Inhibition of Glutamate-Induced Intensification of Free Radical Reactions by Gangliosides: Possible Role in Their Protective Effect in Rat Cerebellar Granule Cells and Brain Synaptosomes

The neurotoxic effect of exposure of rat cerebellar granule cells to glutamate (I00 M) is to a large extent prevented by incubation of neurons not only with micromolar, but even with nanomolar concentrations of gangliosides GM1, GD1b, and GT1b. GM1 was also shown to decrease significantly the per cent of dead neurons in culture after induction of lipid peroxidation. Exposure to glutamate was found to cause a significant decrease of the activity of Na⁺, K⁺-ATP-ase in rat brain cortex synaptosomes, but superoxide dismutase, alpha-tocopherol, or 10–100 nM GM1 practically prevented its action. Other data showing the ability of gangliosides to inhibit the intensification of free radical reactions by glutamate (based on the estimation of methemoglobin formation, SH group content, etc.) have been obtained. The results suggest that gangliosides are able to decrease the glutamate-induced activation of free radical reactions in nerve cells. This effect appears to contribute to their protective action against glutamate neurotoxicity.     

Comparison of the mitochondrial endonucleases from Neurospora crassa and Saccharomyces cerevisiae

The endonucleases from Neurospora crassa and Saccharomyces cerevisiae are not closely related antigenically. They also differ with respect to their activity at pH 8, their degree of hydrophobicity, and their sensitivity to elevated temperatures. However, the two nucleases have similar specific activities, are inhibited by EDTA, and have nearly identical substrate specificities. Since the enzymes also have the same mode of action and intracellular location, these similarities may indicate that they have the same physiological role despite their structural differences.     

Comparison of the phosphatases of Lysobacter enzymogenes with those of related bacteria

Lysobacter enzymogenes ATCC 29487 (UASM 495) produces an outer-membrane-associated phosphatase and an excreted phosphatase. The cell-associated enzyme was compared to phosphatases of nine other Gram-negative gliding bacteria and to that of Escherichia coli. The other three species of the genus Lysobacter also produce a particulate, cell-associated phosphatase. Antiserum prepared against the phosphatase from the outer membrane of L. enzymogenes effectively precipitated the phosphatases of two other L. enzymogenes strains and the enzymes of L. antibioticus, L. brunescens and L. gummosus. Some inhibition of the enzyme by the antiserum also was observed. No significant reaction could be detected between the antiserum and the cell-associated phosphatases of species of Cytophaga johnsonae, 'C. compacta', Myxococcus xanthus, E. coli and the excreted phosphatase of L. enzymogenes. The results indicate that the four species of the genus Lysobacter are closely related despite their physiological differences and that the outer-membrane-associated phosphatases of these organisms have different structural characteristics than the phosphatases of the other Gram-negative bacteria that were used. Furthermore, differences in the amino acid compositions of the cell-associated and the excreted phosphatase of L. enzymogenes confirm the immunological results and are in agreement with the physical and chemical differences noted between the two enzymes.     

The use of Tween 20 in a sensitive turbidimetric assay of lipolytic enzymes

A turbidimetric esterase assay was developed using a Tween 20 solution in the presence of CaCl2 and Lysobacter enzymogenes esterase (EC 3.1.1.1) as the enzyme source. The reaction was followed by measuring the increase in the optical density at 500 nm (OD500) due to the hydrolytic release of the fatty acids from Tween 20 and their precipitation as the calcium salts. Concentrations of 1.8% Tween and 3 mM CaCl2 were found to be optimal for the assay of 0.036 to 0.15 esterase units in a 4-mL reaction mixture over a 30-min period. The esterase reactions were linear with time at least up to 1.2 OD500 and the rate of increase in the OD500 was proportional to the enzyme concentration. Low initial reaction rates were seen with low esterase activity, presumably because of the limited solubility of the fatty acid - calcium salt in a 1.8% Tween solution. This turbidimetric method is much simpler and at least 36 times more sensitive than the titrimetric assay with Tween 20, and at least four times more sensitive than a spectrophotometric assay with p-nitrophenyl palmitate. This assay has been used to determine the activities of cell-associated and excreted esterases produced by Lysobacter enzymogenes and Pseudomonas aeruginosa, and of lipolytic enzymes from porcine liver, Chromobacterium viscosum, Candida cylindracea, and wheat germ.