Levels and syntheses of cerebrosides and sulfatides in subcellular fractions of jimpy mutants

During the period of brain development, the levels of nonhydroxy- and hydroxy-cerebrosides in the cytosol from brains of jimpy mutants were determined by high-performance liquid chromatography and compared to those in the rest of the particulates from the same brains as well as those in the littermate controls. The concentrations of cerebrosides in jimpy brain preparations were much lower than in controls at all ages. In another experiment, [U-¹⁴C]glucose was injected intraperitoneally into jimpy mutants and their littermate controls. The amounts of radioactivity incorporated into cerebrosides and sulfatides in brain cytosol, the microsome-rich fraction, and the rest of the heavier particles were determined. Although the total radioactivity incorporated into these lipids was much lower in jimpy, the specific activities were 2–3 times the control value in all subcellular fractions.     

Enzyme activity and composition of myelin and subcellular fractions in the developing rat brain

1. Subcellular fractions and myelin were isolated from developing and adult rat brain. 2. Measurements of chemical composition and enzyme activities indicate the presence of a second myelin-like fraction mainly in the brain of developing rats. 3. This membrane fraction has a different lipid composition from myelin, but resembles myelin in its content of phosphohydrolase and aminopeptidase activity. 4. It is suggested that the second myelin-like fraction may be a submicrosomal contaminant or it may be derived from oligodendroglial plasma membrane during myelinogenesis.     

Metabolism of cerebrosides and sulfatides in the nervous system ofXenopus tadpole during metamorphosis

Xenopus laevis tadpoles undergoing metamorphosis were used to study the turnover of cerebrosides and sulfatides in the nervous system of the frog. Tadpoles at the beginning of metamorphosis were treated by intraperitoneal injection with [U-¹⁴C]glucose and radioactivity incorporated into galactosphingolipids of brain and tail was measured after various times. The specific activity of brain cerebrosides increased rapidly for the first 24 hr after injection, reached a plateau after 48hr, and then declined 40% by 7 days. The specific activity of sulfatides changed somewhat more slowly. Hydroxy fatty acid-containing galactosphingolipids had nearly twice the specific activity compared with their nonhydroxy counterparts in brain. Despite the complete regression of tail nerve cord, metabolism of glycosphingolipids in this tissue also indicated active synthesis as well as degradation during this period. The specific activities of these lipids were similar and all reached a peak 24 hr after injection. Examination of the components of these galactosphingolipids disclosed that only a small fraction (7–25%) of the radioactivity was in the galactose moiety in both brain and tail. The ratios of the radioactivity in fatty acid to that in the sphingoid base were much higher for hydroxycerebroside and hydroxysulfatide than for the nonhydroxy isomers.Abbreviations used: Cerebroside is N-acyl, 1-0--galactosyl derivative of sphingoid base (D-erythro-2-amino-alkyl-1,3-diol) Sulfatide is the galactose-3-sulfated derivative of cerebroside. The prefixes hydroxy and nonhydroxy indicate cerebroside or sulfatide containing -hydroxy and nonhydroxy fatty acids, respectively     

Metabolism of progesterone in subcellular fractions of rat uterus

The metabolism of progesterone and 5α-pregnane-3,20-dione was studied in subcellular fractions of uterus from untreated and estradiol-17β treated immature rats. The reduction of progesterone to 5α-pregnane-3, 20-dione took place in all the particulate fractions of the uterus. The nuclear 5α-reductase accounted for the greatest fraction of enzymatic activity and was stimulated by estradiol treatment in vivo. The 5α-reductase activity in the mitochondrial and microsomal fractions was not increased after estradiol treatment. The reduction of 5α-pregnane-3,20-dione to 3α-hydroxy-5α-pregnan-20-one occurred mainly in the soluble fraction and was only slightly stimulated by estradiol. It proceeded much more rapidly than the reduction of progesterone to pregnanedione. Progesterone was also reduced to 20α-hydroxy-4-pregnen-3-one by a soluble enzyme whose activity was increased after estradiol-17β treatment.     

