EFFECT OF PYRIDOXINE DEFICIENCY ON AROMATIC l-AMINO ACID DECARBOXYLASE IN THE DEVELOPING RAT LIVER AND BRAIN

—Maternal pyridoxine deficiency begun 2 weeks before mating and continued throughout pregnancy and the nursing period resulted in diminished wt. gains in the brain, the liver and the body in the first 16 days of life, as well as lowered levels of the aromatic l -amino acid decarboxylase in both brain and liver tissue. The fetus was protected from the effect of vitamin B₆ deficiency during pregnancy, since at birth the body wt., organ weights, and decarboxylase levels in these tissues were comparable to those of control litters. The brain was affected less than the liver, both in rate of wt. increase and decarboxylase activity. The cerebellum normally developed measurable decarboxylase activity only during the second week of life. The cortex normally slowly increased its low decarboxylase activity during the first week postnatally, with a more rapid increase during the second week. This rapid increase was primarily in the holoenzyme moiety. The rest of the brain, which had well developed levels of decarboxylase activity at birth, normally showed a sharp increase during the second week of life which was also largely in the holoenzyme portion. When the increasing weights of these tissues were considered, it became obvious that the total amount of apoenzyme as well as the amount of holoenzyme were increasing in the normally developing rat, although the greatest amount of the change was in the holoenzyme form. The liver normally showed a much more rapid increase in decarboxylase activity than did the brain, and showed the increase much earlier. The holoenzyme normally increased rapidly after the first 4 days, whereas the apoenzyme concentration levelled off at this time. The effect of the pyridoxine deficiency on decarboxylase activity was almost entirely on the holoenzyme form of the decarboxylase, since the apoenzyme form generally remained the same in the control and the deficient pups during development. There appeared to be no decarboxylase inhibitor present in pyridoxine deficient tissues, nor any evidence in control tissues for an enzyme required for the activation of the decarboxylase by cofactor.     

OCCURRENCE OF N-ACETYL-l-GLUTAMIC ACID IN THE HUMAN BRAIN

A substance apparently identical with N-acetyl-l -glutamic acid was isolated from an aqueous extract from human brain by a combination of paper and ion exchange chromatography. The isolated substance does not react with ninhydrin reagent but yields glutamic acid upon acid hydrolysis. Acetyl hydrazide was identified by paper chromatography of hydrazinolysates of the isolated substance and N-acetyl-l -glutamic acid. The configuration was determined with l -specific hog kidney acylase.     

REGIONAL DISTRIBUTION OF GLUTAMIC ACID DECARBOXYLASE IN THE DEVELOPING BRAIN OF THE PYRIDOXINE-DEFICIENT RAT

—Glutamic acid decarboxylase was determined in seven brain regions: hypo-thalamus; midbrain; thalamus; corpus striatum; cerebral cortex-hippocampus; medulla-pons; and cerebellum, of suckling rats subjected to Vitamin B₆ deficiency for 2 weeks from birth; of adult rats subjected to the deficiency for 5 weeks and of their respective controls. Large regional variations in the enzyme activity were found in brains of both adult and suckling control rats. The activity of the enzyme (assayed without pyridoxal phosphate) and its saturation with endogenous cofactor were markedly reduced in all brain regions of both suckling and adult pyridoxine-deficient rats. The apoenzyme (activity assayed with pyridoxal phosphate), in adult rat brain, showed no change with the deficiency in all regions except in the cerebellum where it increased slightly. In pyridoxine-deficient suckling rat brain, the apoenzyme increased substantially in all regions suggesting a process of enzyme induction. The increase in apoenzyme varied from region to region.     

OCCURRENCE OF N-ACETYLALANINE IN THE HUMAN BRAIN1

A substance identical with N-acetyl-l -alanine was isolated from an aqueous extract of human brain by a combination of paper and ion-exchange Chromatography. The isolated substance did not react with ninhydrin reagent but yielded alanine upon acid hydrolysis. An acetyl hydrazide was identified by paper chromatography of hydrazinolysates of the isolated substance and N-acetyl-l -alanine. The unknown alanine had the l -configuration. The results of elementary analysis of the isolated compound were in full accord with the analysis calculated for synthetic N-acetyl-l -alanine.     

