A STUDY OF THE MOLECULAR SIZES AND STABILITY OF CHOLINE ACETY LTRANSFER ASE

Aggregation of the enzyme acetyl-CoA: choline-O-acetyltransferase (ChAc, EC 2.3.1.6) which appears to be homogeneous has been observed. The molecular weight of the most abundant form of ChAc was estimated by gel filtration and sucrose gradient centrifugation to be in the range 58,000-62,000. The most frequently encountered aggregates were much larger and eluted in the void volume from Sephadcx® G-100 and G-150 indicating molecular weights in excess of 400,000. In fact, they were subsequently found to be 1.2 × 10⁶ and 1.9 × 10⁶ by sucrose gradient centrifugation. The percentage of activity associated with high molecular weight ChAc increased with purification, but these aggregates disappeared after storage for 2-3 weeks at ?20°C. The loss occurred independently of any fall in enzymic activity in the preparations examined.     

CHOLINE ACETYLTRANSFERASE ACTIVITY OF HUMAN BRAIN TISSUE DURING DEVELOPMENT AND AT MATURITY

Abstract— (1) On analysis of human brain tissue to determine its choline acetyltransferase (ChAc) content the recovery of enzyme from many regions is very poor when the tissue is acetone-dried and then extracted in the standard manner; for this reason the method is unsuitable when quantitative recoveries are required; it is preferable to prepare sucrose homogenates and activate these with ether before incubation.
(2) From measurements made on homogenates of one adult brain the highest concentration of ChAc was found in the putamen and the lowest in the corpus callosum. The caudate nucleus also had a high activity. As in other mammals, the concentration of enzyme in the cerebellum was found to be low. Analogous results were obtained on a nine-year-old brain but the level of ChAc activity was generally higher than in the older brain.
(3) During foetal development up to thirty-two weeks, ChAc is higher in the cerebellum than in the caudate, the thalamus, corpora quadrigemina, medulla and spinal cord. In all regions the concentration and total amount of enzyme rise fairly steadily up to this time; between 24 and 32 weeks, however, its concentration in the cerebellum and corpora quadrigemina falls slightly although the total increases considerably.
(4) Comparison of the results with the data of other authors indicates general agreement between the distribution of the enzyme in the human brain and its distribution in other mammals, especially the rhesus monkey. The corpus callosum may be an exception since in man it contains little ChAc while in lower mammals it seems to have relatively high concentrations of both ACh and ChAc.
(5) In comparing the values for ChAc reported here with the values for AChE reported by others, three tissues, the globus pallidus, substantia nigra and cerebellum are found to be exceptional in that relative to their concentration in the caudate the activity of ChAc is only about one-tenth that of AChE.     

SEPARATION OF APPARENT MULTIPLE FORMS OF HUMAN BRAIN CHOLINE ACETYLTRANSFERASE BY ISOELECTRIC FOCUSING

Abstract— Choline acetyltransferase (acetyl-CoA: choline O -acetyl transferase; EC 2.3.1.6; ChAc) purified from human brain (basal ganglia) and sciatic nerve were separated into apparent multiple enzyme forms by the method of isoelectric focusing (pH gradient 3-10) on acrylamide gel. A preparative separation of enzyme forms of human brain was accomplished by the column method, by using a sucrose gradient. When each separated form was re-electrofocused, only a portion of the ChAc activity was observed in its original pH region while more than one-half of the recovered activity for each fraction appeared at pH 7.8-8. Gel filtration and kinetic studies of separated forms indicated that the more acidic forms might be aggregates, while more basic forms might be configurational isomers. Human ChAc of sciatic nerve did not exhibit acidic forms on electrofocusing, but otherwise yielded an electrofocusing profile similar to that of human brain. ChAc of rabbit brain and sciatic nerve each exhibited only a single form at pH 7.1 ± 0.2. Although ChAc differs among species, the enzyme of brain and sciatic nerve of the same species cannot be clearly distinguished by electrofocusing.     

