Enriched fractions of neuronal and glial cells from rat and human (autoptical specimens of frontal cortex) brains were assayed for thiaminpyrophosphatase (TPPase) activity by isoelectric focusing (IEF). Glial and neuronal fractions yielded about 0.16 and 0.03 g/g of wet tissue respectively. Purity was estimated to be 85-90%. The isolated fractions were homogenized in the presence of 1% Triton X-100, and IEF was carried out on thin layer polyacrylamide gel in a pH range of 3.5-9.5, using Ampholine PAG plates. TPPase activity of the protein bands was assayed by laser-densitometry, after incubation with thiaminpyrophosphate in an appropriate buffer and PbS staining. IEF analysis showed that TPPase activity was present almost exclusively in the neuronal fraction (at levels 16-fold higher than those found in the glial fraction). After IEF, TPPase activity was present in 10 distinct protein bands with different isoelectric points. These preliminary data suggest that TPPase may be involved in the neuronal activity of rat and human brain.

Different distribution of thiaminpyrophosphatase activity in neuronal and glial cell enriched fractions from human and rat brain: an isoelectric focusing investigation.

LAFORENZA, UMBERTO;MAZZARELLO, PAOLO ANGELO;
1990

Abstract

Enriched fractions of neuronal and glial cells from rat and human (autoptical specimens of frontal cortex) brains were assayed for thiaminpyrophosphatase (TPPase) activity by isoelectric focusing (IEF). Glial and neuronal fractions yielded about 0.16 and 0.03 g/g of wet tissue respectively. Purity was estimated to be 85-90%. The isolated fractions were homogenized in the presence of 1% Triton X-100, and IEF was carried out on thin layer polyacrylamide gel in a pH range of 3.5-9.5, using Ampholine PAG plates. TPPase activity of the protein bands was assayed by laser-densitometry, after incubation with thiaminpyrophosphate in an appropriate buffer and PbS staining. IEF analysis showed that TPPase activity was present almost exclusively in the neuronal fraction (at levels 16-fold higher than those found in the glial fraction). After IEF, TPPase activity was present in 10 distinct protein bands with different isoelectric points. These preliminary data suggest that TPPase may be involved in the neuronal activity of rat and human brain.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11571/108729
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