DEVELOPMENT OF GUANINE ANALYZER TO MEASURE ACTIVITY OF GUANYLATE-CYCLASE

A previous analyzer of adenine compounds by high-performance liquid ch romatography was converted for the determination of guanine, its nucle oside and nucleotides by a post-column fluorescence derivatization wit h phenylglyoxal (PGO) in place of bromoacetoaldehyde. The gel filtrati on column (Asahipak GS-320H) was used for separation by a mobile phase consisting of 25 mM sodium citrate buffered (pH 4.0)-150 mM NaCl solu tion and CH3CN (85:15, v/v) containing 15 mM PGO. The separated analyt es reacted with flow through PGO in a reaction coil at 90 degrees C in to fluorescent derivatives. Those derivatives were detected fluorimetr ically, highly selective and quantitatively. The activity of soluble g uanylate cyclase (sGC) in the neuroblastoma N1E-115 cell was measured by tracing the peak height of cGMP synthesized from substrate GTP usin g this guanine analyzer. The sensitivity of the present method was low er than the radioisotope method. However, our modified method was simp ler, safer and quicker than the radioisotope method. Furthermore, this method could trace other guanine compounds simultaneously, allowing m easurement of guanine metabolizing enzymatic activity, Therefore, it w ill be useful for screening of effecters on sGC. (C) 1998 Elsevier Sci ence B.V.