THE DISTRIBUTION OF ACETYL-CoA IN SPECIFIC AREAS OF THE CNS OF THE RAT AS MEASURED BY A MODIFICATION OF A RADIO-ENZYMATIC ASSAY FOR ACETYLCHOLINE AND CHOLINE1

A rapid and sensitive enzymatic assay for measuring picomole quantities of acetyl-CoA, acetylcholine (ACh), and choline from the same tissue extract has been developed. After ACh and choline were extracted into 15% 1 N formic acid/85% acetone, the pellet was further extracted with 5% trichloroacetic acid (TCA) to remove the remaining acetyl-CoA. The two extraction solvents were pooled and lipids, organic solvents, and TCA were removed first by a heptane-chloroform wash followed by an ether extraction. In the acetyl-CoA assay, endogenous ACh and choline were removed by extractions with sodium tetraphenylboron in butenenitrile prior to the enzymatic reactions. The acetyl-CoA remaining in the aqueous phase was then converted enzymatically to labelled ACh in the presence of [Me-¹⁴C]choline using choline acetyltransferase. The unreacted labelled precursor was converted to choline phosphate by the enzyme choline kinase. The [¹⁴C]ACh formed from acetyl-CoA was extracted into sodium tetraphenylboron in butenenitrile and a portion of the organic phase containing the [¹⁴C]ACh was counted in a scintillation counter. Acetylcholine and choline were assayed from the same tissue extracts by a modification of the procedure by SHEA & APRISON (1973). Acetyl-CoA levels in rat whole brain when killed by the near-freezing procedure were found to be 5.50 ± 0.2 nmol/g. The content of acetyl-CoA was the same whether the rats were killed by the near-freezing method or by total freezing in liquid nitrogen. The levels of acetyl-CoA did not change with time after death when the tissue was maintained at a temperature of ?10°C. In the same tissue extracts from rat whole brain killed by the near-freezing method, the content of ACh was 20.6 ± 0.7 nmol/g and choline 58.2 ± 1.2 nmol/g. Although reproducible, the level reported for choline is high when assayed under this condition. The content of choline however after total freezing was found to be 25.2 ± 2.0 nmol/g. The sensitivity (d. p. m. of sample twice blank) is 10 pmol for the acetyl-CoA assay and 25 pmol for the ACh and choline assays. The regional distribution of these three compounds in the brain of rats as well as the content of acetyl-CoA in heart, liver and kidney are presented.     

THE ENZYMIC HYDROLYSIS OF PHOSPHATIDYL INOSITOL BY GUINEA PIG BRAIN:

Abstract—

• 1 Phosphatidylinositol hydrolase activity of homogenates of guinea pig brain was studied by using [2-³H]inositol labelled substrate and measuring the release of radioactivity into the acid soluble fraction.
• 2 Inositol phosphate and diglyceride were found to be the main hydrolysis products. The principal enzyme involved, therefore, is a phosphatidylinositol inositolphosphohydrolase.
• 3 Most of the enzymic activity (61 per cent) was found in the soluble fraction. Osmotic shock of the high speed particulate fraction resulted in release of an additional 23 1 per cent into the soluble fraction. However, as contrasted to lactate dehydrogenase, significant activity remained particulate bound.

     

REGIONAL AND SUBCELLULAR DISTRIBUTION AND KINETIC PROPERTIES OF RAT BRAIN CHOLINE ACETYLTRANSFERASE–SOME FUNCTIONAL CONSIDERATIONS

The kinetic properties of soluble and membrane-bound choline acetyltransferase (ChAc) were determined as a function of homogenization media and solubilization procedure in various regions of rat brain. Treatment of homogenate and/or subcellular fractions with KCl, Triton X-100, or ether dramatically altered the apparent V_max and the degree of solubilization of the enzyme, but no fraction exhibited K_m values substantially different from 12 μM for acetyl-CoA and 200 μM for choline. On the other hand, increasing the ionic strength of the assay medium for a given fraction from 0-02 M to 0-5 M increased both V_max and K_m values for both substrates. The absolute levels and subcellular distribution of ChAc were determined in 11 brain regions to localize cholinergic cell bodies and nerve endings. Levels of ChAc varied from 139 m-units/g tissue in caudate-putamen to 5-7 m-units/g tissue in cerebellum. The fraction of ChAc activity associated with synaptosomes varied from near 75 per cent in caudate-putamen, hippocampus and cortical regions to near 20 per cent in septum, locus coeruleus area and substantia nigra area. The apparent parallel distribution of cholinergic and catecholaminergic nerve endings is discussed in terms of a hypothetical model for the pathophysiology and treatment of Parkinson's syndrome.     

