Joseph Bernsohn

SUBCELLULAR LOCALIZATION OF RAT BRAIN ESTERASES

The localization and properties of soluble and bound esterases of subcellular fractions of rat brain have been investigated. Bound esterases were extracted with 1% Triton X-100 and separated by starch gel electrophoresis. By these means a molecular population of isoenzymes was demonstrable that was quantitatively different from the isoenzyme population of the watersoluble esterase activity. The highest specific activity for α-naphthyl acetate hydrolysis was contained in the microsomal fraction and could be extracted by Triton. In contradistinction to whole brain and to other subcellular fractions, microsomes contained a molecular population of esterase isozymes which was qualitatively distinct from that of water extracts in that the fast moving A-type esterases were absent. In addition, there was present a heavy concentration of slow movinig B-esterase. Acetylcholinesterase could also be extracted from this fraction by Triton, migrated with B-esterase and actively hydrolyzed αnaphthyl acetate. Combined electron microscopic and quantitative chemical analysis of the subcellular fractions suggested that some bound nonspecific esterase may be localized in sub-synaptic membranes. The pI50 values for E 600 of the soluble and insoluble, Triton X-100-extracted and Triton X-100-insoluble esterases are, respectively, 5.3, 7.5, 7.5 and 7.4. It is noted that these results may be determined in part by the participation of acetylcholinesterase in hydrolysis of the substrate. Mitochondria are virtually devoid of esteratic activity. C-type esterase (CMB-activated, E 600-resistant) occurred in both the bound and soluble state. A-, B- and C-type esterases exist in both bound and soluble forms and their chemical properties appear to be independent of their site of localization. Histochemical studies indicate that the B-type esterase is localized predominantly to cytoplasm of neurons and to neuropil. A-esterase is localized to droplets (presumed lysosomes) of neurons and other cell types, e.g., pericytes. The histochemical localization of C-esterase could not be determined.

Essential fatty acid deficiency and CNS myelin: Biochemical and morphological observations

Essential fatty acid (EFA) deficiency was induced by feeding pregnant rats a fat-free diet 10--12 days after impregnation and maintaining the offspring on this diet until 120 days of age. EFA-deficiency rats demonstrated marked alterations in the fatty acid composition of ethanolamine phosphoglycerides (EPGs) from myelin subfractions. A decrease in the fatty acids of the linoleic (n-6) and linolenic (n-3) families was accompanied by an increase in the non-essential fatty acids of the oleic (n-9) family. These alterations decreased the unsaturation index of heavy myelin by 23% and that of light myelin by 10%. The EPG fatty acid composition of heavy myelin from control animals contained a higher percentage of polyunsaturated fatty acids than the light myelin which contained more monounsaturated fatty acids. These differences may be a reflection of distinct anatomical locations or functional properties of the subfractions. The differences between light and heavy myelin EPG fatty acids were not maintained during EFA deficiency. Morphologically, 1 mum thick sections revealed vacuoles within the optic nerve of EFA-deficient rats. Ultrastructurally these vacuoles were identified as fibers undergoing Wallerian degeneration and fibers demonstrating intramyelinic splitting. No qualitative changes were found in oligodendrocytes, astrocytes or vascular elements within EFA-deficient optic nerve. EFA deficiency did not alter the diameter of fibers within the optic nerve. These results show that although there is no apparent decrease in the degree of myelination within the optic nerve, morphological changes do occur in fibers of EFA-deficient optic nerve concomitantly with alterations in the EPG fatty acids of myelin subfractions.

Thin-layer chromatographic separation of adenine nucleotides from erythrocytes☆

Abstract The separation by undimensional thin-layer chromatography, for the purpose of quantitative determination, of high-energy phosphate compounds and some of the glycolytic intermediates in human erythrocytes is described. These data are compared to those obtained after separation of these compounds by column chromatography. The mean erythrocyte content of ATP (20 min incubation) after TLC separation was 101.8 ± 11.5 μmoles/100 ml erythrocytes, and after column separation, 103.3 ± 6.0 μmoles/100 ml erythrocytes. After 120 min incubation values were 111.9 ± 11.4 μmoles/100 ml erythrocytes and 113.0 ± 7.0 μmoles/100 ml erythrocytes, respectively. Within each method the change was significant, but there was no correlation between the two methods for the per cent change in ATP. The primary advantages of the TLC method are the ease with which 2,3-DPG and ATP are separated and the considerable reduction of time required for the separation compared to column chromatography. It was concluded that TLC is a useful screening procedure.

Phospholipid fractions in multiple sclerosis and normal serum.

