NAD+ glycohydrolase, an ecto-enzyme of calf spleen cells

Abstract

By using a sensitive fluorimetric assay of NAD+ glycohydrolase (EC 3.2.2.6), we showed that calf spleen cells are able to hydrolyse 1,N6-etheno-NAD+ given in the medium. The observed rates of substrate hydrolysis and product accumulation in the medium are equivalent. Moreover, the splenocytes are able to cleave the nicotinamide-ribose bond of a water-soluble polymer of NAD+, and their NAD+ glycohydrolase activity is fully inhibited by a high-molecular-weight Blue Dextran. Selective permeation of the cellular membrane digitonin revealed an intracellular pool of NAD+ glycohydrolase, which accounts for 25% of the total activity. We conclude that NAD+ glycohydrolase associated with the splenocytes has the characteristics of an ecto-enzyme.

Molecular cloning and functional expression of bovine spleen ecto-NAD+ glycohydrolase: structural identity with human CD38

“…Moreover, purification to apparent homogeneity of hydrophobic untruncated forms of this class of enzymes was achieved only recently in a few cases [9,10]. We have reported the purification of bovine spleen ecto-NADase [9], a glycoprotein overwhelmingly expressed at the surface of splenocytes [3] and which presents catalytic properties similar to those of CD38 [16]. The present work, which reports the molecular cloning of this enzyme, has yielded the first primary sequence of a ' classical ' ecto-NADase, as well as information on its mode of association with cell membranes and its structural identity with CD38.…”

“…NADase activity was routinely determined with NAD + by the cyanide addition assay [8] or by a sensitive continuous fluorimetric method using 1,N'-etheno-NAD + as substrate [3]. To measure both ADP-ribosyl cyclase (formation of cADPR) and NADase [formation of ADP-ribose (ADPR)] activities, the enzyme fraction was assayed in the presence of [adenosine-"%C]NAD+ (final concentration 20-50 µM) as described previously [16,30].…”

“…At a cellular level, it was first believed that these enzymes were constituents of the endoplasmic reticulum and could therefore play a role in the regulation of intracellular levels of the pyridine nucleotides. However, in organs that are rich in this enzyme, the catalytic activity is far too high to account for such a function, and it was established later that NADases are in fact overwhelmingly markers of the plasma membrane and ecto-enzymes [3], i.e. the active site of the enzyme facing the outer surface of the cells has no access to intracellular NAD + .…”

In Situ Staining for Poly(ADP-Ribose) Polymerase Activity Using an NAD Analogue

“…However, an alternative explanation would be that introduced ⑀-NAD is metabolized during the period before MNNG treatment. Normal mouse macrophages possess high levels of an NADase ectoenzyme (Artman and Seeley 1979), similar to an NADase in calf spleen cells, which can metabolize ⑀-NAD (Muller et al 1983). Furthermore, we found that staining was reduced when higher levels of ATP were used for permeabilization, perhaps because ATP is a substrate for NAD synthesis.…”

Localization of an arginine‐specific mono‐ADP‐ribosyltransferase in skeletal muscle sarcolemma and transverse tubules

“…Small increases in the presence of unmasking agents were also observed for other ecto-enzymes, e.g. acetylcholinesterase [24] and NAD' glycohydrolase [25] (Table 3).…”