Stimulation of choline acetyltransferase activity by ethanol in in vitro preparations of rat cerebrum

Choline acetyltransferase activity in homogenates, or in partially purified extracts of rat brain cerebra, was increased by 11–37% in the presence of ethanol when incubated at 38°C with [¹⁴C] acetyl-CoA, choline chloride and alcohol concentrations of 0.17M to 1.02M. In preincubation experiments with enzyme preparations and ethanol, inactivation of the enzyme by the alcohol, which occurs at incubation times longer than 20 minutes, could be at least partially prevented by the addition of certain components of the incubation mixture to the preincubation mixture.     

SUBCELLULAR DISTRIBUTION AND SOME PROPERTIES OF ACETYL-COENZYME A HYDROLASE IN THE BRAIN

—The detailed subcellular distribution and some properties of acetyl-CoA hydrolase were studied in the rat brain. The brain homogenate (S₁) hydrolysed acetyl-CoA at a rate of approx 2·3 nmol/min/mg of protein at 37°C. The total activity of acetyl-CoA hydrolase was distributed in the following order: soluble > mitochondrial > microsomal, synaptosomal > myelin fraction. The order of the specific activity of the enzyme was: soluble, microsomal > mitochondrial > synaptosomal > myelin fraction. The synaptic vesicle fraction (D) had relatively high specific activity among the intraterminal particulate fractions, having two or three times higher specific activity than that of the synaptic cytoplasmic membrane fraction (F or G). Attempts to de-occlude acetyl-CoA hydrolase in the particulate fraction showed that only the enzyme activity in the myelin fraction was increased markedly by the treatment with ether or Triton X-100. Lineweaver-Burk plots gave straight lines for each subcellular fraction and apparent K_m values for acetyl-CoA were between 0·1 and 0·2 mM. Neither diisopropyl fluorophosphate nor physostigmine at the concentration of 0·1 mm inhibited the enzyme activity.     

Transport of proteins, glycoproteins and cholinergic enzymes in regenerating hypoglossal neurons

The accumulation of [³H]leucine- and [³H]fucose-labelled axonal proteins, acetyl-CoA : choline O-acetyltransferase (ChAc, EC 2.3.1.6) and acetylcholinesterase (AChE, EC 3.1.1.7) was studied proximal to a ligature applied to the hypoglossal nerve of the rabbit at different phases of nerve regeneration. After 1 week of regeneration, the accumulation of rapidly migrating [³H]leucine-labelled proteins, ChAc and AChE was reduced as compared to that of the contralateral nerve. In contrast, the accumulation of [³H]fucose-labelled glycoproteins was markedly increased. After a regeneration period of 4-6 weeks, the accumulation of proteins and glycoproteins in the regenerating nerve was increased whereas the accumulation of ChAc and AChE was almost normal. The results indicate an initial depression of the synthesis and axonal transport of the bulk of rapidly migrating proteins, ChAc and AChE in the chromatolytic hypoglossal neurons whereas the synthesis and transport of rapidly migrating glycoproteins is increased. These initial changes are less pronounced during the subsequent regeneration period.     

