Development of guanine analyzer to measure activity of guanylate cyclase.
A previous analyzer of adenine compounds by high-performance liquid chromatography was converted for the determination of guanine, its nucleoside and nucleotides by a post-column fluorescence derivatization with phenylglyoxal (PGO) in place of bromoacetoaldehyde. The gel filtration 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 solution and CH3CN (85:15, v/v) containing 15 mM PGO. The separated analytes reacted with flow through PGO in a reaction coil at 90 degrees C into fluorescent derivatives. Those derivatives were detected fluorimetrically, highly selective and quantitatively. The activity of soluble guanylate cyclase (sGC) in the neuroblastoma N1E-115 cell was measured by tracing the peak height of cGMP synthesized from substrate GTP using this guanine analyzer. The sensitivity of the present method was lower than the radioisotope method. However, our modified method was simpler, safer and quicker than the radioisotope method. Furthermore, this method could trace other guanine compounds simultaneously, allowing measurement of guanine metabolizing enzymatic activity. Therefore, it will be useful for screening of effectors on sGC.
Isumi
Y
Y
Faculty of Pharmaceutical Sciences, Setsunan University, Osaka, Japan.
Akizawa
T
T
Matsuda
K
K
Umekawa
H
H
Kurose
T
T
Yoshioka
M
M
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006152:Cyclic GMP / Q000096:biosynthesis; D005456:Fluorescent Dyes; D006147:Guanine / Q000032:analysis*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006162:Guanylate Cyclase / Q000032:analysis* / Q000378:metabolism*; D007202:Indicators and Reagents; D009447:Neuroblastoma; D010658:Phenylglyoxal; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D006147:Guanine; D006160:Guanosine Triphosphate; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D010658:Phenylglyoxal