Journal of Neurochemistry

Preti, Augusto; Fiorilli, Amelia; Lombardo, Adriana; Caimi, Luigi; Tettamanti, Guido

doi: 10.1111/j.1471-4159.1980.tb06263.xpmid: 6161218

The possible occurrence of sialyltransferase activity in the plasma membranes surrounding nerve endings (synaptosomal membranes) was studied, using calf brain cortex. The synaptosomal membranes were prepared by an improved procedure which provided: (a) a „nerve ending fraction” consisting of at least 85% well‐preserved nerve endings and containing only small quantities of membranes of intracellular origin; (b) a „synaptosomal membrane fraction” carrying high amounts of authentic plasma membrane markers (Na+‐K+ ATPase, 5′‐nucleotidase, sialidase, gangliosides) with values of specific activity four to fivefold higher than those in the „nerve ending fraction” and very small amounts of cerebroside sulphotransferase, marker of the Golgi apparatus, and of other markers of intracellular membranes (rotenone‐insensitive NADH and NADPH: cytochrome c reductases), the specific activities of which were, respectively, 0.5‐ and 0.7‐fold that in the „nerve ending fraction”. Thus the preparation of synaptosomal membranes used had the characteristics of plasma membranes and carried a negligible contamination of membranes of intracellular origin. The distribution of sialyltransferase activity in the main brain subcellular fractions (microsomes; P2 fraction; nerve ending fraction; mitochondria) resembled most closely that of thiamine pyrophosphatase, the enzyme known to be linked to the Golgi apparatus and the plasma membranes and of acetylcholine esterase, the enzyme known to be linked to either intracellular or plasma membranes. The enrichment of sialyltransferase activity in the „synaptosomal membrane fraction”, referred to the „nerve ending fraction”, was practically the same as that exhibited by authentic plasma membrane markers. All this is consistent with the hypothesis that in calf brain cortex sialyltransferase has two different subcellular locations: one at the level of intracellular structures, most likely the Golgi apparatus (as described by other authors), the other in the synaptosomal plasma membranes. The basic properties (pH optimum, V/S, V/t and V/protein relationships) and detergent requirements of the synaptosomal membrane‐bound sialyltransferase were established. The highest enzyme activities were recorded on exogenous acceptors, lactosylceramide and ds‐fetuin. The Km values for CMP‐NeuNAc were different using lactosylceramide and ds‐fetuin as acceptor substrates (0.57 and 0.135 mm, respectively); the thermal stability of the enzyme acting on glycolipid acceptor was higher than that on the glycoprotein acceptor; the effect of detergents was different when using glycoprotein from glycolipid acceptors; no competition was observed between lactosylceramide and ds‐fetuin. Thus the synaptosomal membranes carry at least two different sialyltransferase activities: one acting on lactosylceramide (and glycolipid acceptors), the other working on ds‐fetuin (and glycoprotein acceptors). Ganglioside GM3 was recognized as the product of synaptosomal membrane‐bound sialyltransferase activity working on lactosylceramide as acceptor substrate.

Edström, Anders; Hanson, Mats; Prus, Karen; Wallin, Margareta

doi: 10.1111/j.1471-4159.1980.tb06264.xpmid: 6108993

The microsomal fraction of frog sciatic nerves was found to contain Ca2+‐ or Mg2+‐dependent hydrolytic activity toward different nucleoside di‐and triphosphates. In the presence of Ca2+ substrate specificity was in the order CTP > UTP > GTP > ATP. When Mg2+ was used, the triphosphates were approximately equally good substrates. ATP hydrolytic activity was very similar with Ca2+ or Mg2+ as the cofactor, whereas Ca2+ was the more potent activator of hydrolysis of the other triphosphates tested. The preparation showed some activity toward the nucleoside diphosphates but none toward the monophosphates or p‐nitrophenylphosphate. The enzymic properties of ATP hydrolysis were more closely studied. The hydrolysis was optimal at 18–24°C in the presence of 1 mm‐Ca2+ or 1 mm‐Mg2+. Ca2+‐ and Mg2+‐ATP hydrolysis displayed pH maxima around 8.0–8.5 and 7.4–8.0, respectively. Vmax values for Ca2+‐ and Mg2+‐ATP hydrolysis were similar: approx. 12 μmol Pi per h per mg protein with a Km value of approx. 0.05 mm. The ATP hydrolysis activity was inhibited by NaF but unaffected by ouabain, vanadate, cytochalasin B, and various drugs known to influence ATPase activity of mitochondria. Zn2+ stimulated the ATP hydrolysis activity at low concentrations (10‐6–10‐5m) and inhibited it at higher concentrations. The possibility that these observations account for stimulation and inhibition of axonal transport in frog sciatic nerves exposed to similar concentrations of Zn2+ is discussed.