DISTRIBUTION OF CHOLINE ACETYLTRANSFERASE AND GLUTAMATE DECARBOXYLASE WITHIN THE SUBSTANTIA NIGRA AND IN OTHER BRAIN REGIONS FROM CONTROL AND PARKINSONIAN PATIENTS

—The distribution of choline acetyltransferase (ChAc, EC 2.3.1.6) and l -glutamate 1-carboxylyase (glutamate decarboxylase, GAD, EC 4.1.1.15) was studied in serial frontal slices of the substantia nigra (SN) (pars compacta, PC; pars reticulata, PR; an intermediate region, IR) as well as in other brain areas from post mortem tissue of control and Parkinsonian patients. Within the SN from control brain ChAc and GAD activities showed a distinctive distribution: ChAc activity in PC was higher than in PR and IR by 427% and 253% respectively and within PC the enzyme activity in the rostral part exceeded that in the control part by 353%. The GAD activity in PC was higher by 41% than that in PR and within PC seemed to be higher in the caudal than in the rostral part. For both enzyme activities there were no significant differences between PR and IR or within these regions. In Parkinsonian brain both ChAc and GAD activities were reduced to 15-25% of controls in all 3 regions of the SN. The distinctive distribution of ChAc and GAD activity found in the SN of control brain was abolished: no difference was observed between the 3 regions. However, within PC the ChAc activity was lower in the medial than in the rostral part. Since nigral ChAc is possibly located in interneurons, the decrease in enzyme activity may be connected with the cell loss observed in the SN of Parkinsonian brain. By contrast, nigral GAD is probably contained in terminals of strio-nigral neurons and the decrease in enzyme activity in Parkinson's disease in the absence of striatal cell loss, may reflect a change in the functional state of these GABA neurons. Among various areas of control brains ChAc activity was highest in caudate nucleus and putamen while GAD was highest in SN. caudate nucleus, putamen and cerebral cortex. In Parkinsonian brain the most severe reduction in ChAc and GAD activities was found in the SN.     

INHIBITION OF CHOLINE ACETYLTRANSFERASE AND ITS HISTOCHEMICAL LOCALIZATION

Abstract— A method for the histochemical identification of choline acetyltransferase has been investigated further by studying the effects of certain inhibitors of the enzyme both on rat brain homogenates and on the localization of the enzyme in tissue sections.
It was confirmed that acetyl-CoA hydrolase activity both in homogenates and in tissue sections is inhibited by preincubation in 1 mM-DFP. The effects of the choline acetyltransferase inhibitors chloro- and bromoacetylcholine on the appearance of histochemical staining were related to their activity in homogenates and tissue slices. Bromoketone was found to inhibit choline acetyltransferase in homogenates and, less efficiently, in tissue sections but it also inhibited the hydrolysis of acetyl-CoA by some other unknown enzyme which is inactivated by 1 mM-DFP.
The results obtained with the choline acetyltransferase inhibitors provide support for the specificity of the histochemical method.     

ENZYMES CATALYZING THE DE NOVO SYNTHESIS OF METHYL GROUPS IN THE BRAIN AND OTHER TISSUES OF THE RAT

—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.     

PURIFICATION OF RAT BRAIN CHOLINE ACETYLTRANSFERASE AND AN ESTIMATION OF ITS HALF-LIFE

—Acetyl-CoA:choline-O-acetyltransferase (ChAc, EC 2.3.1.6) was purified from rat cerebral cortex and its half-life determined. The molecular weight of the enzyme under non-denaturing conditions was estimated by gel filtration to be in the range of 60,000–65,000. On SDS acrylamide gels, the purified enzyme migrated as a single band with a molecular weight estimated as 62,000. The turnover rate of ChAc in the mature rat was determined by the double label method, employing l -[1-¹⁴C]leucine and l -[4,5-³H]leucine. Its half-life under steady-state conditions was estimated to be 5.2 days. As a control, tubulin was isolated from the same preparation and its half-life measured. Under these conditions tubulin exhibited a half-life of 3.8 days.     