Lysophospholipase activity in rat brain subcellular fractions

Lysophospholipase activity in brain subcellular fractions was measured by the release of myristic acid from 1-myristoylglycerophosphocholine or through the formation of [³²P]glycerophosphocholine from [³²P]lysophosphatidylcholine. Although the lysophospholipase activity was highest in microsomes, considerable enzyme activity was also found in other subcellular membrane fractions. The pH optimum for the microsomal enzyme was around 7, whereas the synaptosomes and non-synaptic plasma membranes exhibited a pH maximum around 8. Although the enzyme did not require divalent cations for activity, divalent cations (1 mM) such as Hg²⁺, Cu²⁺, and Zn²⁺ inhibited potently the enzyme activity. Enzyme activity was also partially inhibited by both saturated and polyunsaturated fatty acids (25–200 M), and the inhibition seemed to be greater in the membrane than in the cytosolic fractions. Ionic detergents such as deoxycholate and taurocholate inhibited the lysophospholipase. On the other hand, the effect of Triton X-100 was biphasic, i.e., stimulation at concentrations below 100 g/mg protein and inhibition at higher concentrations. Addition of cholesterol (50–250 g/ml), but not cholesteryl esters, also potently inhibited enzyme activity. The presence of active lysophospholipase(s) in brain is probably an important mechanism for preventing unnecessary accumulation of lysophospholipids which may exert a deleterious effect on the membranes because, of their detergent properties.     

Synthesis and turnover of cerebrosides and phosphatidylserine of myelin and microsomal fractions of adult and developing rat brain.

The synthesis and turnover of cerebrosides and phospholipids was followed in microsomal and myelin fractions of developing and adult rat brains after an intracerebral injection of [U-14C]serine. The kinetics of incorporation of radioactivity into microsomal and myelin cerebrosides indicate the possibility of a precursor-product relationship between cerebrosides of these membranes. The specific radioactivity of myelin cerebrosides was corrected for the deposition of newly formed cerebrosides in myelin. Multiphasic curves were obtained for the decline in specific radioactivity of myelin and microsomal cerebrosides, suggesting different cerebroside pools in these membranes. The half-life of the fast turning-over pool of cerebrosides of myelin was 7 and 22 days for the developing and adult rat brain respectively. The half-life of the slowly turning-over pool of myelin cerebrosides was about 145 days for both groups of animals. The half-life of the rapidly turning-over microsomal cerebrosides was calculated to be 20 and 40 h for the developing and adult animals respectively. The half-life of the intermediate and slowly turning-over microsomal cerebrosides was 11 and 60 days respectively, for both groups of animals. The amount of incorporation of radioactivity into microsomal cerebrosides from L-serine was greatly decreased in the adult animals, and greater amounts of the precursor were directed towards the synthesis of phosphatidylserine. In the developing animals, considerable amounts of cerebrosides were synthesized from L-serine, besides phosphatidylserine. The time-course of incorporation indicated that a precursor-product relationship exists between microsomal and myelin phosphatidylserine. The half-life of microsomal phosphatidylserine was calculated to be about 8 h for the fast turning-over pool in both groups of animals.     

Diacylglycerol kinase and lipase activities in rat brain subcellular fractions

Phosphatidic acid synthesis via diacylglycerol kinase and free fatty acid release via diacylglycerol lipase were investigated in rat brain subcellular fractions using membrane-bound [I-¹⁴C]arachidonoyl-diacylglycerol as substrate. Labeled diacylglycerol was generated by incubating brain membranes containing [I-¹⁴C]arachidonoyl-phosphatidylinositols in the presence of deoxycholate and Ca²⁺. Incubation of the prelabeled synaptosomes enriched in [1-¹⁴C]arachidonoyl-diacylglycerols or incubation of brain subcellular fractions with heat-treated prelabeled membranes resulted in the release of free fatty acids from the diacylglycerols. When incubations were carried out in the presence of ATP, MgCl₂ and NaF, both free fatty acid release and conversion of diacylglycerols to phosphatidic acids were observed. The conversion of diacylglycerols to phosphatidate or their hydrolysis to free fatty acids were linear with time for at least 15 min. In three brain subcellular fractions examined, diacylglycerol kinase activity indicated a pH maximum of 7.4. The free fatty acid release was enhanced slightly by Ca²⁺ (1 mM), but Ca²⁺ (0.5–4 mM) in the presence of Mg²⁺ (10 mM) was inhibitory to the diacylglycerol kinase reaction. Phosphatidate formation was also inhibited by an excessive amount of deoxycholate added to the incubation mixture. Among the brain subcellular fractions, diacylglycerol kinase was more active in synaptic vesicles and cytosol than in the microsomal fraction, whereas diacylglycerol lipase activity was higher in the cytosol fraction than in the membrane fractions. Upon washing the membranes by centrifugation, a substantial portion of the diacylglycerol kinase activity was removed after the first washing, whereas the diacylglycerol lipase activity remained essentially unchanged. The metabolic role of arachidonoyl-diacylglycerols in brain membranes in relation to the biosynthesis of phosphatidate and the release of arachidomic acid is discussed.     

