Enzyme activity and composition of myelin and subcellular fractions in the developing rat brain.
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ABSTRACT:
SUBMITTER: Banik NL
PROVIDER: S-EPMC1185247 | biostudies-other | 1969 Dec
REPOSITORIES: biostudies-other
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Project description:1. The lipids of whole brain and subcellular fractions of the rat were analysed during development. 2. The deposition of cholesterol occurred in two phases, one related to increasing wet weight of the brain and the second to myelination. Cerebroside accumulation was related only to myelination. 3. The composition of myelin isolated from 12-day-old rat brain was different in some respects from that of the adult. In the former there was an increase of phospholipid in relation to cholesterol and a marked deficiency in cerebroside. 4. It is suggested that early myelin is extruded glial plasma membrane, which only later becomes mature myelin.
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Project description:Protein- and lipid-bound sialic acid was assayed in myelin fractions isolated by four different methods from rat brain homogenates. The extent to which non-myelin cellular membranes contaminate these fractions was assessed by electron microscopy and marker-enzyme assays. Small amounts of sialic acid found in the least contaminated myelin fractions are considered to be constituents of axonal and satellite cell plasma membranes known to be present. The data are discussed with reference to the ultrastructural appearance of myelin.
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Project description: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.
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Project description:This study investigated the enantioselective metabolism of benoxacor, an ingredient of herbicide formulations, in microsomes or cytosol prepared from female or male rat livers. Benoxacor was incubated for ≤30 min with microsomes or cytosol, and its enantioselective depletion was measured using gas chromatographic methods. Benoxacor was depleted in incubations with active microsomes in the presence and absence of NADPH, suggesting its metabolism by hepatic cytochrome P450 enzymes (CYPs) and microsomal carboxylesterases (CESs). Benoxacor was depleted in cytosolic incubations in the presence of glutathione, consistent with its metabolism by glutathione S-transferases (GSTs). The depletion of benoxacor was faster in incubations with cytosol from male than female rats, whereas no statistically significant sex differences were observed in microsomal incubations. The consumption of benoxacor was inhibited by the CYP inhibitor 1-aminobenzotriazole, the CES inhibitor benzil, and the GST inhibitor ethacrynic acid. Estimates of the intrinsic clearance of benoxacor suggest that CYPs are the primary metabolic enzyme responsible for benoxacor metabolism in rats. Microsomal incubations showed an enrichment of the first eluting benoxacor enantiomer (E1-benoxacor). A greater enrichment occurred in incubations with microsomes from female (EF = 0.67 ± 0.01) than male rats (EF = 0.60 ± 0.01). Cytosolic incubations from female rats resulted in enrichment of E1-benoxacor (EF = 0.54 ± 0.01), while cytosolic incubations from male rats displayed enrichment of the second eluting enantiomer (E2-benoxacor; EF = 0.43 ± 0.01). Sex-dependent differences in the metabolism of benoxacor in rats could significantly impact ecological risks and mammalian toxicity. Moreover, changes in the enantiomeric enrichment of benoxacor may be a powerful tool for environmental fate and transport studies.
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Project description:1. Protein metabolism of myelin and other subcellular components from developing rat brain was studied for periods from 5h to 210 days after intraperitoneal injection of [(3)H]lysine and [(14)C]glucose. 2. Half-lives for total brain proteins (t(0.5)) were 27 days after [(3)H]lysine and 4 days after [(14)C]glucose injection. 3. Factors accounting for the difference in the turnover rates obtained with different precursors, and the problem of reutilization of the label were investigated. 4. The catabolism of purified myelin proteins was studied and the half-lives of individual myelin proteins were calculated. 5. Myelin basic proteins turned over at two different rates. Half-life of the fast component of myelin basic proteins was 19-22 days and the slow component exhibited a high degree of metabolic stability. 6. Proteolipid protein underwent slow turnover. High-molecular-weight Wolfgram (1966) proteins underwent (relatively) fast metabolism (t(0.5) of 17-22 days).
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Project description:1. The effect of inhibition of cholesterol synthesis by a hypocholesterolaemic drug (AY-9944) was studied in rat brain during development. 2. At 2 weeks after administration of AY-9944 to young rats 7-dehydrocholesterol accounted for half the total sterol of myelin and other subcellular components. 3. At 4 weeks after injection of the drug 7-dehydrocholesterol had disappeared whereas the cholesterol content of myelin had increased by an equivalent amount. Our studies show that purified myelin has low 7-dehydrocholesterol reductase activity and suggest that 7-dehydrocholesterol is largely converted into cholesterol outside the myelin sheath. 4. Resultant cholesterol may be re-incorporated into myelin by an exchange process. 5. The metabolism of sterols in developing brain is discussed.