PURIFICATION AND PROPERTIES OF CHOLINE ACETYLTRANSFERASE FROM OX BRAIN STRIATE NUCLEI

Abstract—

• 1 Choline acetyltransferase was purified from ox brain striate nuclei by an extraction step at pH 5, cation-exchange chromatography, fractional precipitation with ammonium sulphate, and chromatography on Sephadex G-200. The enzyme was obtained free of deacylases and cholinesterases, at specific activities of 01-0-3 μmol acetylcholine formed per min per mg protein.
• 2 The enzyme was found to be a stable and relatively basic protein, with a molecular weight of 65,000.
• 3 In the catalysed reactions, , k₁, was about four times k₂, and the equilibrium constant was approximately 40. For the forward reaction, the Michaelis constant for each substrate was independent of the concentration of the other (choline = 0-75 mM; acetyl-CoA = 10 μM), whereas in the back reaction one substrate increased the affinity for the other (acetylcholine = 0-75-5 MM; CoA = 25-150 μM).
• 4 CoA inhibited acetylcholine synthesis by competing with acetyl-CoA (K₁, = 16 μM). Acetylcholine slightly inhibited the forward reaction (e.g. 45 per cent in 200 mM) without competing with choline or acetyl-CoA. These data indicate an ordered reaction mechanism; acetyl-CoA probably always binds before choline.

     

DEVELOPMENT OF THE UPTAKE AND STORAGE OF L-[3H]NOREPINEPHRINE IN THE RAT BRAIN

The uptake and storage of L-[³H]norepinephrine at various stages of development was examined in homogenates of rat brain. For the adult animal, active uptake accounted for 80 per cent of the total uptake. At 14 days of gestation, no active uptake was demonstrable At 18 days of gestation, saturable uptake of L-[³H]norepinephrine with a K_m of 3 × 10 ^?7m was first demonstrable; the K_m value did not vary during subsequent development. The V_max. of uptake increased five-fold between 18 days of gestation and 28 days postnatally, at which stage it was the same as the adult value. The development of saturable uptake paralleled but preceded the increase in endogenous norepinephrine. When homogenates were incubated with l -[³H]norepinephrine and subjected to centrifugation on linear sucrose gradients, there was a peak of tritium in the synaptosomal fractions; the magnitude of the peak increased with maturation of the brain. The increase in the peak of tritium paralleled the increase in particulate LDH activity and was distinct from the peak of MAO activity. Desipramine, a compound that blocks the initial uptake of norepinephrine, first exhibited inhibition of uptake at 19 days of gestation; the degree of inhibition did not vary during subsequent development. In contrast, reserpine, a compound which inhibits the intra-neuronal storage of norepinephrine, exhibited a progressive increase of inhibition with maturation of the brain at and subsequent to 19 days of gestation.     

THE MEASUREMENT OF TRIGLYCERIDE IN BRAIN AND THE METABOLISM OF BRAIN TRIGLYCERIDE IN VITRO

Abstract—

• 1 Triglyceride has been isolated from brain by thin-layer chromatography and determined by absorption of the carbonyl group at 1740 cm^?1. The means of yields from whole mouse brain, whole rat brain, rat brain grey matter, rat brain stem, and incubated slices of rat brain cortex were 0.15–0.17 μmole/g tissue.
• 2 The distribution of fatty esters varied from preparation to preparation. Palmitate, stearate and oleate usually occurred in greatest amounts. Hydrolysis of a preparation of triglyceride from whole rat brain with pancreatic lipase indicated that palmitate was equally distributed between the α and β esters.
• 3 [1-¹⁴C]Acetate was rapidly incorporated into triglyceride of slices of incubated rat brain cortex. When the resulting triglyceride was hydrolysed with pancreatic lipase the distribution of radioactivity amongst the hydrolysis products was consistent with both the α and β esters of the triglyceride having been radioactively labelled.

