Alterations in brain protein kinase C after experimental brain injury.

Regional activities and levels of protein kinase C were measured after lateral fluid percussion brain injury in rats. At 5 min and 20 min after injury, neither cofactor-dependent nor -independent PKC activities in the cytosol and membrane fractions changed in the injured and contralateral cortices or in the ipsilateral hippocampus. Western blot analysis revealed decreases in the levels of cytosolic PKC alpha and PKC beta in the injured cortex after brain injury. In the same site, a significant increase in the levels of membrane PKC alpha and PKC beta was observed after injury. Although the level of PKC alpha did not change and that of PKC beta decreased in the cytosol of the ipsilateral hippocampus, these levels did not increase in the membrane fraction after injury. The levels of PKC gamma were generally unchanged in the cytosol and the membrane, except for its decrease in the cytosol of the hippocampus. There were no changes in the levels of any PKC isoform in either the cytosol or the membrane of the contralateral cortex after injury. The present results suggest a translocation of PKC alpha and PKC beta from the cytosol to the membrane in the injured cortex after brain injury. The observation that such a translocation occurs only in the brain regions that undergo substantial neuronal loss suggests that membrane PKC may play a role in neuronal damage after brain injury.

Regional concentrations of cyclic nucleotides after experimental brain injury.

Regional concentrations of lactate, glucose, cAMP, and cGMP were measured after lateral fluid percussion brain injury in rats. At 5 min after injury, while tissue concentrations of lactate were elevated in the cortices and hippocampi of both the ipsilateral and contralateral hemispheres, those of glucose were decreased in these brain regions. By 20 min after injury, increases of lactate concentrations and decreases of glucose concentrations were observed only in the cortices and in the hippocampus of the ipsilateral hemisphere. Whereas the cAMP concentrations were unchanged in the cortices and hippocampi of the ipsilateral and contralateral hemispheres at 5 min after injury, decreases were found in the injured cortex and ipsilateral hippocampus at 20 min after injury. The tissue concentrations of cGMP were found to be elevated only in the ipsilateral hippocampus at 5 min after injury. The present observation that tissue glucose decreases in the injured cortex and the ipsilateral hippocampus are consistent with the published findings of increased hyperglycolysis and oxidative metabolism in brain immediately after injury. The present findings that the concentrations of cAMP and cGMP change in the cortex and hippocampus provide biochemical evidence for the neurotransmitter's surge after brain injury.

Hydrolysis of glycosylpyridinium ions by anomeric-configuration-inverting glycosidases.

The hydrolyses of five beta-D-xylopyranosylpyridinium ions by the beta-D-xylosidase of Bacillus pumilus proceed with kcat values 10(8)-10(9)-fold larger than the rates of spontaneous hydrolysis of the same compounds. Log(kcat) values correlate well with aglycon pK(a) [B1g(V) = -0.52, r = 0.99], whereas the correlation of log(kcat/Km) is poor [r = 0.77; beta 1g(V/K) = approximately -0.6]. The (1-->3)-beta-D-glucanase of Sporotrichum dimorphosporum hydrolyses 4-bromo-2-(beta-D-glucopyranosyl)isoquinolinium ion with a rate enhancement of 10(8). The amyloglucosidase II of Aspergillus niger hydrolyses three alpha-D-glucopyranosylpyridinium ions with rate enhancements of 10(5)-10(8). The efficient hydrolysis of glycosylpyridinium ions by these three inverting glycosidases, the catalytic mechanism of which is unlikely to involve a nucleophile from the enzyme, makes it improbable that the hydrolysis of glycosylpyridinium ions by retaining glycosidases, discovered some years ago, is initiated by addition of a catalytic nucleophilic carboxylate group of the enzyme to the pyridinium ring.

Polarimetry and 13C n.m.r. show that the hydrolyses of beta-D-glucopyranosyl fluoride by beta(1-->3)-glucanases from Phanerochaete chrysosporium and Sporotrichum dimorphosporum have opposite stereochemistries.

The time courses of optical rotation and fluoride ion release during hydrolysis of beta-D-glucopyranosyl fluoride by the beta(1-->3)-glucanase of Phanerochaete chrysosporium (J. L. Copa-Patiño and P. Broda, unpublished work) indicated that the initial sugar product was beta-D-glucopyranose. This was confirmed by monitoring the hydrolysis of 1-[13C]beta-D-glucopyranosyl fluoride by this enzyme with 13C n.m.r. (without proton decoupling). The same two techniques were used to confirm that hydrolysis of beta-D-glucopyranosyl fluoride by the exo beta(-->3)-glucanase of 'Basidiomycete QM 806' (identified as Sporotrichum dimorphosporum) yielded alpha-glucopyranose as first sugar product, in accordance with previous results using laminarin as substrate [Parrish and Reese (1963) Carbohydr. Res. 3, 424-429; Nelson (1970) J. Biol. Chem. 245, 869-872].