Concentration and fatty acid composition of cerebrosides and sulfatides in mature and immature human brain

The fatty acid composition of cerebrosides and sulfatides from frontal lobe gray and white matter was determined for five fresh and four formalinized adult brains and for eight infants. Fatty acid patterns were unaffected by formalinization, but varied considerably from one another in the proportion of saturated to unsaturated fatty acids. The percentages of 24:0 and 24:1 increased with age. Cerebrosides obtained from areas such as the brainstem and cerebellum, where myelination was more advanced, tended to have a larger proportion of long-chain fatty acids than samples extracted from frontal or parietal lobe white matter. Hydroxy fatty acids showed an adult pattern in all instances in which amounts sufficient for accurate quantification could be isolated. Lipid hexose, cerebroside + sulfatide hexose, and methanoleluted hexose were measured in the brains of 12 infants ranging in age from a 4 month fetus to 2 yr. In the most immature, the majority of lipid hexose was in the form of glycolipids more polar than cerebrosides and sulfatides. These have tentatively been identified as hematosides and globosides. With maturation, cerebrosides and sulfatides increased progressively, but the amounts of the more polar glycolipids remained constant in relation to the total lipid content of tissue.     

Metabolism of triacylglycerol in developing rat brain

Metabolism of triacylglycerol (TAG) in developing brain has been examined. TAG is a relatively minor fraction of brain lipid in both suckling and adult rats and cannot be accounted for as entrapped blood. When glycerol tri[1-¹⁴C]oleate and [2-³H]glycerol trioleate were simultaneously injected intracerebrally into suckling rats, both labels appeared in diacylglycerol and the major phospholipids; acyl chain label was incorporated more extensively at early time points, with choline phosphoglycerides being most actively labeled. With [1-¹⁴C]fatty acids and [2-³H] glycerol administration, the specific activity of TAG was much greater than that of the more abundant phospholipids. Although direct acyl exchange between TAG and phospholipids was not demonstrated, relationships of TAG to selective mechanisms of phosphoglyceride synthesis were indicated.Abbreviations used TAG triacylglycerol - DAG diacylcerol - HPLC high performance liquid chromatography - CoA coenzyme A - BSA bovine serum albumin - TLC thin layer chromatography - DPM disintegrations per minute - ATP adenosine triphosphate - GLC gas liquid chromatography - PC choline, phosphoglyceride - PE ethanolamine phosphoglyceride - PS serine phosphoglyceride - PI inositol phosphoglyceride     

Activity of calpain in subcellular fractions of the rat brain

We studied the activity of a calcium-dependent proteinase, calpain, in subcellular fractions obtained from rat brain tissue. The rates of calpain-mediated hydrolysis of fluorescein isothiocyanate (FITC)-labeled substrates, casein and fodrin, were comparable; in the former case the rate was higher. This fact stipulated the choice of fluorescent-labeled casein as an adequate substrate. The greatest enzyme activity of calpain (87% of total) was found in the cytoplasmic fraction. At the same time, quite detectable enzyme activities were observed in the investigated membrane fractions obtained from rat brain tissue (coarse mitochondrial fraction, microsomes, and myelin). The highest specific calpain activity was registered in the cytoplasmic fraction. The enzyme activity was efficiently suppressed in the presence of calpain inhibitor I and increased after purification of the preparations from an endogenous calpain inhibitor, calpastatin.Neirofiziologiya/Neurophysiology, Vol. 36, No. 4, pp. 265–271, July–August, 2004.This revised version was published online in April 2005 with a corrected cover date.     