     

THE SYNTHESIS OF ACETYLCHOLINE FROM ACETYL-CoA, ACETYL-DEPHOSPHO-CoA AND ACETYLPANTETHEINE PHOSPHATE BY CHOLINE ACETYLTRANSFERASE

Abstract— The synthesis of ACh by choline acetyltransferase (ChAc) has been examined using acetyl-CoA, acetyl-dephospho-CoA and acetylpantetheine phosphate. At pH 7.5 K_m values of 25.7 μ m for acetyl-CoA, 54.8 μ m for acetyl-dephospho-CoA and 382 μ m for acetylpantetheine phosphate were obtained and are similar to those at pH 6.0. This indicates that the 3-phosphate may not be required for binding the substrate to the enzyme unlike carnitine acetyltransferase.
Inhibitor constants ( K_i ) for CoA, dephospho-CoA and pantetheine phosphate were also measured and when considered with the K_m values obtained for the acetyl derivatives it is concluded that acetyl-dephospho-CoA could be a successful acetyl donor in the synthesis of ACh.
Acetyl-dephospho-CoA was found to be less satisfactory as a substrate for citrate synthase.     

PROVENANCE OF THE ACETYL GROUP OF ACETYLCHOLINE AND COMPARTMENTATION OF ACETYL-CoA AND KREBS CYCLE INTERMEDIATES IN THE BRAIN IN VIVO

—The origin of the acetyl group in acetyl-CoA which is used for the synthesis of ACh in the brain and the relationship of the cholinergic nerve endings to the biochemically defined cerebral compartments of the Krebs cycle intermediates and amino acids were studied by comparing the transfer of radioactivity from intracisternally injected labelled precursors into the acetyl moiety of ACh, glutamate, glutamine, ‘citrate’(= citrate +cis-aconitate + isocitrate), and lipids in the brain of rats. The substrates used for injections were [1-¹⁴C]acetate, [2-¹⁴C]acetate, [4-¹⁴C]acetoacetate, [1-¹⁴C]butyrate, [1, 5-¹⁴C]citrate, [2-¹⁴C]glucose, [5-¹⁴C]glutamate, 3-hydroxy[3-¹⁴C]butyrate, [2-¹⁴C]lactate, [U-¹⁴C]leucine, [2-¹⁴C]pyruvate and [³H]acetylaspartate. The highest specific radioactivity of the acetyl group of ACh was observed 4 min after the injection of [2-¹⁴C]pyruvate. The contribution of pyruvate, lactate and glucose to the biosynthesis of ACh is considerably higher than the contribution of acetoacetate, 3-hydroxybutyrate and acetate; that of citrate and leucine is very low. No incorporation of label from [5-¹⁴C]glutamate into ACh was observed. Pyruvate appears to be the most important precursor of the acetyl group of ACh. The incorporation of label from [1, 5-¹⁴C]citrate into ACh was very low although citrate did enter the cells, was metabolized rapidly, did not interfere with the metabolism of ACh and the distribution of radioactivity from it in subcellular fractions of the brain was exactly the same as from [2-¹⁴C]pyruvate. It appears unlikely that citrate, glutamate or acetate act as transporters of intramitochondrially generated acetyl groups for the biosynthesis of ACh. Carnitine increased the incorporation of label from [1-¹⁴C]acetate into brain lipids and lowered its incorporation into ACh. Differences in the degree of labelling which various radioactive precursors produce in brain glutamine as compared to glutamate, previously described after intravenous, intra-arterial, or intraperitoneal administration, were confirmed using direct administration into the cerebrospinal fluid. Specific radioactivities of brain glutamine were higher than those of glutamate after injections of [1-¹⁴C]acetate, [2-¹⁴C]acetate, [1-¹⁴C]butyrate, [1,5-¹⁴C]citrate, [³H]acetylaspartate, [U-¹⁴C]leucine, and also after [2-¹⁴C]pyruvate and [4-¹⁴C]acetoacetate. The intracisternal route possibly favours the entry of substrates into the glutamine-synthesizing (‘small’) compartment. Increasing the amount of injected [2-¹⁴C]pyruvate lowered the glutamine/glutamate specific radioactivity ratio. The incorporation of ¹⁴C from [1-¹⁴C]acetate into brain lipids was several times higher than that from other compounds. By the extent of incorporation into brain lipids the substrates formed four groups: acetate > butyrate, acetoacetate, 3-hydroxybutyrate, citrate > pyruvate, lactate, acetylaspartate > glucose, glutamate. The ratios of specific radioactivity of ‘citrate’ over that of ACh and of glutamine over that of ACh were significantly higher after the administration of [1-¹⁴C]acetate than after [2-¹⁴C]pyruvate. The results indicate that the [1-¹⁴C]acetyl-CoA arising from [1-¹⁴C]acetate does not enter the same pool as the [1-¹⁴C]acetyl-CoA arising from [2-¹⁴C]pyruvate, and that the cholinergic nerve endings do not form a part of the acetate-utilizing and glutamine-synthesizing (‘small’) metabolic compartment in the brain. The distribution of radioactivity in subcellular fractions of the brain after the injection of [1-¹⁴C]acetate was different from that after [1, 5-¹⁴C]citrate. This suggests that [1-¹⁴C]acetate and [1, 5-¹⁴C]citrate are utilized in different subdivisions of the ‘;small’ compartment.     

