Guanylate cyclase: purification, properties, free radical activation, radiolabeling, and preparation of hybridoma antibodies.
In summary, we have succeeded in purifying soluble guanylate cyclase from several rat tissues. These methods are being used to purify the enzyme from other tissues and other species. We have also been able to purify partially the particulate form of guanylate cyclase 100- to 200-fold. We expect that our experience with the soluble enzyme and with the antibodies that we are developing should enable us to purify the particulate form of the enzyme in the near future. We can activate the purified enzyme with nitric oxide and other agents such as hydroxyl radical and unsaturated fatty acids (5). Both the activation and the reversal to the basal state can occur without the presence of other macromolecules, suggesting that these activators interact directly and reversibly with the protein. Both basal activity and the capacity to activate with nitric oxide require free sulfhydryl groups on the enzyme. The pure enzyme can be radiolabeled with either [35S] or 125I, and labeled enzyme has assisted us in various ways including purification, examination of the enzyme's subunit structure, and screening sera and media from hybridoma cultures for antibodies to the protein. Thus, as we anticipated several years ago, the availability of purified guanylate cyclase has allowed us to develop a number of new techniques and approaches to examine the regulation and function of guanylate cyclase and cyclic GMP. We expect that there will be much new information and exciting progress in the field forthcoming as a result of these and other new methods.
D000818:Animals; D000911:Antibodies, Monoclonal; D004789:Enzyme Activation; D005609:Free Radicals; D006162:Guanylate Cyclase / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D006825:Hybridomas / Q000276:immunology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008168:Lung / Q000201:enzymology; D008970:Molecular Weight; D009569:Nitric Oxide / Q000494:pharmacology; D051381:Rats
D000911:Antibodies, Monoclonal; D005609:Free Radicals; D009569:Nitric Oxide; D006162:Guanylate Cyclase