Morphological and Biochemical Correlates of Cerebral Microsomes

Abstract

Microsomal fractions, both homogeneous in appearance and functionally operative, were isolated from a homogenate of rat cerebral cortex by fractionation in water. The preparations thus obtained contain the membranous elements of the endoplasmic reticulum, synaptic vesicles, and ribosomes. Esterase, ATPase, and glutamine synthetase were found to be present and fully functional in the microsomal fractions isolated in water. The contamination of the water-isolated microsomal fractions by mitochondria and lysosomes was found to be considerably lower than in microsomal pellets isolated in sucrose. The contamination by nerve ending particles, as judged by electron microscopy and by the levels of soluble lactic dehydrogenase entrapped in the cytoplasm of the particles, was also low. Most of the contamination by mitochondria and nerve ending particles could be removed by treatment of the microsomal pellet with 150 mM NaC1. Resistant to elution by this treatment is the lysosomal contamination as well as microsomal esterase and ATPase. Glutamine synthetase, on the other hand, was almost totally solubilized. Microsomal preparations isolated in water are also shown to contain amounts of protein, RNA, phospholipid, and ganglioside comparable to those found in microsomal preparations isolated in sucrose.The microsomal fraction isolated from mammalian tissues by high speed centrifugation has not been unequivocally characterized, for it has often been found to consist of disrupted fragments of intracellular materials, including the cellular envelope, that connot readily be compared with, or identified with, intact structures (1, 2). This is particularly true for fractions isolated from brain, owing to the complex and heterogeneous organization of this organ. In contrast to brain is the liver, for instance, from which fractions can be isolated which are much more homogeneous as a eonsequence of the less complex organization of this organ. Palade and Siekevitz (3) have demonstrated that in rat liver the microsomal fraction consists preponderantly of vesicular and tubular fragments of the intracellular endoplasmic reticulum and of ribosomal granules. Hanzon and Toschi (4) described a similar composition for the microsomal fraction of brain tissue. However, more recent studies by de Robertis et al. (5) and by Whittaker (6) have revealed that the microsomal fraction of brain, as usually isolated in sucrose media, consists of a rather heterogeneous popula-69

MORPHOLOGICAL AND BIOCHEMICAL CORRELATES OF CEREBRAL MICROSOMES–II. *†

“…The differential susceptibility of glutamine synthetase to solubilization by 15 mMand 150 mM-NaC1 was noted previously (DE BALBIAN VERSTER et al, 1965). In the present studies, where a wide range of Na+ concentrations was tested, a polyphasic effect of this cation on the solubilization of the enzyme was noted, and the interesting observation made that Na+, when used between 10 and 60 mM, actually facilitates the retention of the enzyme in particulate form (Fig.…”

“…These structures were not seen in the other subfractions in equally abundant numbers. That the presence of these structures does not reflect disaggregation or dissociation of native phospholipid-protein bonds which may have occurred as a result of the suspension of Fraction P, in 15 mM-NaC1 (to yield Fraction SP) is supported by the fact that identical structures could be readily seen in electron micrographs of the unfractionated pellet (Fraction P,) (DE BALBIAN VERSTER et al, 1965).…”

“…Fractionation of rat cerebral cortex in distilled water. The preparation of the cerbral cortex homogenate in distilled water and the centrifugation procedure used for the isolation of the microsomal fraction ('slow' procedure of DE BALBIAN VERSTER et uf., 1965) were described in detail in previous publications (SBLLINGER and DE BALBIAN VERSTER, 1962a;DE BALBIAN VERSTER et al, 1965). Briefly, male rats (150-250 g, Holtzman Co., Madison, Wisconsin) were decapitated, and the brains rapidly excised.…”

Relative activities of glutamine synthetase and glutaminase in mammalian synaptosomes

“…Glutamine synthetase (EC 6.3.1.2) is predicted by this hypothesis to be mainly glial in localisation, and glutaminase (EC 3.5.1.2) mainly neuronal (VAN DEN BERG & GARFINKEL, 1971;BALAZS et al, 1973). Histochemical studies of many brain areas and of retina strongly indicate a glial site for glutamine synthetase (BELL & NORENBURG, 1977;REIPE & NORENBURG, 1977) and previous subcellular fractionation studies (SELLINGER & DE BALBIAN VERSTER, 1962;DE BALBIAN VERSTER et al, 1965;SALGANICOFF & DE ROBERTIS, 1965) have indicated a microsomal or soluble fraction localisation and enrichment, which would be consistent with a glial localisation. The extent to which this enzyme is present in synaptosome fractions is obviously an important consideration in view of the concentration of glutaminase present.…”