Sturman, J. A.; Rassin, D. K.; Gaull, G. E.; Cote, L. J.

doi: 10.1111/j.1471-4159.1980.tb06265.xpmid: 7452261

The concentrations of taurine in all regions of fetal and neonatal rhesus monkey brain are greater than in the same regions of adult monkey brain. (35S)Taurine injected into pregnant rhesus monkeys is accumulated by the fetus. This process occurs rapidly in most tissues, but occurs slowly in fetal brain. Neonatal rhesus monkey brain also accumulates (35S)taurine slowly compared with other tissues after i.v. injection, and continues to accumulate (35S)taurine for a long period of time. These results suggest that the accumulation and exchange of taurine in developing rhesus monkey brain is slow, as found in neonatal rats, and that if there is a period of development at which rapid exchange of brain taurine occurs in the rhesus monkey, it is before the rapid brain growth spurt.

Dirks, B.; Hanke, J.; Krieglstein, J.; Stock, R.; Wickop, G.

doi: 10.1111/j.1471-4159.1980.tb06266.xpmid: 7452262

An isolated rat brain preparation was perfused using glucose‐free (=aglycemic) media. The high‐energy phosphates, substrates of the glycolytic pathway, free atnino acids, acetylcholine as well as the intracellular distribution of hexokinase activity were determined in brain tissues. The EEG was evaluated visually. The levels of glycolytic substrates, glutamate, and glutamine in cortical tissue decreased after aglycemic perfusion, whereas the aspartate level increased and the GABA level remained unchanged. The high‐energy phosphate content seemed to be unaffected for about 15 min of aglycemic perfusion and fell significantly after 20 min. The EEG of the isolated brain changed rapidly after starting aglycemic perfusion and became isoelectric after 12–15 min. Hyperglycemic perfusion (35 mmol glucose per liter perfusion medium) did not alter the energy metabolism of the isolated brain. The breakdown of cerebral energy metabolism and of EEG activity was postponed when thiopental was added to the perfusion medium. The soluble hexokinase activity measured in cortical tissue was reduced after aglycemic perfusion and was enhanced after thiopental. Hyperglycemic perfusion did not influence the intracellular hexokinase distribution. The acetylcholine level in the striatum of the isolated rat brain was significantly decreased by aglycemia and was increased in hypothalamus by thiopental. It was suggested that hexokinase bound to the mitochondrial membrane may play an important role in the relationship of energy metabolism and neuronal activity.

Jope, Richard S.; Jenden, Donald J.

doi: 10.1111/j.1471-4159.1980.tb06267.xpmid: 7452263

Acetylcholine synthesis in rat brain synaptosomes was investigated with regard to the intracellular sources of its two precursors, acetyl coenzyme A and choline. Investigations with α‐cyano‐4‐hydroxycinnamate, an inhibitor of mitochondrial pyruvate transport, indicated that pyruvate must be utilized by pyruvate dehydrogenase located in the mitochondria, rather than in the cytoplasm, as recently proposed. Evidence for a small, intracellular pool of choline available for acetylcholine synthesis was obtained under three experimental conditions. (1) Bromopyruvate competitively inhibited high‐affinity choline transport, perhaps because of accumulation of intracellular choline which was not acetylated when acetyl coenzyme A production was blocked. (2) Choline that was accumulated under high‐affinity transport conditions while acetyl coenzyme A production was impaired was subsequently acetylated when acetyl coenzyme A production was resumed. (3) Newly synthesized acetylcholine had a lower specific activity than that of choline in the medium. These results indicate that the acetyl coenzyme A that is used for the synthesis of acetylcholine is derived from mitochondrial pyruvate dehydrogenase and that there is a small pool of choline within cholinergic nerve endings available for acetylcholine synthesis, supporting the proposal that the high‐affinity transport and acetylation of choline are kinetically coupled.

Daly, J. W.; McNeal, E.; Partington, C.; Neuwirth, M.; Creveling, C. R.

doi: 10.1111/j.1471-4159.1980.tb06268.xpmid: 6256481

Norepinephrine, histamine, adenosine, glutamate, and depolarizing agents elicit accumulations of radioactive cyclic AMP from adenine‐labeled nucleotides in particulate fractions from Krebs‐Ringer homogenates of guinea pig cerebral cortex. The particulate fractions contain sac‐like entities, which apparently are associated with a significant portion of the tnembranal adenylate cyclase. Particulate fractions from sucrose homogenates are a less effective source of such responsive entities. Activation of the adenine‐labeled cyclic AMP‐generating systems by norepinephrine is by means of α‐adrenergic receptors, while activation by histamine is through H1‐ and H2‐histaminergic receptors. Adenosine responses are potentiated by the amines and are antagonized by alkylxanthines. Glutamate and depolarizing agents appear to elicit accumulations of cyclic AMP via „release” of endogenous adenosine. It is proposed, based on the virtual absence of an α‐adrenergic or H1‐histaminergic response in the presence of a combination of potent adenosine and H2‐histaminergic antagonists, that α‐adrenergic and H1‐histaminergic receptor mechanisms do not activate adenylate cyclase directly in brain slices or Krebs‐Ringer particulate fractions, but merely facilitate activation by β‐adrenergic, H2‐histaminergic, or adenosine receptors.

