Journal of Neurochemistry

Yamamura, H. I.; Snyder, S. H.

doi: 10.1111/j.1471-4159.1973.tb06022.xpmid: 4771436

—The accumulation of (3H)choline into synaptosome‐enriched homogenates of rat corpus striatum, cerebral cortex and cerebellum was studied at (3H)choline concentrations varying from 0.5 to 100 μm. The accumulation of (3H)choline in these brain regions was saturable. Kinetic analysis of the accumulation of the radiolabel was performed by double‐reciprocal plots and by least squares iterative fitting of a substrate‐velocity curve to the data. With both of these techniques, the data were best satisfied by two transport components, a high affinity uptake system with Km. values of 1.4 μM (corpus striatum), and 3.1 μM (ceμ(cerebral cortex) and a low affinity uptake system with respective Km. values of 93 and 33 μM for these two brain regions. In the cerebellum choline was accumulated only by the low affinity system. When striatal homogenates were fractionated further into synaptosomes and mitochondria and incubated with varying concentrations of (3H)choline, the high affinity component of choline uptake was localized to the synaptosomal fraction. The high affinity uptake system required sodium, was sensitive to various metabolic inhibitors and was associated with considerable formation of (3H)acetylcholine. The low affinity uptake system was much less dependent on sodium, and was not associated with a marked degree of (3H)acetylcholine formation. Hemicholinium‐3 and acetylcholine were potent inhibitors of the high affinity uptake system. A variety of evidence suggests that the high affinity transport represents a selective accumulation of choline by cholinergic neurons, while the low affinity uptake system has some less specific function.

Vanier, M. T.; Holm, M.; Månsson, J. E.; Svennerholm, L.

doi: 10.1111/j.1471-4159.1973.tb06023.xpmid: 4797802

—Gangliosides and allied neutral glycosylceramides were isolated from human infant (2‐24 months of age) cerebral cortex and white matter. The individual glycolipids were separated quantitatively by a combination of column and thin‐layer chromatographic methods on silica gel, DEAE‐cellulose and Sephadex G‐25. In cerebral cortex GD1a and GM1 were the major fractions and constituted more than 70 per cent of the total gangliosides. The concentrations of neutral glycolipids, except for galactosylceramides, were very low: lactosylceramide and glucosylceramide comprised 30 and 5 nmol/g wet weight, respectively. In white matter their concentrations were 10 times higher. The ganglioside concentration was only 50 per cent of that in cerebral cortex: the difference was accounted for mainly by the much lower content of the major di‐ and trisialogangliosides. Stearic acid was the predominant fatty acid of all brain gangliosides. GM3, and GD3 had a considerable content of the very long‐chain fatty acids, C22‐C24, particularly in the white matter. Glucosylceramide and lactosylceramide had almost identical fatty acid patterns between each other in cerebral cortex and white matter. In the cerebral cortex stearic acid and in the white matter the very long‐chain acids predominated. d20:1 Sphingosine comprised more than 20 per cent of total sphingosine in all the gangliosides of the Gl‐ and G2‐series. GM3, and GD3 like lactosylceramide contained significantly less of d20:1 sphingosine. The findings suggest the existence of separate compartments for the biosynthesis of the gangliosides. Glucosylceramides and lactosylceramides of white matter have the same ceramide composition as the galactosylceramides with normal fatty acids and are thus unlikely to be intermediates in the metabolism of the major brain gangliosides which have a completely different fatty acid composition.

Wajda, I. J.; Manigault, I.; Hudick, J. P.; Lajtha, A.

doi: 10.1111/j.1471-4159.1973.tb06024.xpmid: 4203809

—Homogenates of corpus striatum, cerebral cortex and hypothalamus excised from rat brain were fractionated on discontinuous Ficoll and sucrose density gradients, and the distribution of choline acetyltransferase (ChAc) in the mitochondrial and synaptosomal fractions was determined. In the hypothalamic and cortical regions the fractions enriched in synaptosomes showed much higher activity of ChAc than those containing mainly mitochondria. On the other hand, the corpus striatum showed an equal distribution of ChAc activity in those two fractions. The localization of ChAc was also studied in the postnuclear supernatants obtained from three brain regions, using continuous sucrose density gradients. The distribution of ChAc was compared to that of monoamine oxidase (MAO), potassium and protein. When the pellets obtained from the fractions collected from the gradient were suspended in sucrose, the peak of ChAc activity was close to that of MAO in all three brain regions. When 0.1 mm EDTA +1% butanol was used in order to liberate the occluded form of ChAc, the maximum liberation occurred in lighter fractions, resulting in a shift of the activity peak toward the top of the gradient. This was found with fractions from hypothalamic and cortical regions. In the striatum, the liberated ChAc remained in the same fractions as the occluded enzyme. The results indicate that ChAc is liberated only in those fractions where it is present in synaptosomes. In agreement with the results on the discontinuous gradients this occurs in particles of lower density than mitochondria in cortex and hypo‐thalamus, but in particles of similar density to mitochondria in the corpus striatum, indicating regional differences in the distribution of ChAc in the brain. K+ containing particles centrifuged in less dense fractions than those containing ChAc, indicating that synaptosomes are heterogeneous with respect to these two marker substances.

