Turnover of ribosomal RNA in the rat brain

Ribosomal RNA from rat brain has been partially purified by sodium dodecyl-sulphate treatment of microsomes, and density gradient centrifugation. The apparent half-life of ribosomal RNA after labelling with a pyrimidine precursor was 6 days. Classes of ribosomal RNA with longer half-lives could not be excluded. There was evidence for re-utilization of the labelled precursors.     

Demonstration of a new methylase in cell membranes of rat brain

In Rat brain membranes, two methylases occurred which converted phosphatidylethanolamine to phosphatidylcholine. We have pointed out another methylase which gave chloroform extractable methylated products; its Km was nearly 10 fold lower than that of the other methylases.     

Turnover rate of molecular species of sphingomyelin in rat brain

Turnover rate of individual molecular species of sphingomyelin of adult rat brain myelin and microsomal membranes was determined after an intracerebral injection of 100 Ci of [C³H₃]choline. Myelin and microsomal membrane sphingomyelins were isolated from the rest of the lipids. The individual molecular species of benzoylated sphingomyelin were separated and quantitated by reversed-phase high performance liquid chromatography. All individual major molecular species of microsomal and myelin sphingomyelin had maximum incorporation at 6 and 15 days, respectively, after the injection. The specific radioactivity of all the various molecular species of both myelin and microsomal sphingomyelin declined at a similar rate after reaching a maximum. There was no significant difference in the turnover rate of short chain (16:0, 18:0) and long chain (>22:0) fatty acid containing sphingomyelin. The average apparent turnover rate of myelin and microsomal sphingomyelin molecular species was about 14–16 days for the fast pool and about 45 days for the slow pool. It is concluded that individual molecular species of sphingomyelin of myelin and microsomal membranes turned over at a similar rate. Thus, turnover rate of sphingomyelin in myelin and microsomal membranes is not affected by the fatty acyl composition of the lipid.     

Molecular species of phosphatidylcholine and phosphatidylethanolamine in subcellular membranes from rat liver.

Four subfractions of phosphatidycholine and phosphyatidylethanolamine according to the degree of unsaturation of their fatty acids have been separated from lipid extracts of microsomes, and inner and outer mitochondrial membranes. The predominant species found in the three membranes contained one saturated and one unsaturated fatty acid. In microsomes completely saturated species of both phosphatidylcholine and phosphatideylethanolamine were practically nonexistent. In outer mitochondrial membranes species with two unsaturated fatty acids were absent. In the inner mitochondrial membranes, however, disaturated species and those with two unsaturated fatty acids were found.     

Turnover of ethanolamine phosphoglycerides in different brain areas of adult and aged rats

The turnover of ethanolamine glycerophospholipids (EGP) has been determined in six different cerebral areas of 4-month and 22-month-old rats, by injecting [³H]glycerol together with [¹⁴C]ethanolamine into the lateral ventricle of the brain. The areas examined behave quite differently in respect to their utilization of the most simple precursors of phosphoglyceride biosynthesis. The incorporation of both glycerol and ethanolamine is already complete as early as 2–4 hours and then reutilization begins, at least for the so called fast pools of phosphoglycerides. The different slopes of the specific activity of the two precursors in EGP suggest a high degree of variance among catabolic rates in the different brain regions. In aged rats the utilization of the water-soluble precursors of EGP synthesis decreases in all brain areas and these data suggest that aging may have a different effect on the catabolic activities as well as phospholipid biosynthesis.     

