Abstract
Abstract A convenient, simple and reliable HPLC assay method for membrane-bound enzymes, such as guanylate Cyclase and 5′- nucleotidase, were developed. The membrane enzymes of guanylate cyclase had been assayed by determination of the product cyclic GMP by radioimmunoassay after column chromatographical separation of cyclic GMP, and 5′-nucleotidase had been assayed by colorimetrical phosphate assay method. The use of radioisotope is dangerous and requires a special laboratory, and photometrical assay method on membrane enzymes is difficult to be carried out reliably because membrane samples exhibit turbidity. RP-HPLC assay method for guanylate cyclase directly measures the product of cyclic GMP fluorimetrically, which afford a sensitive detection for cyclic GMP at pmol level. RP-HPLC assay method for 5′-nuc1eotidase also directly the product of aderosine by UV detection at 260 nm. Reliable stoichiometry was observed in both of enzyme assay, i. e., decrease in substrate of GTP and increase in product of cyclic GMP for guanylate cyclase, decrease in substrate of AMP and increase in product of adenine for 5′-nucleotidase. These assay method are successfully applied onto membrane samples and also onto serum samples.
Published Version
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