Illingworth, D. R.; Glover, J.

doi: 10.1111/j.1471-4159.1973.tb06025.xpmid: 4771437

—The incorporation of an orally administered mixture of (9,10‐3H2joleic acid and (1‐14C)linoleic acid into the brain and spinal cord lipids was maximal after 24 h compared with 4 h for extraneural tissue. In the latter, both acids were utilized equally well for triglyceride biosynthesis, but linoleate entered phosphatidylcholine more rapidly than oleate. Oleic acid was preferentially incorporated into newly synthesized cholesterol esters although 4 h after dosing most cholesterol esters present in serum were formed preferentially from linoleate presumably by the action of lecithin‐cholesterol acyl transferase. In neural tissue, a considerable amount of (1‐14C)linoleate was metabolized to higher polyunsaturated fatty acids, whereas in the case of oleate, 90 per cent of the tritium activity remained in monoenic acids at all time periods studied. Both acids were initially incorporated most rapidly into the lecithin fraction of brain and spinal cord, but after 7 days diacyl phosphatidylethanolamine had the highest specific activity. These data are consistent with the view that the uptake of labelled fatty acids by the brain takes place principally as free acids but that some uptake of esterified forms, probably largely as phosphatidylcholine, also occurs. The low linoleate content of the brain and probably also of cerebrospinal fluid cannot be explained on the basis of a selective restriction on the uptake of this lipid from plasma.

Chasin, M.; Mamrak, F.; Samaniego, Sylvia G.; Hess, S. M.

doi: 10.1111/j.1471-4159.1973.tb06026.xpmid: 4149181

—Five areas of guinea pig brain were examined to determine the properties of the receptor sites mediating increases in (3H)adenosine 3′,5′‐monophosphate (cyclic AMP). Both epinephrine and histamine were effective in causing increases in cyclic AMP in slices derived from cerebral cortex, hippocampus or amygdala, but not in diencephalon or brainstem. Stimulation of slices of cerebral cortex by either epinephrine or histamine resulted in a small, but reproducible, decrease in specific radioactivity of the (3H)‐cyclic AMP produced, as did stimulation of the hippocampus by epinephrine. The catecholamine receptor was an α‐adrenergic receptor in all three areas where epinephrine was effective; α‐adrenergic stimulation, but not β‐adrenergic stimulation, increased levels of (3H)‐cyclic AMP. Furthermore, α‐, but not β‐adrenergic blocking agents, prevented the epinephrine‐ induced increase of both (3H)‐ and total cyclic AMP in cerebral cortex and hippocampus. Only antihistaminic agents were capable of antagonizing the histamine‐induced increase of both (3H)‐ and total cyclic AMP in these two brain areas. The catecholamine receptor in the amygdala also appeared to be an α‐adrenergic receptor. The effects of histamine and epinephrine together were far greater than the sum of effects of either hormone alone in both cerebral cortex and hippocampus.

Skaper, S. D.; Das, S.; Marshall, F. D.

doi: 10.1111/j.1471-4159.1973.tb06027.xpmid: 4358880

—An enzyme from rat brain catalysing the synthesis of the histidine‐containing dipeptides carnosine and homocarnosine (l.‐histidine: β‐alanine ligase (AMP) (EC 6.3.2.11)) was purified about 30‐40‐fold from a 100,000 g supernatant. Assays were conducted by measuring the incorporation of L‐(14C)histidine into carnosine and homocarnosine isolated by paper electrophoresis from the incubation mixture. The ratios of specific activities for the formation of carnosine and homocarnosine were not significantly different for the various purification steps. This was taken as evidence of one enzyme synthesizing both dipeptides. In studying the properties of this enzyme, a pH optimum of 7.4 was shown for carnosine synthesis. The concentrations of amino acid substrates giving maximal synthesis of both dipeptides were in the physiological range found for rat brain. An apparent requirement for ATP, Mg2+, and DPN was seen for dipeptide synthesis. A substrate dependent, enzymecatalysed 32PPi‐ATP exchange reaction was observed, suggesting the formation of an aminoacyl‐AMP intermediate. Certain other nucleoside triphosphates could substitute for the ATP; this effect showed a specificity toward the dipeptide being synthesized. The apparent requirement for DPN was quite specific, with a number of related compounds having no effect. The stoichiometry of enzyme‐catalysed carnosine synthesis was studied. A one to one relationship between carnosine formed and ATP hydrolysed was demonstrated. However, the ratio between carnosine synthesized and DPN hydrolysed was about 6 to 1, indicating a catalytic role for the DPN. The breakdown of DPN did not occur with enzyme alone but was dependent on the presence of substrate.

