Subcellular distribution of thiamine pyrophosphatase in rat cerebral cortex

L. SEIJO; G. RODRÍGUEZ DE LORES ARNAIZ

BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES

ELSEVIER SCIENCE BV

Año: 1970 vol. 211 p. 595 - 598

0005-2736

In the present note the results of the study of the content of thiamine pyrophosphatase in several fractions of the rat cerebral cortex and particularly in those fractions commonly prepared for the isolation of synaptic structures are presented. Thiamine pyrophosphatase was determined by a microtechnique based on a method described for histochemical assay. The enzyme incubation was made at 37° for 6o min using buffer substrate and the homogenized fractions. The composition of the mixture in final concentration was 55 mM Tris maleate (pH 7.2), 6.6 mM MnCl2 and 3.6 mM thiamine pyrophosphate (Sigma Chemical Co. St. Louis, Mo.). After incubation, the reaction was stopped by the addition of 30 °o (w/v) trichloroacetic acid to achieve 7.5 % (w/v) and the orthophosphate was assayed. A tissue blank, prepared by adding triehloroacetic acid before the addition of the homogenate, was used in each case. The unit of activity, was defined as the amount of enzyme which catalyzed the liberation of 1 micromole of Pi per h. Proteins were determined by the method of LOWRY et al. a with bovine plasma albumin as standard. Groups of 4 6 Wistar rats (about IeO g body weight) were decapitated and the cerebral cortices were homogenized in 0.32 M sucrose adjusted to pH 7.0 with Tris base. When the primary fractions were isolated according to the methods previously described in this laboratory, most of the thiamine pyrophosphatase was associated with the fractions containing the isolated nerve endings.