INCORPORATION OF PHENYLALANINE, TYROSINE AND TRYPTOPHAN INTO PROTEIN OF HOMOGENATES FROM DEVELOPING RAT BRAIN: KINETICS OF INCORPORATION AND RECIPROCAL INHIBITION

The kinetics of the incorporation into protein of [³H]phenylalanine, [³H]tyrosine and [³H]tryptophan were studied with homogenates prepared from whole brain of 1-, 7-, 21- and 60-day-old rats. The maximal velocities (V_max)of incorporation of phenylalanine and tyrosine decreased and the apparent Michaelis-constants (K_m) for all three amino acids increased with increasing age of the rats. Tyrosine had the smallest and tryptophan the largest K_m values in all age groups. Phenylalanine competitively inhibited the incorporation of tyrosine, but tyrosine inhibited non-competitively the incorporation of phenylalanine. Tryptophan inhibited competitively the incorporation of phenylalanine, but at least partially non-competitively the incorporation of tyrosine. Phenylalanine and tyrosine did not significantly affect the incorporation of tryptophan in homogenates from 60-day-old rats. In 1-day-old rats only a very large excess of phenylalanine or tyrosine inhibited detectably. The K_i for phenylalanine in the incorporation of tyrosine was significantly smaller in 1- than in 60-day-old rats. In every case the inhibition presumably occurred at a single rate-limiting step in the complicated process of incorporation of amino acids into protein.     

REGIONAL TRANSPORT OF TRYPTOPHAN IN RAT BRAIN

Abstract— Tryptophan uptake was studied in brain slices and synaptosomes prepared from regions known to vary in the numbers of serotoninergic cell bodies and nerve endings that they contain. The rate of tryptophan uptake was highest in hypothalamus for both types of preparation. Differences among the regions were much more pronounced in isolated nerve endings (synaptosomes). Loading with tryptophan did not affect the uptake into tissue slices. Tryptophan accumulation in hypothalamus synaptosomes was reduced after intraventricular injection of 5,7–dihydroxytryptamine whereas no change was observed in synaptosomes prepared from cerebellum under the same conditions; accumulation by synaptosomes prepared from the hypothalamic and hippocampal regions was reduced after raphe lesions.     

THE INFLUENCE OF HIGH PHENYLALANINE AND TYROSINE ON THE CONCENTRATIONS OF ESSENTIAL AMINO ACIDS IN BRAIN

—High circulating levels of phenylalanine caused depletions of threonine, valine, methionine, isoleucine, leucine, histidine, tryptophan, and tyrosine in immature and adult rat brains. The branched-chain amino acids were most affected. Their reductions ranged between 38–64 per cent of control values when phenylalanine was administered either parenterally or in the diet. The pattern of cerebral amino acid depletions found in phenylalanine-injected infant rats was similar to that of the adults. Phenylalanine loading caused depletions in serum amino acid levels in adult rats, but in infant rats the serum levels were either unchanged or somewhat elevated. Tyrosine, when administered to adult rats either parenterally or via the diet, caused cerebral depletions in essential amino acids, but the depletions were not as striking as with phenylalanine. In both the infant and adult rat, brain-blood ratios of most of the essential amino acids were significantly reduced by phenylalanine loading.     

INCREASED CONCENTRATIONS OF GLYCINE IN HYPOXANTHINE-GUANINE PHOSPHORIBOSYLTRANSFERASE-DEFICIENT MOUSE NEUROBLASTOMA CELLS1

—The intracellular concentrations of a number of amino acids were compared in a parental line of mouse neuroblastoma cells, a line selected for gross deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and in a revertant line containing the enzyme selected from the deficient line. Most prominent was the increased concentration of free glycine and to a lesser extent glutamic acid in enzyme-deficient cells. The increase in glycine was eliminated after restoration of the HGPRT activity.     