Ordóñez, L. A.; Wurtman, R. J.

doi: 10.1111/j.1471-4159.1973.tb06028.xpmid: 4771438

—Rat brain contains all three of the enzymes required for de novo synthesis of the methyl group of methionine (serine transhydroxymethylase, methylene reductase, and (B12)transmethylase) in activities comparable to those found in liver and kidney. The activities of methylene reductase in female kidney, and of (B12)transmethylase in female brain and kidney, are higher than in the corresponding male tissues. Liver and kidney extracts contain an inhibitor of methylene reductase not present in brain extracts. This inhibitor differs from S‐adenosylmethionine (SAM), which also inhibits methylene reductase in both liver and brain homogenates. The administration of l‐DOPA to rats, which has been previously shown to deplete brain S‐adenosylmethionine, also reduces the activity of brain (B12)transmethylase if assayed without added SAM. Since SAM is required for activity of this enzyme, its decreased activity probably results from the decline in brain SAM concentration. De now synthesis of methyl groups could be a mechanism by which the brain maintains its level of methionine in the face of increased methyl group utilization after administration of l‐DOPA.

Oravec, M.

doi: 10.1111/j.1471-4159.1973.tb06029.xpmid: 4771439

—Methyl albumin kieselguhr chromatography (MAK) has been employed to separate rat brain nuclear RNA, labelled in vivo with (3H)uridine, into three major fractions. The first fraction (QI RNA) is ribosomal in nature for it has a high G + C/U ratio and is methylated by (methyl‐3H) methionine. The other two fractions (Q2 RNA and TD RNA) are DNA‐like for they exhibit a low G + C/U ratio and are labelled minimally by methionine. Pure ribosomal RNA chromatographs almost entirely in the Q1 RNA fraction. Labelling studies indicate that ribosomal RNA and DNA‐like RNA behave differently. Initially, the label in the DNA‐like RNA fractions increases rapidly and in a linear fashion for the first 30 min, but thereafter decreases rapidly and reaches a steady state level by 1 h and remains so up to at least the 2 h period. In contrast, the labelling of ribosomal RNA is much slower than that of DNA‐like RNA during the first 30 min; however, unlike DNA‐RNA, the labelling of ribosomal RNA still continues to increase linearly thereafter. Thus, during longer labelling periods, ribosomal RNA is labelled more rapidly than DNA‐like RNA. It appears that the labelling of ribosomal RNA relative to DNA‐like RNA is more rapid in liver than in brain.

Chaconas, G.; Finch, C. E.

doi: 10.1111/j.1471-4159.1973.tb06030.xpmid: 4771440

—The effect of ageing on RNA/DNA was investigated in brain regions and liver of a healthy subpopulation of C57BL/6J male mice. A decrease of 10% in RNA/DNA was found in senescent striatum in contrast to absence of significant change in the hypothalamus, cerebellum, hippocampus, septum and liver.

Tjiong, H. B.; Seng, P. N.; Debuch, H.; Wiedemann, H.‐R.

doi: 10.1111/j.1471-4159.1973.tb06031.xpmid: 4771441

—Lipids of frontal lobe grey and white matter were examined in parallel from a normal and a diseased child (M. Niemann‐Pick), both nine years of age. In the grey matter of the pathological case the following changes, although small, were found: a slight increase in all phospholipids and decreased values for nervonic acid in cerebrosides and for hydroxy fatty acids in sulphatides. White matter seemed much more affected by the disease: water content was about 6 per cent higher which corresponds to an approx. 20 per cent loss of dry substance compared with the normal brain. Further increases were observed in ‘ganglioside’ fraction and in all phosphatides. Cerebroside and sulphatide levels appeared decreased owing to destruction of myelin. In all of the glycerophosphatides oleic acid portions were lowered whereas in sphingolipids mainly nervonic acid values were reduced. Aldehyde content of both tissues seemed lowered in the disease, however, changes in composition were observed only in white matter, where the stearaldehyde portion of ethanolamine glycerophospholipid increased at the expense of palmitaldehyde and oleinaldehyde.