A novel assay for 5 ' - nucleotidase using 1 , N 6 - etheno - AMP as substrate , and comments on the properties of the reaction product ,

5'-Nucleotidase (EC 3.1.3.5) is an ectoenzyme in many tissues concerned with the conversion of AMP into adenosine. This activity is probably part of a metabolic pathway for removing extracellular adenine nucleotides released during processes such as neurotransmission (Burnstock, 1981), strenuous exercise (Forrester & Lind, 1969), platelet thrombus formation (Gaarder et al., 1961) and shock (Trams et al., 1980). In turn, besides being further metabolized, the product adenosine has effects at Aior A2-type adenosine receptors on various tissues, including vasculature [a possible role of 5'-nucleotidase in the regulation of blood flow has been proposed for several years; see, e.g., Baer et al. (1966); Baer & Drummond (1968); Nakatsu & Drummond (1972)]. 5'Nucleotidase is interesting in other respects. As a cellsurface glycoprotein it undergoes various stages of posttranslational processing (Wada et al., 1986; van den Bosch et al., 1986), appears to circulate between the cell surface and an intracellular pool (Stanley et al., 1980; Wilcox et al., 1982; Widnell et al., 1982), possibly interacts with elements of the cytoskeleton (Mannherz & Rohr, 1978; Carraway et al., 1979), and is attached to the plasma membrane either as a short-stalked integral membrane protein (Baron et al., 1986) or through a glycosyl-phosphatidylinositol lipid anchor (Low et al., 1986; Low, 1987). In addition, in white adipose tissue 5'-nucleotidase activity shows sex differences and adaptivity in several pathophysiological states (Green et al., 1981; Vernon et al., 1983; Newsholme et al., 1985; Jamal & Saggerson, 1987). Over the past two decades 5'-nucleotidase has been assayed in various ways. These include measurement of orthophosphate release (colorimetric or radiochemical; Widnell, 1974), spectrophotometric measurement by coupling to adenosine deaminase (Widnell & Unkeless, 1968; Belfield & Goldberg, 1968; Burger & Lowenstein, 1970), or measurement of the conversion of radiolabelled AMP into adenosine. In the last method, product and substrate have been separated by ion-exchange chromatography (Glastris & Pfeiffer, 1974), by paper chromatography (Widnell & Unkeless, 1968) or by precipitation of AMP with ZnSO4 + Ba(OH)2 (Avruch & Wallach, 1971; Newby et al., 1975). Here we demonstrate a novel simple assay of 5'nucleotidase that exploits the fluorescent properties of etheno-AMP and ethenoadenosine, and we also make some comments on the metabolism and pharmacological action of ethenoadenosine.