AXOPLASMIC TRANSPORT OF AROMATIC l-AMINO ACID DECARBOXYLASE (EC 4.1.1.26) AND DOPAMTNE β-HYDROXYLASE (EC 1.14.2.I) IN RAT SCIATIC NERVE

The axoplasmic transport of aromatic l -amino acid decarboxylase and dopamine β-hydroxylase, two enzymes involved in the biosynthesis of catecholamines, was studied in rat sciatic nerve. The two enzymes exhibited markedly different axoplasmic flow characteristics, since dopamine β-hydroxylase activity accumulated on the proximal side of a ligation nearly three times as fast as aromatic l -amino acid decarboxylase activity. Distally dopamine β-hydroxylase activity remained essentially constant for 24 h, whereas aromatic l -amino acid decarboxylase activity fell precipitously. Evidence was obtained to rule out the possibility that differences in the rate of inactivation of the two enzymes could account for the different rates of accumulations observed. The conclusion, that aromatic L-amino acid decarboxylase and dopamine β-hydroxylase are transported in sympathetic nerve at different rates is discussed in relation to the biosynthesis of norepinephrine.     

PROPERTIES AND REGIONAL DISTRIBUTION OF HISTIDINE DECARBOXYLASE IN RAT BRAIN

—Properties of the histamine-forming enzyme in rat brain were studied, utilizing a sensitive fluorometric assay. The optimum pH was related to substrate concentration and found to be6·4 at 10^?2m -histidine; the apparent Km was about 4·10^?4m ; enzyme activity was inhibited by α-hydrazino -histidine and brocresine but was not affected by α-methyl DOPA or benzene. These different data suggest that the 'specific’histidine decarboxylase (EC 4.1.1.22)—and not the aromatic l -aminoacid decarboxylase—is involved. Determination of enzyme activity and histamine level in different areas of the rat brain revealed important regional differences, the two values being roughly parallel.     

CHOLINE ACETYLTRANSFERASE, ACETYLCHOLINESTERASE AND AROMATIC l-AMINO ACID DECARBOXYLASE IN SINGLE IDENTIFIED NERVE CELL BODIES FROM SNAIL HELIX ASPERSA

—The distribution of choline acetyltransferase, aromatic l -amino acid decarboxylase and acetylcholinesterase in the nervous system of Helix aspersa has been studied using homogenates of whole ganglia, microdissection from freeze-dried sections and dissection of single neurons from fresh tissue. Choline acetyltransferase was found in both the cell body and neuropil layers of all the Helix ganglia. The enzyme was not specifically localized to any ganglion or region of ganglion. Between 10 and 30 per cent of the isolated single cell bodies contained the enzyme. The enzymic activity corresponded to 50–200 mmol ACh/1 cell bodies/h. Choline acetyltransferase is probably a specific marker for cholinergic cells in this species. Aromatic l -amino acid decarboxylase was more selectivity localized and its distribution corresponded well with that of monoamine containing cells as visualized by the fluorescence histochemical technique. A large proportion of cell bodies were localized in the boundary between the visceral and right parietal ganglia and in the pedal ganglion. The other ganglia contained few such cells. The activity of aromatic l -amino acid decarboxylase corresponded 10–50 mmol dopamine/1 cell bodies/h. A method was developed to measure the enzyme activity towards 5-hydroxytryptophan and DOPA in single cells simultaneously. The ratio between the activity towards both substrates did not vary significantly for the different cells. The enzyme is probably a specific marker for monoamine cells, but cannot be used to differentiate between the different monoamine cells. Acetylcholinesterase was uniformly distributed in the ganglia and was probably present in all nerve cells.     

