High yield purification of soluble guanylate cyclase from bovine lung

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• 作者：Karl J. Mathis ; Thomas L. Emmons ; Daniel F. Curran ; Jacqueline E. Day ; Alfredo G. Tomasselli
• 关键词：Soluble guanylate cyclase (sGC) ; Protein purification ; 3-Ethyl-3-(ethylaminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-12)
• 刊名：Protein Expression and Purification
• 出版年：2008
• 出版时间：July 2008
• 年：2008
• 卷：60
• 期：1
• 页码：58-63
• 全文大小：301 K

文摘

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 g src=""http://www.sciencedirect.com/scidirimg/entities/204e.gif"" alt=""greek small letter alpha"" title=""greek small letter alpha"" border=""0""> (M_r = 77,532) and β (M_r = 70,500) and had an A_280nm/A_430nm of g src=""http://www.sciencedirect.com/scidirimg/entities/223c.gif"" alt=""not, vert, similar"" title=""not, vert, similar"" border=""0"">1 indicating one heme per heterodimer. The yield of enzyme was 8–10 mg from 4 to 5 kg bovine lungs. V_max and K_m of non-stimulated sGC were 22 nmol/mg/min and 180 μM, 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 μM. 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.