Soluble guanylate cyclase from rat lung exists as a heterodimer.
The soluble form of guanylate cyclase (EC 4.6.1.2) from rat lung has been purified to homogeneity by a one-step immunoaffinity chromatographic procedure. The purified soluble guanylate cyclase has specific activities of 432 and 49.1 nmol of cyclic GMP formed per min/mg protein with manganese and magnesium ions as a cofactor, respectively. This represents a purification of approximately 2,000-fold with a 50% recovery. The native enzyme has a molecular weight of 150,000 and a Stokes radius of 4.8 nm as determined on Spherogel TSK-G3000SW gel permeation chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results in two protein-staining bands with molecular weights of 82,000 and 70,000. The purified soluble guanylate cyclase was also subjected to native polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, ion exchange chromatography, and GTP-agarose affinity chromatography. These additional purification procedures confirmed the presence of a single protein peak coincident with enzyme activity. The two subunits separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were shown to have different primary structures by immunoblotting with monoclonal and polyclonal antibodies prepared against purified soluble guanylate cyclase and by peptide mapping with papain or Staphylococcus aureus V8 protease treatment. These data demonstrate that soluble guanylate cyclase purified from rat lung is a heterodimer composed of 82,000- and 70,000-dalton subunits with different primary structures.
Kamisaki
Y
Y
Saheki
S
S
Nakane
M
M
Palmieri
J A
JA
Kuno
T
T
Chang
B Y
BY
Waldman
S A
SA
Murad
F
F
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006162:Guanylate Cyclase / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D008168:Lung / Q000201:enzymology*; D051379:Mice; D008970:Molecular Weight; D051381:Rats; D012995:Solubility
D000911:Antibodies, Monoclonal; D006162:Guanylate Cyclase