REGIONAL DISTRIBUTION OF MONOAMINES AND THEIR METABOLITES IN THE HUMAN BRAIN

Abstract— Noradrenaline (NA), dopamine (DA). 5-hydroxytryptamine (5-HT), 4-hydroxy, 3-methoxy-phenylethylene glycol (MHPG), homovanillic acid (HVA), 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindolylacetic acid (5-HIAA) were measured in twenty areas of post-mortem brain from ten psychiatrically and neurologically normal patients. There was a marked difference, which did not appear to be related to sex, medication, cause of death or time between death and dissection, in amine and metabolite concentrations between brains. In the cortex, 5-HT, MHPG, HVA. DOPAC and S-HIAA were approximately even in their distribution; NA and DA could not be detected. In sub-cortical areas there were clear differences in the distribution of the three amines accompanied by less marked differences in the distribution of their respective metabolites.     

INTERACTION OF CATECHOLAMINE AND AMINO ACID METABOLISM IN BRAIN: EFFECT OF PARGYLINE AND l-DOPA

Abstract— The combination of l -DOPA and pargyline caused a decrease in level of aspartate and an increase in that of glutamine in vivo in cerebral cortex, cerebellum, brain stem, hypothalamus, neostriatum and cervical cord of rat. There was also a decreased incorporation of radioactivity from [1-¹⁴C]acetate into amino acids in vivo , most notably in cerebellum and brain stem. The labelling of glutamine was especially affected. In addition, cortical slices were prepared from guinea pigs which had been pretreated with pargyline. These slices were incubated with and without 1 m m l -DOPA in media containing [1-¹⁴C]acetate. Pargyline alone caused a stimulation of the labelling of glutamate and aspartate but not glutamine and GABA; the levels of aspartate and GABA were greater than in control slices. The addition of l -DOPA to slices from pargylinized animals caused a severe decrease in glutamine labelling but not in that of glutamate or aspartate; the level of glutamine was increased while that of glutamate was decreased. The results are discussed in terms of the known biochemical and morphological compartmentation of amino acids in brain. It is suggested that catecholamines, in the process of functioning as transmitters, may also function as metabolic regulators of other transmitters, e.g. amino acids, as well as of the energy required for balanced neuronal function.     

IMMUNOLOGICAL QUANTITATION OF GLUTAMIC ACID DECARBOXYLASE IN DEVELOPING MOUSE BRAIN1

Abstract— l -Glutamic acid decarboxylase (GAD) was isolated from bovine cerebellum and purified approx 32-fold by a combination of DEAE-Sephadex chromatography and gel filtration. This preparation was purified electrophoretically. Rabbit antiserum against the electrophoretically purified bovine GAD was found to react with the decarboxylase of bovine cerebellum and mouse brain. Examination of GAD enzyme specific activity at various postnatal ages of developing mouse brain showed that an initial rise in GAD activity occurs at 6 days postnatally. followed by a rapid increase in enzymatic activity which reaches a maximum at 28 days postnatally. Quantitative immunoprecipitation of mouse GAD by rabbit anti-GAD antisera indicated that the amount of GAD per brain increases 10-fold over the period between 1 and 28 days postnatally. This increase coincides closely with the GAD enzyme activity profile. Therefore, the increase in GAD enzyme specific activity during the postnatal development of mouse brain represents an increase in the absolute amount of GAD enzyme protein.     

DISTRIBUTION OF N-ACETYL-l-ASPARTIC ACID IN RAT BRAIN

The distribution of N-acetyl-l -aspartic acid in the rat brain has been studied by means of a new gas-chromatographic method. The results obtained concern twelve different brain areas.     

AROMATIC ACID METABOLITES OF PHENYLALANINE IN THE BRAIN OF THE HYPERPHENYLALANINEMIC RAT: EFFECT OF PYRIDOXAMINE

Abstract— Phenylalanine levels approaching those found in clinical phenylketonuria were produced in the brain of suckling rats by injections of p -chlorophenylalanine and ^L-phenylalanine. The predominant aromatic acid metabolite found in the brain of these animals was phenylacetic acid with decreasing amounts of phenylpyruvic, phenyllactic, and mandelic acids.
The disposition of [³H]pyridoxamine in the brain of normal and hyperphenylalaninemic animals was found to be similar. Pyridoxamine was rapidly phosphorylated in the brain, and excess vitamer was converted mainly to pyridoxal. Pyridoxamine, when injected repeatedly, was effective in significantly reducing the amount of phenylacetate that accumulated in the brain over a period of 6 h. The significance of these findings is discussed.     

