Partial purification and characterization of particulate guanylate cyclase from rat liver after solubilization with trypsin.
Publication/Presentation Date
1-1-1982
Abstract
Guanylate cyclase from 105,000 X g particulate fractions of rat liver homogenates (20 pmoles of cyclic GMP formed/min/mg protein) was solubilized in the absence of detergents by incubating fractions 12 min at 37 degrees with 5 ug/ml trypsin. Optimal solubilization was dependent upon trypsin and particulate preparation concentrations. Virtually no activation of particulate guanylate cyclase was observed at any time point or trypsin concentration tested. Guanylate cyclase solubilized with trypsin was purified about 500-fold (9.4 nmoles/min/mg protein) using ammonium sulfate precipitation, GTP-affinity chromatography, and preparative polyacrylamide gel electrophoresis. Activity eluted as a single peak on Sepadex G-200 (Stokes radius = 40 A) and migrated as a single peak on sucrose density gradients (S20,w = 4.6). Thus, the tryptic fragment was estimated to be about 80,000 daltons (Mr) with a frictional ratio (f/fo) of 1.4. These partially purified preparations exhibited linear double reciprocal plots with Mn-GTP and Hill coefficients of 1.0. This is in contrast to the crude membrane-associated enzyme which had a Hill co-efficient of 1.5. Membrane-bound and trypsin-solubilized guanylate cyclase were activated 3- and 4-fold with nitric oxide and were inhibited with 1mM cystamine. Cystamine inhibition could be partially reversed with 7.5 mM dithiothreitol. These studies indicate that particulate guanylate cyclase solubilized by limited proteolysis is amenable to purification by "classical" chromatographic techniques. The partially purified fragment contains the catalytic site, the site for nitric oxide activation, and at least one sulfhydryl group required for activity.
Volume
8
Issue
6
First Page
359
Last Page
370
ISSN
0095-1544
Published In/Presented At
Waldman, S. A., Lewicki, J. A., Brandwein, H. J., & Murad, F. (1982). Partial purification and characterization of particulate guanylate cyclase from rat liver after solubilization with trypsin. Journal of cyclic nucleotide research, 8(6), 359–370.
Disciplines
Business Administration, Management, and Operations | Health and Medical Administration | Management Sciences and Quantitative Methods
PubMedID
6136535
Department(s)
Administration and Leadership
Document Type
Article