THE INHIBITION OF PHENYLALANINE AND TYROSINE HYDROXYLASES BY HIGH OXYGEN LEVELS

Abstract— The K _m for oxygen for rat liver phenylalanine hydroxylase depended on the structure of the reduced pterin cofactor. When the synthetic cofactor, 6,7-dimethyltetrahydropterin, was employed, the apparent K _m for oxygen was 20%. When the natural cofactor, tetrahydrobiopterin, was used, the apparent K _m for oxygen was 0.35 %. Substrate inhibition (40 per cent inhibition at 43% oxygen) was observed with the natural cofactor but not with the synthetic cofactor. Oxygen also caused substrate inhibition with bovine adrenal medulla and brain tyrosine hydroxylases. The inhibition was more dramatic in the presence of the natural cofactor than with the synthetic cofactor. Substrate inhibition by oxygen of brain tyrosine hydroxylase may explain the lowered brain levels of norepinephrine and dopamine observed after treatment of animals with hyperbaric oxygen.     

EFFECTS OF CHRONIC PORTO-CAVAL ANASTOMOSIS ON BRAIN TRYPTOPHAN, TYROSINE AND 5-HYDROXYTRYPTAMINE

—Three weeks after porto-caval anastomosis, tryptophan and 5-hydroxyindolylacetic acid concentrations were-greatly increased in rat brain regions. 5-Hydroxytryptamine showed smaller increases. Midbrain tyrosine and muscle tyrosine and tryptophan concentrations were also increased. Striatal dopa-mine concentration was not significantly changed. Unlike previous results from acute liver failure, brain tryptophan changes in this chronic study did not simply reflect plasma-free tryptophan changes. Midbrain tryptophan/plasma-free tryptophan ratio and midbrain tyrosine/plasma tyrosine ratio both rose, suggesting increased effectiveness of uptake of these amino acids from plasma by brain. Corresponding muscle/plasma ratios were unaltered by the porto-caval anastomosis. Uptake of tryptophan from buffer by cerebral cortex slices was unaffected. Results on control animals illustrate the importance of plasma-free tryptophan in the normal physiological control of brain tryptophan.     

COMPARTMENTATION OF GLUTAMATE METABOLISM IN THE DEVELOPING BRAIN: EXPERIMENTS WITH LABELLED GLUCOSE, ACETATE, PHENYLALANINE, TYROSINE AND PROLINE

—(1) Phenylalanine, proline and presumably tyrosine are precursors of the small glutamate pool in brain. This follows from the finding that with these precursors the specific radioactivity of glutamine is higher than the specific radioactivity of glutamate. (2) Glucose is not as efficient a precursor of glutamate and related amino acids in the brain of 10-day-old mice as it is in the adult brain. (3) Acetate, phenylalanine, tyrosine and proline are incorporated to about the same extent in glutamate, aspartate and glutamine in the brains of 10-day-old and adult mice. (4) The results suggest that the brain of the immature animal uses substrates other than glucose, relative to glucose better than the brain of adult animals.     

REGULATION OF TYROSINE HYDROXYLASE ACTIVITY IN MOUSE NEUROBLASTOMA CLONE N1E-115

Abstract— Mouse neuroblastoma (clone N1E-115) cells in the logarithmic growth phase were incubated for 12 days. From early log phase to late stationary phase, the specific activity of tyrosine hydroxylase (EC 1.14.3a) increased greater than 30-fold. The increase in tyrosine hydroxylase per cell and per dish was 12- and 2700-fold, respectively. When cell division was stopped by removing serum or by adding 0.1 m m -5-fluorodeoxyuridine and 0.1 m m -uridine, the enzyme activity was also found to increase. These results show that tyrosine hydroxylase is regulated in neuroblastoma clone N1E-115.     

TRYPTOPHAN 5-HYDROXYLASE: APPROXIMATION OF HALF-LIFE AND RATE OF AXONAL TRANSPORT

—The half-life of tryptophan 5-hydroxylase (EC 1.14.3) in rats was estimated from the return of enzyme activity after administration of p-chlorophenylalanine and from the decline of enzyme activity in spinal cord after transection or an intraspinal injection of colchicine. The half-life was 2–3 days. Axonal transport of enzyme, estimated from the reappearance of activity in consecutive portions of spinal cord after treatment with p-chlorophenylalanine, was of the order of 5–7 mm/day. This rate is characteristic of 'slow’axonal flow. Our results suggest that changes in the synthesis of new enzyme are probably not responsible for acute changes in the turnover of serotonin.     

