Studies on the submicrosomal fractions of bovine oligodendroglia: lipid composition and glycolipid biosynthesis.

Oligodendroglia were isolated from bovine brain, and a "crude" microsomal fraction obtained from cell homogenates was subfractionated into myelin (MP), plasma membranes (PM), Golgi (GF), smooth (SER) and rough (RER) endoplasmic membranes using discontinuous-sucrose gradient centrifugation. The submicrosomal fractions were characterized by ultrastructural examination and analysis of the specific organelle markers. The myelin and plasma membrane rich fractions contained characteristically the highest amounts of the lipid with lower mole percentages of total phospholipids and phosphatidylcholine, and higher concentrations of phosphatidylethanolamine (+ plasmalogens), cholesterol and galactolipids. Considerable amounts of the typical myelin galactolipids (galacto-cerebrosides, sulfatides and monogalactosyl diglycerides) were also found in the Golgi fraction (GF). The GF fraction had the greatest enrichment of glycolipid-forming galactosyltransferases, and the distribution of these enzymes correlated well with that of the Golgi marker enzymes. The results give evidence that intracellular Golgi apparatus of oligodendroglia is rich in the myelin-specific lipids, and suggest its involvement in the synthesis and processing of myelin lipids.

Polyphosphoinositide mono- and diphosphoesterases of three subfractions of rat brain myelin.

Phosphomonoesterase and diesterase that cleave phosphatidylinositol-4-phosphate (diphosphoinositide, DPI) and phosphatidylinositol-4,5-bisphosphate (triphosphoinositide, TPI) were detected in three subfractions of purified rat brain myelin, and some properties of the enzymes were studied. Monoesterase activity was stimulated by KCl, maximally at a concentration of 25 mM, and inhibited at KCl concentrations above 50 mM. Addition of boiled pH 5 supernatant of rat brain homogenate doubled the enzymic activity; EDTA was inhibitory. The specific activities were nearly equal in the "low density", "medium density", and "heavy density" myelin fractions but about 30% lower than in whole brain homogenate. The monophosphatase could be solubilized by extraction with 0.2% Triton X-100. The phosphodiesterase activity was inhibited by EDTA and EGTA and not stimulated by KCl or pH 5 supernatant. Specific activities were nearly equal in whole brain and myelin but were by about 60 percent elevated in the "heavy density" over the "low density" myelin fractions. These results show that hydrolases operative in the fast turnover of the inositide phosphate groups are distributed over the entire myelin structure.

Rapid incorporation in vivo of intracerebrally injected 32Pi into polyphosphoinositides of three subfractions of rat brain myelin.

At intervals ranging from 1 to 10 min after injection of 32Pi into rat brain, myelin was prepared and separated into three subfractions: heavy, medium, and light. The radioactivity of total phospholipids and polyphosphoinositides (PPI) was then determined. There was rapid incorporation of 32Pi into PPI, which contained 50-70% of the radioactivity among total brain lipids and more than 70% among myelin lipids. The myelin fraction had incorporated 32Pi into total recovered PPI in the order of medium greater than heavy greater than light fraction; however, the order of relative specific radioactivities was heavy greater than light greater than medium. Labeling of the PPI precursors, phosphatidic acid (PA) and phosphatidylinositol (PI), was considerably lower in the purified myelin than in total brain. The di- (DPI) and triphosphoinositides (TPI) in heavy myelin exchanged 32Pi rates 2 to 3 times faster than those in medium and light myelin. DPI of all subfractions of myelin exchanged much faster than TPI. The results show that the most active phosphate turnover of myelin PPI occurs in the heavy myelin fraction (probably largely consisting of myelin appurtenant regions). However, medium and light myelin (most probably representing the closely packed layers of myelin sheaths) also showed rapid turnover of PPI.

Distribution of phosphoinositides among subfractions of rat brain myelin.

Rat brain myelin was separated into three subfractions, heavy, medium, and light, and the concentrations of phosphatidic acids (PA), phosphatidylinositol (PI), di- (DPI), and triphosphoinositide (TPI) in these fractions were determined. PI was evenly distributed among the fractions, and PA, DPI, and TPI occurred in highest concentrations in the "light" myelin. This result indicates that these fast metabolizing lipids play an important role in the tightly packed central lamellae of the myelin sheath.

Isolation and characterization of an enriched Golgi fraction from rat brain.

A fraction rich in membranes of the Golgi apparatus was isolated from rat brain by discontinuous density gradient centrifugation. The fraction sedimented at the characteristic Golgi density of 1.11--1.15 (g/cm3, 5 degrees C) and had specific activities of Golgi-marker enzymes (N-acetyllactosaminyl synthetase, glycoprotein (Fetuin) galactosyltransferase, thiamine pyrophosphatase), 6--7 times over those of the original homogenates. The recovery of the enzyme activities in this fraction ranged from 17 to 31 %. The incorporation [3H]fucose into glycoproteins was 3-fold higher than in homogenate. Recovery and relative specific activities of marker enzymes for other subcellular organelles were low. Electron microscopic analysis of the fraction revealed in the presence of Golgi structures, namely, large sacs or plates with attached tubules and "blebbing" of the tubules into the vesicles.