| S-EPMC1176844 | biostudies-other
Project description:1. Inorganic [(32)P]phosphate, [U-(14)C]glycerol and [2-(14)C]ethanolamine were injected into the lateral ventricles in the brains of adult rats, and the labelling of individual phospholipids was followed over 2-4 months in both a microsomal and a highly purified myelin fraction. 2. All the phospholipids in myelin became appreciably labelled, although initially the specific radioactivities of the microsomal phospholipids were somewhat higher. Eventually the specific radioactivities in microsomal and myelin phospholipids fell rapidly at a rate corresponding to the decline of radioactivity in the acid-soluble pools. 3. Equivalent experiments carried out in developing rats with [(32)P]phosphate administered at the start of myelination showed some persistence of phospholipid labelling in the myelin, but this could partly be attributed to the greater retention of (32)P in the acid-soluble phosphorus pool and recycling. 4. It is concluded that a substantial part of the phospholipid molecules in adult myelin membranes is readily exchangeable, although a small pool of slowly exchangeable material also exists. 5. A slow incorporation into or loss of labelled precursor from myelin phospholipids does not necessarily give a good indication of the rate of renewal of the molecules in the membrane. As presumably such labelled molecules originate by exchange with those in another membrane site (not necessarily where synthesis occurs) it is only possible to calculate the turnover rate in the myelin membrane if the behaviour of the specific radioactivity with time of the phospholipid molecules in the immediate precursor pool is known.
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Project description:The concentration of taurine and the activities of cysteinesulphinate decarboxylase and glutamate decarboxylase have been measured in rat brain. During development, taurine exhibited a decrease in concentration unrelated to the activity of cysteinesulphinate decarboxylase which increased during the same period. The distribution of taurine in subcellular fractions of adult and 7-day-old rat brain was typical of most amino acids, whereas half of the cysteinesulphinate decarboxylase activity was found in the nerve-ending cytoplasm. In anatomical distribution, taurine displayed great regional heterogeneity but both cysteinesulphinate decarboxylase and glutamate decarboxylase were more evenly distributed. Hypertaurinaemia was shown to have no effect on the entry of glycine into the brain or on its utilization in protein synthesis.
| S-EPMC1176845 | biostudies-other
Project description:Protein-bound N-acetylneuraminic acid and hexosamine, including the sialomucopolysaccharides, occur mainly in the least dense particles sedimented in the microsomal fraction from rat whole brain. Particles rich in protein-bound N-acetylneuraminic acid and hexosamine are also found in the subcellular fraction separated as a layer between 0.8m- and 1.2m-sucrose after centrifuging the crude mitochondrial preparation in a density gradient. This distribution is similar to that of the gangliosides and suggests an association of all of these substances in the same subcellular structures. It is postulated that the sialomucopolysaccharides, as well as the gangliosides, are components of cell membranes. Evidence is presented that indicates that there are quantitative differences between distribution of the gangliosides on the one hand, and protein-bound N-acetylneuraminic acid and hexosamine on the other. The ratio of protein-bound N-acetylneuraminic acid (and hexosamine) to gangliosidic N-acetylneuraminic acid (and hexosamine) present in individual subcellular fractions obtained by density-gradient centrifugation tends to increase with increasing particle density. Exposure of the crude mitochondrial fraction to osmotic ;shock' before density-gradient centrifugation causes a shift of the protein-bound N-acetylneuraminic acid and gangliosides to the less dense fractions. In some experiments, a selective shift of the protein-bound N-acetylneuraminic acid was observed.
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Project description:Elongation of fatty acids by microsomal fractions obtained from rat brain was measured by the incorporation of [2-14C]malonyl-CoA into fatty in the presence of palmitoyl-CoA or stearoyl-CoA. 2. Soluble and microsomal fractions were prepared from 21-day-old rats; density gradient centrifugation demonstrated that the stearoyl-CoA elongation system was localized in the microsomal fraction whereas fatty acid biosynthesis de novo from acetyl-CoA occurred in the soluble fraction. The residual activity de novo in the microsomal fraction was attributed to minor contamination by the soluble fraction. 3. The optimum concentration of [2-14C]malonyl-CoA for elongation of fatty acids was 25 mum for palmitoyl-CoA or stearoyl-CoA, and the corresponding optimum concentrations for the two primer acyl-CoA esters were 8.0 and 7.2 muM respectively. 4. Nadph was the preferred cofactor for fatty acid formation from palmitoyl-CoA or stearoyl-CoA, although NADH could partially replace it. 5. The stearoyl-CoA elongation system required a potassium phosphate buffer concentration of 0.075M for maximum activity; CoA (1 MUM) inhibited this elongation system by approx. 30%. 6. The fatty acids formed from malonyl-CoA and palmitoyl-CoA had a predominant chain length of C18 whereas stearoyl-CoA elongation resulted in an even distribution of fatty acids with chain lengths of C20, C22 and C24. 7. The products of stearoyl-CoA elongation were identified as primarily unesterified fatty acids. 8. The developmental pattern of fatty acid biosynthesis by rat brain microsomal preparations was studied and both the palmitoyl-CoA and stearoyl-CoA elongation systems showed large increases in activity between days 10 and 18 after birth.
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