A METHOD FOR THE STUDY OF CHOLESTEROL BIOSYNTHESIS IN THE CENTRAL NERVOUS SYSTEM

Abstract—

• 1 After intraperitoneal injection, there is negligible incorporation of [2-¹⁴C]-mevalonic lactone into the CNS of the adult rat.
• 2 Mevalonic lactone injected into the CSF is quickly transferred to blood.
• 3 Mevalonic lactone injected in the cistema magna or the lateral ventricle of the brain does not diffuse readily into the whole CSF. Spinal cord cholesterol is most heavily labelled after intracisternal injection, as is brain cholesterol after intraventricular administration.
• 4 After intraventricular perfusion, the diffusion of mevalonic lactone into the ventricle opposite the side of the injection is increased when the rate of perfusion is doubled from 5 to 10 μ1/hr. After injection, optimal homogeneity is obtained if a large volume (70μl) is administered.
• 5 An increase in the volume of injection from 70 μl to 130μl does not alter the distribution of activity between the left and right ventricles, nor does it increase the diffusion of mevalonic lactone from ventricle to spinal cord CSF.
• 6 The mean yield of mevalonic lactone incorporation into brain cholesterol is much higher after injection than after perfusion of precursor into the lateral cerebral ventricle.

     

EFFECT OF THYROID HORMONE ON THE BIOCHEMICAL MATURATION OF RAT BRAIN: CONVERSION OF GLUCOSE-CARBON INTO AMINO ACIDS

• 1 The rapid and extensive conversion of glucose-carbon into amino acids is an index of the final coordination of the mechanisms underlying energy metabolism in the adult brain. This phenomenon develops in the rat during a short period extending from 10 to about 19 days after birth. The underlying factors have been analysed.

• 2 The development of the pattern of distribution of glucose-carbon characteristic of the adult brain was markedly influenced by the thyroid state of the animals. The age-curve for the conversion of glucose-carbon into brain amino acids was displaced to the left after treatment with thyroid hormone (T₃) in infancy thus indicating an accelerated maturation. Conversely, neonatal thyroidectomy resulted in a significant retardation in the conversion of glucose-carbon into amino acids.

• 3 The specific radioactivity of glutamate increased five-fold in the brain of normal rats from the 10th to the 19th day of age. The values (as a percentage of those for littermate controls) were 220 in the case of the 10 day-old thyroid treated rats and about 30 for the 19 day-old thyroid deficient animals. At the age of 10 days neither treatment affected the concentration of glutamate which was also only slightly less than the control values in the brain of 19 day-old thyroid deficient animals (–17 per cent).

• 4 Specific pool(s) of glutamate associated with the formation of GABA can be demonstrated in the brain of 19 day-old rats after administration of [U-¹⁴C]glucose as a result of anoxia post mortem. These pools did not develop in the brain of 10 day-old animals. Neonatal thyroidectomy retarded the development of these glutamate pools.

• 5 Evidence is summarized which indicates that the development of the rapid conversion of glucose-carbon into amino acids reflects the enlargement, during maturation, of the metabolic compartments which are associated with neuronal processes.