Distribution of protein kinase activities in subcellular fractions of rat brain

The subcellular distribution of histone and phosvitin kinase activities in brain has been studied and the ability of the various fractions to catalyse the phosphorylation of their endogenous proteins (intrinsic protein kinase activity) also examined. Synaptosome membrane fragments have little or no histone or phosvitin kinase activity but contain the highest concentration of cyclic AMP-stimulated intrinsic protein kinase activity. Homogenisation of the membrane fragments in Triton X-100 increased the histone kinase activity but on centrifugation it was all recovered in the supernatant, while the insoluble material contained all the intrinsic protein kinase activity. These results indicate that the intrinsic protein kinase activity of cerebral membrane fragments is due to the presence of a kinase enzyme which is specific to certain membrane proteins. The intrinsic protein kinase activity of synaptosome membrane fragments is a rather slow reaction which takes several minutes to saturate all the acceptor proteins.     

Distribution of physostigmine and metabolites in brain subcellular fractions of the rat

The distribution of 3H-physostigmine (Phy) has been studied in the rat brain subcellular fractions at various time intervals following i.v. injection. 3H-Phy or its metabolites rapidly accumulate into the cytoplasm of cells and penetrates the intracellular compartments. Kinetic studies of the subcellular distribution of radioactivity (RA) per gm of rat brain following i.v. injection of 3H-Phy show peak concentrations at 30 min in all subcellular fractions with the exception of mitochondria. In the mitochondrial fraction the RA levels continue to rise from 4682 +/- 875 DPM/gm at 5 min to 27474 +/- 2825 DPM/gm at 60 min (P less than .05). The cytosol contains the highest RA: 223341 +/- 21044 DPM/gm at 30 min which declined to 53475 +/- 3756 DPM/gm at 60 min. RA in synaptosome, microsomes and myelin increases from 5 to 30 min, and declines at 60 min. In vitro studies did not show a greater uptake of RA by the mitochondrial or synaptosomal fractions. The finding of relatively high concentrations of RA in the mitochondrial fraction at 60 min increases the likelihood that Phy or its metabolites could interfere with the physiological function of this organelle.     

Effect of undernutrition on DNA and RNA synthesis in subcellular fractions from different regions of the developing rat brain

The effect of undernutrition on the incorporation of [methyl-³H]thymidine into DNA and of 5-[³H]uridine into RNA of cerebral hemispheres, cerebellum, and brain stem was studied in vivo and in vitro in rats. The labeling of DNA from nuclei and mitochondria and of RNA from nuclei, mitochondria, microsomes, and soluble fractions, was also measured in vitro. The results demonstrate that nucleic acid synthesis is impaired and delayed during undernutrition. Specific effects were observed for the different brain regions and subcellular fractions: at 10 days nuclear and mitochondrial DNA and RNA synthesis was impaired, whereas at 30 days only the mitochondrial nucleic acid synthesis was affected.The delay of DNA and RNA labeling, caused by undernutrition, was most evident in the cerebellum, probably due to its intense cell proliferation during postnatal development. The specific sensitivity of mitochondria as compared to other subcellular fractions, may be due to the intense biogenesis and/or turnover of nucleic acids in brain mitochondria not only during postnatal development, but also in the adult animal.     

Protein synthesis rates in rat brain regions and subcellular fractions during aging

In vivo protein synthesis rates in various brain regions (cerebral cortex, cerebellum, hippocampus, hypothalamus, and striatum) of 4-, 12-, and 24-month-old rats were examined after injection of a flooding dose of labeled valine. The incorporation of labeled valine into proteins of mitochondrial, microsomal, and cytosolic fractions from cerebral cortex and cerebellum was also measured. At all ages examined, the incorporation rate was 0.5% per hour in cerebral cortex, cerebellum, hippocampus, and hypothalamus and 0.4% per hour in striatum. Of the subcellular fractions examined, the microsomal proteins were synthesized at the highest rate, followed by cytosolic and mitochondrial proteins. The results obtained indicate that the average synthesis rate of proteins in the various brain regions and subcellular fractions examined is fairly constant and is not significantly altered in the 4 to 24-month period of life of rats.A preliminary report of these results was previously presented at: WFN-ESN Joint Meeting on: Cerebral Metabolism in Aging and Neurological Disorders, Baden, August 28–31, 1986.