype linked to phosphoinositide hydrolysis (Larocca et al, 1987a,b), (3) A 1 adenosine receptors alleged to modify microviscosity in myelin (Casadó et al, 1991), and (4) receptors containing guanylyl cyclase activity whose agonist is not yet known (Chakraborty and Ledeen, 1993;Grabow et al, 1996). The presence of several GTP-binding proteins of the heterotrimeric and low M r types (Boulias and Moscarello, 1989;Braun et al, 1990;Larocca et al, 1991;Berti-Mattera et al, 1992) provides additional evidence for the signaling capability of myelin.…”

“…The discovery of high-affinity muscarinic receptors in myelin (Larocca et al, 1987a) that were subsequently shown to be linked to phosphoinositide phosphatidase (Larocca et al, 1987b) provided a rationale for the previously reported presence in this membrane of enzymes involved in the metabolism and synthesis of polyphosphoinositides (Shaikh and Palmer, 1976;Deshmukh et al, 1978Deshmukh et al, , 1982Saltiel et al, 1987). Several other enzyme activities associated directly or indirectly with signal transducing reactions have been shown to occur in myelin, including adenylyl cyclase (Larocca et al, 1987a), guanylyl cyclase (Chakraborty and Ledeen, 1993;Grabow et al, 1996), and various protein kinases (for review, Ledeen, 1992).…”