Guanine nucleotides allow the trypsin solubilization of an active Mr = 68,000 guanylate cyclase.
It has been previously shown that trypsin treatment of rat liver plasma membranes causes the solubilization of a guanylate cyclase of Mr = 140,000 (Lacombe, M. L., Haguenauer-Tsapis, R., Stengel, D., Ben Salah, A., and Hanoune, J. (1980) FEBS Lett. 116, 79-84). In this study, we observed that addition of Mn-GTP during this step greatly protected the enzyme from proteolytic degradation. This effect was specific for guanine nucleotides, being weaker for other nucleotides triphosphate and GDP, and absent for cyclic GMP and GMP. Metal-GTP complex was required with a strict specificity for Mn2+. In addition to the Mr = 140,000 enzyme, trypsin solubilization in the presence of Mn-GTP led to the formation of a small and active form of guanylate cyclase. Based on its behavior on Ultrogel AcA 34 and sucrose gradients, its apparent Mr was calculated to be 68,000. Both forms could be well separated by high performance liquid chromatography and were shown to be sequentially solubilized (the larger appearing before the smaller species). Mr = 140,000 species, but not the cytosolic enzyme, was able to generate the Mr = 68,000 enzyme upon tryptic treatment in the presence of Mn-GTP. The Mr = 140,000 and 68,000 enzymes exhibited Michaelis-Menten kinetics (Hill coefficient = 1) with Km for Mn-GTP of 130 and 70 microM, respectively. The proteolytically solubilized enzymes were strickingly heat labile and highly protected by Mn-GTP. These results support the hypothesis that the rat liver membrane-bound guanylate cyclase has a dimeric structure similar to that of the cytosolic enzyme. They also suggest a possible role for GTP in limiting the degradation rate of membrane guanylate cyclase in vivo and, thus, in regulating the active enzyme concentration.
D000818:Animals; D002499:Centrifugation, Density Gradient; D002851:Chromatography, High Pressure Liquid; D006150:Guanine Nucleotides / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D051381:Rats; D012995:Solubility; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism*
D006150:Guanine Nucleotides; C037047:manganese GTP; D006160:Guanosine Triphosphate; D014357:Trypsin; D006162:Guanylate Cyclase