McNeal, E. T.; Creveling, C. R.; Daly, J. W.

doi: 10.1111/j.1471-4159.1980.tb06269.xpmid: 6256482

The accumulations of radioactive cyclic AMP elicited by adenosine, norepinephrine, and histamine in adenine‐labeled vesicular entities of a particulate fraction from guinea pig cerebral cortex are greatly reduced as a result of prolonged preincubation. The presence of adenosine deaminase during preincubations largely prevents the loss of adenosine, norepinephrine and histamine responses. Adenosine deaminase was inactivated by deoxycoformycin prior to stimulation of cyclic AMP accumulation by adenosine or amines. If adenosine deaminase is not inactivated, responses to norepinephrine are not significant and histamine responses are reduced by 50%. Adenosine deaminase cannot restore responsiveness of the cyclic AMP‐generating systems. It is proposed that, in participate fractions of guinea pig cerebral cortex, low levels of adenosine cause a slow loss of receptors and/or of coupling of receptors to cyclic AMP‐generating systems.

Kuriyama, Kinya; Kurihara, Etsuo; Ito, Yoshihisa; Yoneda, Yukio

doi: 10.1111/j.1471-4159.1980.tb06270.xpmid: 6256483

The effect of intrastriatal microinjection of kainic acid (KA) on specific binding of (3H)muscimol to the particulate fractions obtained from corpus striatum (CS), globus pallidus (GP), substantia nigra (SN), and cerebral cortex (CC) was examined. Seven days after the unilateral intrastriatal microinjection of KA, the amount of specifically bound (3H)muscimol was significantly increased at the injected site, whereas no significant alteration of (3H)muscimol binding was found in GP, SN, or CC. Scatchard analysis of striatal binding revealed that microinjection of KA significantly increased the affinity (KD) of GABA receptors on the injected (lesioned) side of the CS without affecting the total number of binding sites (Bmax) therein. This significant increase in (3H)muscimol binding, however, was eliminated by pretreating particulate fractions from the CS with Triton X‐100, a non‐ionic detergent. No statistically significant difference in amounts of (3H)muscimol binding was detected when the preparations from the KA‐treated and non‐treated CS were preincubated with 0.05% Triton X‐100, respectively. Scatchard analysis using CS preparations treated with 0.05% Triton X‐100 revealed that the affinity of the GABA receptor was increased by treatment with Triton X‐100, while the total number of binding sites (Bmax) was unchanged by this treatment. These results suggest that neuronal degeneration produced by KA in vivo and pretreatment of particulate preparations with Triton X‐100 in vitro may increase the amount of specifically bound (3H)muscimol to CS preparations by a similar molecular mechanism.

Logan, J. G.

doi: 10.1111/j.1471-4159.1980.tb06271.xpmid: 6256484

(Na+‐K+) ATPase is present in synaptosomal preparations and it is assumed to represent the sodium‐potassium pump. 10 μm‐noradrenaline activates (Na+‐K+) ATPase approximately 100%, but 50 μm‐noradrenaline does not stimulate the rate of 22Na extrusion from synaptosomes. The results suggest that it is unlikely that the noradrenaline stimulation of (Na+‐K+) ATPase is part of a feedback mechanism whereby released noradrenaline can influence the activity of the presynaptic sodium pump.

Hoffman, Paula L.; Wermuth, Bendicht; Wartburg, Jean‐Pierre von

doi: 10.1111/j.1471-4159.1980.tb06272.xpmid: 6778961

Human brain contains multiple forms of aldehyde‐reducing enzymes. One major form (AR3), as previously shown, has properties that indicate its identity with NADPH‐dependent aldehyde reductase isolated from brain and other organs of various species; i.e., low molecular weight, use of NADPH as the preferred cofactor, and sensitivity to inhibition by barbiturates. A second form of aldehyde reductase („SSA reductase”) specifically reduces succinic semialdehyde (SSA) to produce γ‐hydroxybutyrate. This enzyme form has a higher molecular weight than AR3, and uses NADH as well as NADPH as cofactor. SSA reductase was not inhibited by pyrazole, oxalate, or barbiturates, and the only effective inhibitor found was the flavonoid quercetine. Although AR3 can also reduce SSA, the relative specificity of SSA reductase may enhance its in vivo role. A third form of human brain aldehyde reductase, AR2, appears to be comparable to aldose reductases characterized in several species, on the basis of its activity pattern with various sugar aldehydes and its response to characteristic inhibitors and activators, as well as kinetic parameters. This enzyme is also the most active in reducing the aldehyde derivatives of biogenic amines. These studies suggest that the various forms of human brain aldehyde reductases may have specific physiological functions.