The Distribution of Radioligand Binding Activity and 5′‐Nucleotidase Activity in Bovine Caudate Nucleus Subcellular Fractions

198

The subcellular distribution of the ectoenzyme, 5'-nucleotidase, in cerebral cortex and cerebellum of the rat was studied both biochemically and cytochemically. The fractions were characterized biochemically by marker enzymes. The localization of 5'-nucleotidase activity was also investigated cytochemically in the myelin, synaptosomal, mitochondrial, and microsomal fractions. Biochemically 5'-nucleotidase was found to be enriched in the membrane-containing fractions, i.e., myelin, synaptosomal, and microsomal fractions. Cytochemistry showed the reaction product in the myelin fraction to be associated with myelin profiles. In the synaptosomal fraction reaction product could occasionally be seen at synaptosomal membranes, although it could not be attributed unequivocally to the synaptosome itself, since in positions with reaction product unidentifiable membrane structures could always be seen attached. Mitochondria were virtually without any reaction product. In the microsomal fraction 5'-nucleotidase activity was associated with unidentifiable membrane structures. It is concluded that 5'-nucleotidase is associated with myelin profiles and that the high activity found in the synaptosomal fraction is probably not associated with nerve ending plasma membranes.

Section: Discussionsupporting

confidence: 91%

Subcellular Localization of 5′‐Nucleotidase in Rat Brain

Heymann¹,

Reddington²,

Kreutzberg³

1984

198

“…The cavitation lysis method permitted us to obtain a majority of RSO vesicles in which the active sites of acetylcholinesterase and 5'-nucleotidase were accessible to the medium. The cellular distribution of 5'-nucleotidase from caudate nucleus was similar to that observed by Withy et al (1982). By contrast, it appeared somewhat different from that observed in the bovine cortex, where 94.5% of the activity occurs in the cytoplasm (Montero and Fes, 1982).…”

Section: Discussionsupporting

confidence: 64%

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

Méflah¹,

Harb²,

Duflos³

et al. 1984

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.

“…Since this enzyme may be of considerable importance to synaptic transmission (e.g., in the production of the neuromodulator adenosine) (Phillis et al, 1979), and in the salvage of purines released during chemical transmission (Dowdall, 1978;Zimmerman et al, 1979), its distribution on presynaptic terminals was thought to be of interest. Subcellular fractionation of mammalian brain has shown 5'-nucleotidase to be present in all the major particulate fractions (Banik and Davidson, 1969;Withy et al, 1982), and its presence in myelin has received attention in recent years (Cammer and Zimmerman, 1981). Cytochemical results, however, have yielded equivocal results, since some workers have shown 5'-nucleotidase to be present on synaptic membranes (Bernstein et al, 1978), whereas Kreutzbert et al (1978 detected the enzyme solely on glial membranes in rat brain.…”

mentioning

confidence: 99%

Presynaptic Distribution of the Cholinergic‐Specific Antigen Chol‐1 and 5′‐Nucleotidase in Rat Brain, as Determined by Complement‐Mediated Release of Neurotransmitters

Richardson

1983

Nerve terminals prepared from rat cortex and hippocampus were loaded with seven radioactive putative neurotransmitters (serotonin, noradrenaline, dopamine, gamma-aminobutyric acid, aspartate, glutamate, and taurine). The release of these transmitters, choline acetyltransferase, 3,4-dihydroxyphenylalanine decarboxylase, enolase, and lactate dehydrogenase was monitored during complement-mediated lysis. Three antisera were used: anti-5'-nucleotidase, anti-Chol-1, and anti-rat cerebrum. Anti-5'-nucleotidase serum did not cause the release of any labelled transmitter or of any of the enzymes studied. Anti-Chol-1 serum released choline acetyltransferase and small amounts of enolase and lactate dehydrogenase. Anti-rat cerebrum caused the release of all seven transmitters, choline acetyltransferase, and small amounts of the other three enzymes. It was concluded that 5'-nucleotidase was not present on any of the terminals studied, and that Chol-1 is only present on cholinergic terminals.