INCORPORATION OF PHENYLALANINE AS A SINGLE UNIT INTO RAT BRAIN PROTEIN: RECIPROCAL INHIBITION BY PHENYLALANINE AND TYROSINE OF THEIR RESPECTIVE INCORPORATIONS

Abstract— Of seven amino acids studied, glutamic acid and phenylalanine were incorporated in highest amounts into the hot-TCA-insoluble material of the 100,000 g supernatant fraction of rat brain homogenate. The system for incorporation of phenylalanine was RNase-insensitive and required ATP (apparent K_m = 0.64 m m ), KC1 (apparent K_m = 14 m m ) and MgCl₂ (optimal concentration range 4-15 m m ). The apparent K_m for phenylalanine was 2.9 m m . [¹⁴C]Phenylalanine did not undergo modification before incorporation. Tyrosine and phenylalanine inhibited the incorporation, respectively, of [¹⁴C]phenylalanine and [¹⁴C]tyrosine when incubated simultaneously or successively. The K_m and K_t (3.3 m m ) values for phenylalanine in the incorporation reaction and as inhibitor of the incorporation of [¹⁴C]tyrosine were similar. We suggest that both the enzyme and the acceptor for the incorporation of these two amino acids are the same. [¹⁴C]Phenylalanine and [¹⁴C]tyrosine entered into COOH-terminal positions in the reactions described. Brain exhibited a 25- to 100-fold higher capacity to incorporate phenylalanine than that of liver, kidney or thyroid. The acceptor capacity in rat brain rapidly decreased from day 5 to day 15 of postnatal age and then slowly until age 150 days.     

KINETIC ANALYSIS OF PHENYLALANINE-INDUCED INHIBITION IN THE SATURABLE INFLUX OF TYROSINE, TRYPTOPHAN, LEUCINE AND HISTIDINE INTO BRAIN CORTEX SLICES FROM ADULT AND 7-DAY-OLD RATS

—An attempt was made to isolate the saturable uptake from the unidirectional influx of amino acids into tissue slices and to estimate the transport constants and maximal velocities of saturable transport. The method was applied to studies on the inhibition of phenylalanine in the saturable influx of tyrosine, tryptophan, histidine and leucine into brain cortex slices from adult and 7-day-old rats. In both age groups phenylalanine inhibited the influx of the other amino acids, and vice versa. The apparent transport constants of the other amino acids increased in the presence of phenylalanine more noticeably in the slices from 7-day-old rats than in those from adult rats, whereas the concomitant influx of phenylalanine was inhibited less in the slices from 7-day-old rats. In immature animals in vivo competition between amino acids may play a more marked role in the supply of amino acids from plasma to brain, as the transport systems in brain slices from 7-day-old rats become saturated with extracellular amino acids more readily than do the transport systems in brain slices from adult rats.     

THE SOLUBILITY OF TYROSINE IN ACID AND IN ALKALI

Measurements have been made of the solubility at 25°C. of tyrosine in hydrochloric acid and in sodium hydroxide solutions varying from 0.001 to 0.05 M, and also in distilled water. The pH of the saturated solutions was measured with the hydrogen electrode. The following values for the ionization constants of tyrosine have been obtained from the measurements: k_b = 1.57 x 10^–12, k_a1 = 7.8 x 10^–10, k_a2 = 8.5 x 10^–11. The changes in solubility with pH can be satisfactorily explained by the use of these ionization constants.     