Summary Determinations on the serum of normal and multiple sclerosis serum for ceph-alin? lecithin and sphingomyelin, showed normal values for these fractions of 9.4%, 65.0′% and 19.7% of the total phospholipids. The multiple sclerosis group exhibited a similar distribution of these components, though the total phospholipids tended to be lower than in the control group.

Incorporation of [1-14C]linoleic acid into central nervous system of the adult cat after intracisternal administration

Summary The incorporation of [1-14C]linoleic acid into the CNS of the adult cat was studied after intracisternal administration of the fatty acid. About 23% of the isotope was found in the CNS after 24 h compared to the amount incorporated 14 days after the injection. About 1% of the isotope injected was taken up by the CNS. Most of the radioactivity was localized in the phosphoglyceride fraction with both choline glycerophosphatides and ethanolamine glycerophosphatides equally labeled. Subcellular fractionation of the cervical cord showed that the myelin and the ‘enriched’ synaptic membrane fraction incorporated the major activity in that tissue. Fatty acid analysis of the phosphoglyceride fraction indicated that about 35% of the radioactivity was in linoleic acid per se and that the adult central nervous tissue can apparently carry out de novo fatty acid synthesis, acyl transferase, elongation, saturation, desaturation and fatty acid synthesis to a limited extent under the experimental conditions. The implication of these findings to myelin metabolism are discussed. A major phosphoglyceride fatty acid in adult cat CNS was found to be 20: 1.

Electrophoretic fractionation of serum protein in multiple sclerosis.

Summary 1. Electrophoretic fractionation of serum proteins in 27 cases of multiple sclerosis showed an increase in the a2-globulin moiety with a concomitant reduction in the albumin component. This resulted in a lowered A/G ratio in the pathological cases. These differences were statistically significant. 2. In 15 out of the 27 cases an electrophoretic heterogeneity in the a2-globulin fraction was observed which was characterized by a “double” peak.

Nonspecific esterases of human peripheral nerve and centrum ovale. A comparison and observations on esteratic activity of myelin fractions.

Nonspecific esterases (NsE) of human PNS and CNS occur in free (hydrosoluble) and bound (triton X-100 extractible) forms. Free and bound NsE were studied in whole tissue and in myelin fractions by starch gel electrophoresis (zymograms) and chemical assay, using as substrates alpha-naphthyl acetate, (NA), alpha-naphthyl propionate (NP) and alpha-naphthyl butyrate (NB). By chemical assay whole homogenates of CNS and PNS differ thusly: (1) NP/NA and NB/NA hydrolysis ratios are greater in PNS; (2) NsE activity, whether expressed per gram wet weight or specific activity, is several-fold greater in CNS; (3) 76–86% of NsE activity of PNS occurs in free form while CNS free esterase constitutes less than 20% of the total. NA zymograms of free NsE of PNS and CNS are alike, and differ from other tissues and contain predominantly E-600-resistant A-type esterase, with a minor C-type component. Certain molecular forms of NsE preferentially hydrolyzing NP and NB (and the valerate ester) are characteristic of cerebral and cerebellar white matter and are absent from PNS, liver, kidney, skeletal muscle and leptomeninges. CNS myelin fractions, prepared as described in this publication, contain NsE of predominately B-type (E-600-sensitive enzyme). Respectively, 6.7, 8.0 and 9.1% of the NA-, NP- and NB-hydrolyzing capacity of centrum ovale are recovered in the myelin fraction. PNS myelin lacks NsE. These results, though suggestive, do not prove an intrinsic association of NsE with human CNS myelin. Autolysis, among other factors, may cause displacement of enzyme from in vivo sites. Nonetheless results reported here and in earlier papers support a role for NsE in myelin metabolism, especially of those CNS NsE that cleave preferentially 3–5 carbon esters.

Differential trypsin effect upon (1-14C) acetate incorporation into choline and ethanolamine glycerophosphatides of rat brain and liver

Preincubation of rat brain and liver slices in a medium (5% glucose, 5% fructose, 1% albumin, 1% trypsin, 10 mM phosphate buffer pH 6.0) used to pretreat brain tissue for the separation of cell types was found to uncouple the incorporation of (1-14C) acetate into ethanolamine phosphoglycerides from that of the choline phosphoglycerides. Incorporation into ethanolamine phosphoglycerides was stimulated in both brain (330%) and liver (780%) slices, while the incorporation of (1-14C) acetate into choline phosphoglycerides was reduced for both brain (71%) and liver (63%) slices, compared to control values from nonpreincubated material. With (1-14C) linolenic acid as a precursor, no significant differences were found in incorporation into ethanolamine phosphoglycerides and choline phosphoglycerides.