cause no continuous assay for the detection of free phos-A new continuous coupled uv-spectrophotometric phate had been developed that is sensitive yet also conassay is described for two phosphate-releasing envenient and cost effective. Of the continuous assays zymes, aspartate transcarbamylase and ATPase of herthat have been reported, many of these require multipes simplex virus (HSV). Phosphate release is coupled ple coupling enzymes and/or substrates and are useful to the phosphorolysis of the nucleoside analog 7-methonly under limited conditions (1, 2).
ylinosine (m 7 Ino) catalyzed by purine nucleoside phos-
Recently, purine nucleoside phosphorylase (PNPase, 2 phorylase. When this reaction is monitored at 291 nm, EC 2.4.2.1) has been used in a continuous phosphate assay the coupled assay can readily detect 10 nmol P i rewhich uses only one enzyme and one substrate. PNPase leased/min. Our method offers advantages over a reis an enzyme in the purine salvage pathway of both eucently reported continuous assay devised for measurkaryotes and prokaryotes (3). Bacterial PNPase catalyzes ing aspartate transcarbamylase activity using the the reversible phosphorolysis of adenine, hypoxanthine, or nucleoside analog methylthioguanosine (MESG) as the guanine (deoxy)ribonucleosides yielding the purine base linking substrate. In contrast to MESG, m 7 Ino is easily and (deoxy)ribose 1-phosphate (Fig. 1) (4). This lack of and inexpensively synthesized and is also commersubstrate specificity initially generated interest in PNPase cially available. The spectrophotometric signal at 291 as a tool in synthetic organic chemistry by allowing the nm, produced by the difference in the extinction coefassembly of synthetic purine nucleosides from substituted ficients between nucleoside substrate and the base purine bases and (deoxy)ribose 1-phosphate (5). However, product, is significant over a much wider pH range the spectral characteristics of some of the synthetic subthan the signal difference between MESG and its phosstrates soon led to the development of both fluorescent phorolysis product at 360 nm. Saturation curves for and spectrophotometric PNPase-coupled phosphate aspartate and carbamyl phosphate and pH rate profiles have been reproduced using the purine nucleo-assays. side phosphorylase/m 7 Ino coupled assay. Initial