PURIFICATION OF L-GLUTAMIC ACID DECARBOXYLASE FROM CATFISH BRAIN

—L-Glutamic acid decarboxylase (GAD) from brain of the channel catfish (Ictalurus punctatus) has been purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, calcium phosphate gel and preparative polyacrylamide gel electrophoresis. The purity of the enzyme preparation was established by showing that on both 7.5% regular and 3.7–15% gradient polyacrylamide gel electrophoresis the enzyme migrated as a single protein band which contained all the enzyme activity. The molecular weight of the purified GAD was estimated by gel filtration and gradient polyacrylamide gel to be 84,000 ± 2000 and 90,000 ± 4000, respectively. SDS-polyacrylamide gel electrophoresis revealed three major proteins with molecular weights of 22,000 ± 2000, 40,000 ± 5000 and 90, 000 ± 6000 which may represent a monomer, dimer, and tetramer. Antibodies against the purified enzyme were obtained from rabbit after four biweekly injections with a total of 80 μg of the enzyme. A double immunodiffusion test using these antibodies and a crude extract from catfish brains showed only a single, sharp precipitin band which still retained the enzyme activity, suggesting that the precipitin band was indeed a GAD-anti-GAD complex. In an enzyme inhibition study, a maximum inhibition of 60–70% was obtained at a ratio of GAD protein/anti-GAD serum of about 1:1.6. Furthermore, the precipitate from the GAD-anti-GAD incubation mixture also contained the enzyme activity, suggesting that the antibody was specific to GAD and that the antigen used was homogeneous. Advantages and drawbacks of the purification procedures described here and those used for mouse brain preparations are also discussed.     

SUBCELLULAR DISTRIBUTION OF d-AMINO ACID OXIDASE AND CATALASE IN RAT BRAIN

—Rat brain d -amino acid oxidase was found to be confined to the hindbrain, distributed more or less equally between cerebellum and medulla. Histochemical staining showed the enzyme to be localized largely in the molecular layer of the cerebellum. After fractionation by means of several distinct density gradient centrifugation procedures exploiting differences in sedimentation coefficient or in density or in both, the enzyme was found to be entirely or almost entirely associated with cytoplasmic particles with a median diameter of the order of 0·2 μm, and a median equilibrium density in aqueous sucrose of 1·18. Comparison with the behavior of cytochrome oxidase and of N-acetyl-β-glucosaminidase indicates that these particles are not mitochondria and are unlikely to be lysosomes. They also differ significantly from the bulk of the catalase-containing particles, which on an average appear to be somewhat smaller. The possibility that they might contain some catalase activity, and thereby qualify as ‘peroxisomes’, can however not be excluded. In any case, they differ profoundly from the peroxisomes of liver or kidney.     

DIFFERENCES IN THE NUCLEAR RIBONUCLEIC ACID CONTENT IN DIFFERENT AREAS OF THE HUMAN ADULT AND FOETAL BRAIN*

Abstract— Four different areas of ten brains from adults in the age group between 44 and 74 years and two areas of six foetal brains were studied. The cellular density was similar in the different areas of the brain of adult man; it averaged 11.9 × 10⁷/g wet tissue. The number of neurons per g wet tissue varied from 2.22 × 10⁷ in the cortex to 0.15 × 10⁷ in the centrum semiovale. In the brain of adults the overall RNA content of nuclei was higher for the cortex than for the corpus callosum or the centrum semiovale. The RNA content of neuronal nuclei was higher than that of non-neuronal nuclei. In the brain of foetuses the nuclear density was higher than in adults. The DNA content of a nucleus in foetal brain was 2–3 times as high as in adult brain and it approached adult values with increasing maturity.