Glucose is the major source of brain energy and is essential for maintaining normal brain and neuronal function. Hypoglycemia causes impaired synaptic transmission. This occurs even before significant reduction in global cellular ATP concentration, and relationships among glycolysis, ATP supply, and synaptic transmission are not well understood. We demonstrate that the glycolytic enzymes glyceraldehyde phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate kinase (3-PGK) are enriched in synaptic vesicles, forming a functional complex, and that synaptic vesicles are capable of accumulating the excitatory neurotransmitter glutamate by harnessing ATP produced by vesiclebound GAPDH/3-PGK at the expense of their substrates. The GAPDH inhibitor iodoacetate suppressed GAPDH/ 3-PGK-dependent, but not exogenous ATP-dependent, Glycolysis plays a vital role in maintaining normal brain function. Glucose is known to serve as the major substrate for cerebral energy under normal conditions (1). Recent evidence suggests a direct correlation between glucose utilization and cognitive function (2). Reduction of glucose levels results in pathophysiological states and abnormal electrophysiological activity; however, this occurs long before significant alteration in tissue ATP levels is detected (3-7). Substitution of pyruvate for glucose does not support normal evoked neuronal activity, although tissue ATP level returns to normal (8 -10). Abnormal synaptic transmission caused by hypoglycemia occurs in part if not entirely by a presynaptic mechanism (7,11,12). Fleck et al. (7) have shown that substantial reduction of extracellular glucose results in a decrease in stimulus-evoked Glu release, with no changes in ATP levels. These studies together suggest that glycolysis or glycolytic intermediate(s) are necessary for normal synaptic transmission independent of global cellular ATP levels.In an attempt to reveal the underlying mechanism of hypoglycemia-induced aberrant synaptic transmission, we previously explored the possibility that glycolytic intermediates could modify proteins localized in the nerve ending (13,14). 3-Phosphoglycerate (3-PG) 1 was demonstrated to stimulate phosphorylation of 155-and 72-kDa proteins. The latter was identified as glucose-1,6-bisphosphate synthetase, and 1,3-bisphosphoglycerate (1,3-BPG) was found to serve as the direct substrate for phosphorylation of this enzyme, by donating 1-phosphate. Both of these phosphorylated proteins are enriched in the synaptosomal (nerve ending preparation) as well as cell body soluble fractions, but the significance of these modifications in synaptic transmission remains unclear.In this paper, we show that the glycolytic intermediate 1,3-BPG forms an acyl-enzyme intermediate with vesicle-bound glyceraldehyde phosphate dehydrogenase (GAPDH), that vesicle-bound GAPDH exists in a complex with 3-phosphoglycerate kinase (3-PGK), and that activation of vesicle-associated GAPDH and 3-PGK is sufficient to support vesicular uptake of Glu. Glutamate is now recognized as the major ex...

No abstract

Hypocholesterolemic activity of dietary polyunsaturated fatty acids is observed after relatively short-term but not long-term feedings, and their long-term feedings are suspected to accelerate aging through tissue accumulation of lipid peroxides and age pigments (lipofuscin). To define the long-term effects of fats and oils in more detail, female mice were fed a conventional basal diet supplemented with lard (Lar), high-linoleic (n-6) safflower oil (Saf), rapeseed oil (Rap), high-alpha-linolenic (n-3) perilla oil (Per), or a mixture of ethyl docosahexaenoate and soybean oil (DHA/Soy) from 17 weeks to 71 weeks of age. The DHA/Soy and Per groups had decreased serum cholesterol levels compared with the Lar and Saf groups, but the difference between the Lar and Saf groups was not significant. The 3-hydroxy-3-methyglutary-CoA (HMG-CoA) reductase activity in the liver was also significantly lower in the Per and DHA/Soy groups. However, no significant difference in lipofuscin contents in the brain and liver was observed among the 5 dietary groups, despite significant differences in peroxidizability indices of the dietary and/or tissue lipids. These results indicate that n-3 fatty acid-rich oils are hypocholesterolemic by suppressing hepatic HMG-CoA reductase activity compared with animal fats and high-linoleic (n-6) oil, but tissue lipofuscin contents are not affected by a long-term feeding of fats and oils with different degree of unsaturation in mice.

We purified a novel alpha-glucosidase to homogeneity from an Escherichia coli recombinant transformed with the alpha-glucosidase gene from thermophilic Bacillus sp. SAM1606. The enzyme existed as mono- and multimeric forms of a promoter protein with a relative molecular weight of 64,000 and isoelectric point of 4.6. We isolated a monomeric form of the enzyme and characterized it. The enzyme was unique among the known alpha-glucosidases in both broad substrate specificity and high thermostability. The enzyme hydrolysed a variety of O-alpha-D-glucopyranosides such as nigerose, maltose, isomaltose, sucrose, and trehalose efficiently. The molecular activity (k0) and the Michaelis constant (Km) values at 55 degrees C and pH 6.0 for sucrose were 54.6 s-1 and 5.3 mM, respectively. The optimum pH and temperature for hydrolysis were pH 5.5 and 75 degrees C, respectively. The enzyme exhibited a high transglucosylation activity: it reacted with 1.8 M sucrose at 60 degrees C for 70 h to yield oligosaccharides containing theanderose in a maximum yield of 35% (w/w). High thermostability of the enzyme (stable up to 65 degrees C at pH 7.2 for 10 min) permits the transglucosylation reaction at high temperatures, which would be beneficial for continuous production of oligosaccharides from sucrose.

No abstract