Turnover and uptake of double-labelled high-density lipoprotein sphingomyelin in the adult rat

Rat HDL containing [stearic acid-14C, (methyl-3H)choline]sphingomyelin was prepared by incubating labelled sphingomyelin liposomes with serum. HDL was then separated by ultracentrifugation and purified by gel-filtration chromatography. The maximum transfer was reached when 1.5 microliter sphingomyelin was incubated in the presence of 1 ml of serum at 37 degrees C for 1 h. When transfer was limited to a 5-7% increase in HDL mass, no significant change was observed in the HDL electrophoretic pattern, and rats could therefore be injected with this type of HDL under physiological conditions. Plasma radioactivity decay was followed for 24 h, and the recovery of both isotopes in 11 tissues was studied 24 h after the injection. The decay in plasma of both isotopes followed three exponential phases. During the first two phases, both isotopes disappeared with the same velocity (t1/2 = 12.8 and 98-105 min for the first and second phases, respectively). 10 h after injection, 3H had disappeared more slowly than 14C (t1/2 = 862 and 502 min for 3H and 14C, respectively) and 24 h after injection, only 1.5% of 14C and 2.5% of 3H remained in the plasma. This radioactivity was located mainly in HDL (80-85% for 3H and 14C), with a 3H/14C ratio close to that of injected sphingomyelin, and in VLDL, with the same isotopic ratio as that of liver lipids. Some 3H was associated with non-lipoprotein proteins. 17.5% of 3H and 23.4% of 14C were recovered in the liver, 1.6% of each isotope in erythrocytes, and 1.4% of 3H and 0.6% of 14C in kidney. Less than 1% of each isotope was recovered in each of the other tissues. Phosphatidylcholine was the lipid most labelled, and in several tissues sphingomyelin had a 3H/14C ratio close to that of injected sphingomyelin, showing an uptake without prior hydrolysis.     

A novel pathway for phosphatidylcholine catabolism in rat brain homogenates.

Rat brain homogenates incubated with exogenous [32-P] phosphatidylcholine liberated: LYSO[32-P] phosphatidylcholine, sn-glycero-3-[32-P] phosphorylcholine, [32-P] phosphorylcholine, sn-gleycero-3-[32-P] phosphate and 32-Pi. Further investigation showed that [32-P] phosphorylcholine was released exclusively from sn-glycero-3-[32-P] phosphorylcholien by a novel diesterase activity. We propose that the enzyme be termed L-3-glycerylphosphinicocholine cholinephosphohydrolase (EC 3.1.4.-). Parallel experiments on rat liver homogenates and a P815Y mouse mastocytoma cell-lysate, revealed no diesterase activity.     

Hydrolysis of exogenous [3H]phosphatidylcholine by brain membranes and cytosol

Phosphatidylcholine, in addition to the widely studied inositol phospholipids, is cleaved to produce second messengers in neuronal signal transduction processes. Because of the difficulty in labelling and measuring the metabolism of endogenous phosphatidylcholine in brain tissue, we investigated the utility of measuring the hydrolysis of exogenous labelled substrate incubated with rat cerebral cortical cytosol and membrane fractions as has been successful in studies of phosphoinositide hydrolysis. In the cytosol [³H]phosphatidylcholine was hydrolyzed at a linear rate for 60 min of incubation and GTPS stimulated hydrolysis by 63%. The products of phospholipase C and phospholipase D, phosphorylcholine and choline, contributed only 44% of the [³H]phosphatidylcholine hydrolytic products in the cytosol, with phospholipase D activity slightly predominating. GTPS stimulated cytosolic phospholipase C and reduced phospholipase D activity. [³H]Phosphatidylcholine was hydrolyzed much more slowly by membranes than by cytosol. In membranes the production of [³H]phosphorylcholine and [³H]choline were approximately equal, contributing 27% of the total [³H]phosphatidylcholine hydrolysis, and GTPS only caused a slight stimulation of phospholipase C activity. Chronic lithium treatment (4 weeks) appeared to slightly reduce [³H]phosphatidylcholine metabolism in the cytosol and in membranes, but no statistically significant reductions were achieved. Cytosol and membrane fractions from postmortem human brain metabolized [³H]phosphatidylcholine slowly, and GTPS had no effects. In summary, exogenous [³H]phosphatidylcholine was hydrolyzed by brain cytosol and membranes, and this was stimulated by GTPS, but the complex contributions of multiple metabolic pathways complicates the application of this method for studying individual pathways, such as phospholipase D which contributes only a fraction of the total processes hydrolyzing exogenous [³H]phosphatidylcholine.     

