High yield purification of soluble guanylate cyclase from bovine lung.
Soluble guanylate cyclase (sGC), the main target of nitric oxide (NO), is a cytosolic, heme-containing, heterodimeric enzyme that catalyzes the conversion of guanosine 5'-triphosphate (GTP) to 3,5'-cyclic guanosine monophosphate (cGMP) and pyrophosphate (PPi) in the presence of Mg2+. Cyclic GMP is then involved in transmitting the NO activating signals to a variety of downstream effectors such as cyclic-nucleotide-gated channels, protein kinases, and phosphodiesterases. In this work, sGC has been purified from bovine lung. The lungs were subjected to grinding and extraction with buffer at physiological pH followed by centrifugation. The resulting solution was subjected to successive column chromatography on DEAE- and Q-Sepharose, Ceramic Hydroxyapatite, Resource Q, and GTP-agarose. The purified enzyme migrated as a two-band protein on SDS-PAGE corresponding to sGC subunits alpha (M(r)=77,532) and beta (M(r)=70,500) and had an A(280 nm)/A(430 nm) of approximately 1 indicating one heme per heterodimer. The yield of enzyme was 8-10mg from 4 to 5 kg bovine lungs. V(max) and K(m) of non-stimulated sGC were 22 nmol/mg/min and 180 microM, respectively. Upon stimulation with the NO donor 3-ethyl-3-(ethylaminoethyl)-1-hydroxy-2-oxo-1-triazene, the V(max) increased to 1330 nmol/mg/min while the K(m) dropped to 43 microM. The quality and quantity of enzyme make it suitable for studies to probe the structure and catalytic mechanism of this enzyme and for research related to drug discovery.
Mathis
Karl J
KJ
Global Research and Development, St. Louis Laboratories, Pfizer Inc., 700 Chesterfield Parkway West, Mail Stop: BB4D, Chesterfield, MO 63017, USA.
Emmons
Thomas L
TL
Curran
Daniel F
DF
Day
Jacqueline E
JE
Tomasselli
Alfredo G
AG
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D006152:Cyclic GMP / Q000378:metabolism*; D006162:Guanylate Cyclase / Q000302:isolation & purification* / Q000378:metabolism; D008168:Lung / Q000201:enzymology*; D018160:Receptors, Cytoplasmic and Nuclear / Q000302:isolation & purification* / Q000378:metabolism; D000071756:Soluble Guanylyl Cyclase
D018160:Receptors, Cytoplasmic and Nuclear; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; D006152:Cyclic GMP