PHOTOSYNTHESIS OF GLYCINE AND SERINE IN GREEN PLANTS

1. The effects of "carbonyl" reagents on the photosyntheticin-corporation of ¹⁴CO₂ into the assimilation products of tobaccoand spinach leaves were studied. The presence of "carbonyl"reagents causes an increase in the ratio of ¹⁴CO₂ incorporatedin glycine and a decrease in serine. The incorporation of ¹⁴Cfrom glycolate-1-¹⁴C and glycolaldehyde-2-¹⁴C into glycine andserine was also affected by "carbonyl" reagents, as in the caseof ¹⁴CO₂-experiment. 2. The feeding experiments of glycine-1-¹⁴C and serine-1-¹⁴Cin the presence and in the absence of "carbonyl" reagents revealedthat these reagents inhibit the conversion of glycine to serine. 3. The results obtained above, together with the effects ofthiols on ¹⁴CO₂ incorporation presented in this paper, supportthe assumption that glycine and serine are formed via glycolateand glyoxylate during photosynthesis in green plants. 4. Comparison of ¹⁴C incorporation in malate from ¹⁴CO₂, glycolate-1-¹⁴C,glycine-1-¹⁴C and serine-1-¹⁴C in the presence and in the absenceof "carbonyl" reagents suggested the occurrence of the pathwayof the malate formation via glycolate and glyoxylate, not passingthrough glycine and serine, during photosynthesis. ¹ A part of this paper was presented at the Symposium on "Nitrogenand Plant" by the Japanese Society of Plant Physiologists, inOctober, 1963 ² Present address: Radiation Center of Osaka Prefecture, Sakai,Osaka     

REGIONAL ACCUMULATION OF TRYPTOPHAN AND SEROTONIN METABOLISM FOLLOWING TRYPTOPHAN LOADING IN DIABETIC RATS

Abstract— Streptozotocin-induced diabetes in rats reduces brain tryptophan but is without effect on the central levels of 5-hydroxytryptamine (5-HT) or 5-hydroxyindoleacetic acid (5-HIAA). The present work investigates the effect of diabetes on the accumulation of brain tryptophan, 5-HT and 5-HIAA in various brain regions following a systemic tryptophan load. The results indicate that diabetes severely restricts the uptake of tryptophan by brain but that the tryptophan that is accumulated is normally converted to 5-HT and 5-HIAA. Possible mechanisms which might explain the apparent resistance of 5-HT metabolism to decreased precursor levels in diabetics are discussed.     

苯丙氨酸合成的关键酶基因aroG与pheA串联表达

aroG和pheA是与苯丙氨酸合成有关的两个重要基因。在大肠杆菌(Escherichia coli)中,aroG基因编码脱氧阿拉伯糖型庚酮糖磷酸合酶(DS),该酶催化由糖代谢中心途径分流出来的磷酸烯醇丙酮酸(PEP)和赤鲜糖4磷酸(E4P)缩合形成脱氧阿拉伯糖型庚酮糖磷酸(DAHP)的反应;pheA基因编码一个双功能酶蛋白,它同时催化两步关键反应,即具有分枝酸变位酶(CM)和预苯酸脱水(PD)的两种功能。采用PCR技术分别从两个不同品系的大肠杆菌染色体DNA中扩增到aroG和pheA。当这两个基因串联在一个质粒上导入大肠杆菌P2392中进行表达时,它们编码的酶DS、CM和PD活性分别提高43、44和22倍;导入短杆菌(Brevibacterium)2731中表达时,相应的酶活性分别提高123、23和56倍。两基因的串联表达能大幅度地提高工程菌株的苯丙氨酸发酵产量。     

CAPABILITY OF TUBULIN AND MICROTUBULES TO INCORPORATE AND TO RELEASE TYROSINE AND PHENYLALANINE AND THE EFFECT OF THE INCORPORATION OF THESE AMINO ACIDS ON TUBULIN ASSEMBLY

Abstract— Incorporation of [¹⁴C]tyrosine into the C-terminal position of α-tubulin of rat brain cytosol was 10-fold higher for non-assembled than for assembled tubulin. The incorporation into tubulin from disassembled microtubules was higher than into non-assembled tubulin; therefore, the low incorporation into microtubules was not due to a lower acceptor capacity of their tubulin constituent.
[¹⁴C]Tyrosine was released from assembled and non-assembled [¹⁴C]tyrosinated tubulin by the action of an endogenous carboxypeptidase. Release from non-assembled tubulin was shown by incubating a tubulinyl-[¹⁴C]tyrosine preparation in the presence of CaCl₂ at a concentration that abolished microtubule formation. Release from microtubules was inferred from the observation that the percentages of [¹⁴C]tyrosine released and the decrease of the specific radioactivity of the recovered microtubules were practically identical and did not change after a 10-fold dilution of the incubated microtubules.
[³H]Phenylalanine was released from a preparation of tubulinyl-[³H]phenylalanine also by an enzymatic activity.
The capacity of a tubulin preparation to incorporate tyrosine was increased 43% by pre-treatment with endogenous carboxypeptidase.
Tubulin tyrosinated in vitro was assembled to the same extent as native tubulin. After a mixture of tubulinyl-[¹⁴C]tyrosine and tubulinyl-[³H]phenylalanine was partially assembled, the ratio of ¹⁴C/³H found in the microtubules was the same as in the non-assembled tubulin fraction.     

