ELSEVIER Journal of Chromatography B, 690 (1997) 343-347 JOURNALOF CHROMATOGRAPHY B Short communication Assay of soluble guanylate cyclase activity by isocratic high- performance liquid chromatography • a ~ .a P.G. Pletta " , P.L. Maurl , C. Gardana b, L. Benazzi a alTBA-CNR, Via Ampbre, 56, 20131 Milan, Italy bDipartimento di Scienze e Tecnologie Biomediche, Via Celoria, 2, 20133 Milan, Italy Received 25 March 1996; revised 26 August 1996; accepted 6 September 1996 Abstract A HPLC method alternative to labelled or unlabelled procedures was developed tbr the assay of guanylate cyclase (GC) activity. The substrate (GTP) and the product (cGMP) of the enzymatic reaction were separated in the isocratic mode on a txBondapak C~8 column. The activity of GC was linearly dependent on the amount of cGMP produced in the presence of sodium nitroprusside. This approach was applied to follow the purification of GC from bovine lung and to evaluate its stability in different storage conditions. Keywords: Guanylate cyclase; Enzymes 1. Introduction Guanylate cyclase (EC 4.6.1.2) catalyzes the formation of 3',5'-cyclic GMP (cGMP) from 5'-GTP in the presence of a divalent cation [1]. cGMP is considered a mediator in vascular smooth muscle relaxation, platelet anti-aggregation, intestinal secre- tion and retinal phototransduction [2,3]. For these reasons, the study of the guanylate cyclase system is very important in therapeutic approaches to hyper- tension, enterotoxigenic diarrhoea and vasospasm. Guanylate cyclase exists in two main isoenzyme forms: soluble or cytosolic and particulate or mem- brane-bound; the relative amounts of each form differ with type of tissue and physiological state [2]. The soluble enzyme is a heme protein and consists of *Corresponding author. two subunits with molecular mass of about 70 000 and 75 000 [4]; this form is activated by nitric oxide and/or NO-containing compounds, as sodium nitro- prusside [5]. Guanylate cyclase can be assayed by radioim- munoassay [6]; however, these methods require caution, because of the use of radioactivity and are quite laborious. In recent years, high-performance liquid chromatography (HPLC) has been reported as a valuable alternative for the assay of enzyme activity [7]. This approach is based on the assess- ment of a chromatographic separation of the sub- strate and products of the enzymatic reaction, which has to be performed under time and reaction stopping conditions compatible with HPLC runs. This method has been used for measuring the activity of different classes of enzymes [8,9], including also retinal guanylate cyclase [10]. The described procedure is 0378-4347/97/$17.00 Copyright © 1997 Elsevier Science B.V. All rights reserved Pll S0378-4347(96)00414-8