5′‐Nucleotidase from Bovine Caudate Nucleus Synaptic Plasma Membranes: Specificity for Substrates and Cations; Study of the Carbohydrate Moiety by Glycosidases

Abstract

We studied 5'-nucleotidase in preparations of synaptic plasma membranes from bovine caudate nucleus. The best substrates for this membrane-bound enzyme were purine nucleotides, particularly 5'AMP. Effects of metal cations and chelating agents suggest that 5'-nucleotidase is a metalloprotein. Optimal conditions for solubilization of the 5'-nucleotidase were found by using a low concentration of the zwitterionic detergent sulfobetaine 14. In contrast, another membrane-bound enzyme, acetylcholinesterase, was not solubilized under these conditions, but only in the presence of Triton X-100. The effects of lectins (concanavalin A, Lens culinaris agglutinin, wheat germ agglutinin, and Limulus polyphemus agglutinin) showed that both enzymes are glycoproteins. Sequential hydrolysis with specific glycosidases produced modifications of the effect of lectins on these enzymes. The results suggest the presence of a complex-type glycosylation, with a fucose residue on the internal N-acetyl-D-glucosamine of the pentasaccharide core.

Mass spectrometry study of ecto‐5′‐nucleotidase from bull seminal plasma

“…Preliminary analysis of its glycidic components was reported by Meflah et al . [23], who combined the specific interaction of different vegetal lectins with subsequent hydrolysis with N ‐glycosidases. The complex nature of the glycidic moiety was recognized, however, no further details on the structure of the N‐linked oligosaccharides were obtained.…”

5'-Nucleotidases, Nucleosides and their Distribution in the Brain: Pathological and Therapeutic Implications

“…The substrate specificity of e5'NT is broad, as it can hydrolyze both ribo-and deoxyribonucleoside monophosphates. However, e5'NT exhibits the highest affinity for Ado monophosphate (AMP) (low K m -nucleotidase: K m for AMP is 1-50 M) [82,86,[103][104][105][106]. Its optimal pH is approximately 6.8-9.0 depending on the type of tissue [81,82].…”

“…Its optimal pH is approximately 6.8-9.0 depending on the type of tissue [81,82]. Both Mg 2+ and Mn 2+ increase the activity of e5'NT [82,103,105], whereas Ado diphosphate (ADP) and Ado triphosphate (ATP) (and to a lesser extent Urd diphosphate/UDP, Urd triphosphate/UTP, Guo diphosphate/GDP, Guo triphosphate/GTP, cytidine (Cyd) diphosphate/CDP, Cyd triphosphate/CTP, concanavalin A and , -methyleneadenosine-5'-diphosphate (APCP)) inhibit e5'NT activity [82,103,105,107]. Ecto-5'NT plays a role in (i) the extracellular generation of nucleosides [56][57][58]86] and, as a consequence, nucleoside (e.g., Ado) receptor activation, neurotransmission modulation, and the regulation of learning and memory, sleep, psychomotor coordination, cardiac and renal function, platelet aggregation, nociception and so forth [7, 12, 82-84, 101, 108-116]; (ii) the termination of nucleoside di-and triphosphates activity on their receptors [63,82,117,118]; (iii) the uptake of extracellular nucleosides [56][57][58]; (iv) T-cell activation (as a coreceptor) [119][120][121]; (v) cell-cell adhesion and cell proliferation [60,62,66,121,122]; (vi) synaptic malleability/arrangements and information processing, neuroglial/cell-matrix interactions and synaptogenesis [61,[63][64][65][66][67][68]82]; (vii) neuronal regeneration and development …”