HETEROGENEITY OF ACETYLCHOLINESTERASE IN NEUROBLASTOMA

Abstract– Multiple forms of acetylcholine hydrolyzing activity have been observed in Triton X-100 treated homogenates of mouse neuroblastoma cells. All these forms appear to be the true acetylcholinesterase, AChE (EC 3.1.17): they are substrate-inhibited; hydrolyze acetylcholine > acetyl-β-methylcholine ≫ butrylcholine and are preferentially inhibited by specific AChE inhibitors. Almost all of the cell AChE activity is membrane associated, but readily 'solubilized' by Triton X-100 and as such appears free of membrane contamination. With the aid of affinity chromatography the 'solubilized' neuroblastoma AChE has been partially purified (490-fold) to a specific activity of 34,300 nmol/min/mg protein.
Four active neuroblastoma AChE species appear upon acrylamide gel electrophoresis (with MWs of 64,000; 116,000; 186,000 and 284,000) while three species (4S, 6S and 9.6S) have been found upon sucrose gradient sedimentation analysis. We have determined that the 4S form migrates on acrylamide as the 116,000 MW species and the 9.6S form contains, in equal amounts, the 186,000 and 284,000 MW acrylamide species. Numerous active AChE forms are seen on Sepharose 6B chromatography. From comparing the crude, 4S, 9.6S and partially purified AChEs on acrylamide gels, sucrose gradients and Sepharose, mouse neuroblastoma appears to contain active AChE units which are capable of multiple types of dissociation and reassociation. An attempt is made to correlate all the observed AChE forms as well as relate this data to that known about AChE obtained from other sources.     

DIFFERENCES IN THE REGULATION OF TYROSINE AMINOTRANSFERASE IN BRAIN AND LIVER

Abstract— l -Tyrosine:2-oxoglutarate aminotransferase (EC 2.6.1.5) activity in rat brain is not regulated in the same way as in rat liver. No diurnal rhythm in the activity of the cerebral enzyme was found in rats fed ad lib. although there was a marked diurnal variation in the activity of the hepatic enzyme. In adrenalectomized rats, hydrocortisone and glucagon induced the enzyme in liver but had no effect on the enzyme in brain. In normal rats, treatment with reserpine or exposure to cold elevated the activity of the hepatic enzyme without affecting the enzyme in brain. Thus, the tyrosine aminotransferase of brain differed from the enzyme in liver since it did not exhibit diurnal variations of activity and was not affected by hormones, drugs, or stress.     

MEGASPOROGENESIS AND MEGAGAMETOGENESIS IN SOYBEAN,GLYCINE MAX

Megasporogenesis and megagametogenesis were examined in Glycine max with light, fluorescence, and electron microscopy. Megasporogenesis results in a linear tetrad of four megaspores. Megagametophyte development is of the Polygonum type, with the functional chalazal megaspore undergoing three successive mitotic divisions to produce an eight-nucleate, seven-celled mature megagametophyte. The central cell becomes packed with starch. At fertilization, the antipodals are degenerate, the polar nuclei have fused, starch is diminished, and the egg occupies most of the micropylar portion of the megagametophyte. Several pollen tubes were occasionally observed at each micropyle, yet only one was involved in fertilization. Pollen tube entry occurs through a slightly reduced, viable synergid cell. Endosperm development precedes embryo growth. These results describing normal development allow important comparison with genetic mutants of soybean that affect female fertility.