Mechanism of polymorphic transformations in phosphatidylcholine membranes

Based on the 31P NMR, PMR, and EPR data on the thermally induced behavior of water dispersions of natural and synthetic phospholipids in the presence of membranotropic agents: the neuropeptide adrenocorticotropic hormone and beta-(4-oxy,3,5-ditretbutyl-phenylpropionic acid), a new mechanism of the interaction of membranotropic substances with the molecules of hydrate shells of membrane phospholipids was proposed, which underlies polymorphic transitions in phosphatidylcholine membranes.     

Two active Na+/K+-ATPases of high affinity for ouabain in adult rat brain membranes

The degree of heterogeneity of active Na+/K(+)-ATPases has been investigated in terms of ouabain sensitivity. A mathematical analysis of the dose-response curves (inhibition of Na+/K(+)-ATPase) at equilibrium is consistent with the putative existence of three inhibitory states for ouabain two of high (very high plus high) and one of low affinity. The computed IC50 values are: 23.0 +/- 0.15 nM, 460 +/- 4.0 nM and 320 +/- 4.6 microM, respectively. The relative abundance of the three inhibitory states was estimated as: 39%, 36% and 20%, respectively. Direct measurements of [3H]ouabain-binding at equilibrium carried out on membrane preparations with ATP, Mg2+ and Na+ also revealed two distinct high affinity-binding sites, the apparent Kd values of which were 17.0 +/- 0.2 nM (very high) and 80 +/- 1 nM (high), respectively. Dissociation processes were studied at different ouabain concentrations according to both reversal of enzyme inhibition and [3H]ouabain release. The reversal of enzyme inhibition occurred at three different rates, depending upon the ouabain doses used (10 nM, 2 and 100 microM). When the high-affinity sites were involved (ouabain doses lower than 2 microM) the dissociation process was biphasic. A similar biphasic pattern was also detected by [3H]ouabain-release. The time-course of [3H]ouabain dissociation (0.1 microM) was also biphasic. These data indicate that the three catalytic subunits of rat brain Na+/K(+)-ATPase alpha 1, alpha 2 and alpha 3 (Hsu, Y.-M. and Guidotti, G. (1989) Biochemistry 28, 569-573) are able to hydrolyse ATP and exhibit different affinities for cardiac glycosides.     

Time-dependent cadmium-neurotoxicity in rat brain synaptosomal plasma membranes

• 1.1. The modulation of lipid dynamics and lipid protein interactions were studied in rat brain synaptosomal plasma membranes (SPM) up to 24 hr after exposure to cadmium (Cd).
• 2.2. The activity of acetylcholinesterase and adenylate cyclase showed a considerable decrease after 6 hr of Cd exposure, followed by a progressive increase up to 24 hr.
• 3.3. SPM chemiluminescence showed a maximum decrease at 12 hr, demonstrating a considerable increase in lipid peroxidation.
• 4.4. SPM of Cd-exposed animals showed a statistical significant increase in fluorescence anisotropy parameter [(r₀/r) — 1]⁻¹ at 18 and 24 hr compared to SPM of the control, indicating a decrease of membrane fluidity.

     

Turnover of myelin and other structural proteins in the developing rat brain

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).     

Quantitative evaluation of two pathways for phosphatidylcholine biosynthesis in rat brain in vivo

[Me-³H] choline and [³²P] orthophosphate were injected intraventricularly into adult female rats. After variable intervals from injection (1–10 min) the animals were sacrificed by means of a microwave apparatus, and phosphorylcholine and choline phosphoglycerides extracted from brain and counted for radioactivity content after separation. The kinetic constants (K) for phosphorylcholine incorporation into lipids were determined both for [³²P] and [³H] labeling. From the data obtained by these procedures it is concluded that base-exchange reactions for choline incorporation into lipids are operating in rat brain in vivo and that they represent a rapidly equilibrating system.