COMPARTMENTATION OF AMINO ACID METABOLISM IN THE RAT POSTERIOR PITUITARY

—Isolated rat posterior pituitary glands were incubated with [¹⁴C]glucose or [¹⁴C]acetate and the incorporation of radioactivity into several amino acids was followed. The results indicated that radioactivity was incorporated from [¹⁴C]glucose into a large pool of glutamate which appeared to be responsible for a large proportion of GABA synthesis in the gland. The specific activity of glutamine was always less than that of glutamate when [¹⁴C]glucose was the precursor employed, whereas [¹⁴C]acetate labelled a glutamate pool which had approximately the same specific activity as that of glutamine. The results are discussed with reference to the compartmentation of amino acid metabolism in the nervous system.     

CARBOHYDRATE AND AMINO ACID METABOLISM IN RAT CEREBRAL CORTEX IN MODERATE AND EXTREME HYPERCAPNIA

—The time course of changes in glycolytic and citric acid cycle intermediates and in amino acids was studied in acute and steady state hypercapnia. Experiments on unanaesthetized animals exposed to 10% CO₂ for 10, 20 and 60s showed that there was a transient decrease in glycogen concentration, progressive increases in glucose-6-phosphate and fructose-6-phosphate and decreases in pyruvate and lactate. During this time the levels of amino acids and Krebs cycle intermediates did not change, except for a small fall in malate at 60s. The results indicate that there was a decrease in glycolytic flux due to an inhibition of the phosphofructokinase reaction. Since the tissue levels of phosphocreatine, ATP, ADP and AMP were unchanged inhibition of phosphofructokinase was probably due to the fall in pH. Anaesthetized animals were exposed to about 5% CO₂ (for 2, 5, 15, 30 and 60 min) or to about 45% CO₂ (for 5 and 15 min). Except for succinate, which increased, all citric acid cycle metabolites analysed (citrate, α-ketoglutarate, fumarate and malate) decreased with the rise in CO₂-tension. The sum of the amino acids analysed (glutamate, glutamine, aspartate, asparagine, alanine and GABA) decreased at extreme hypercapnia. The results suggest that Krebs cycle intermediates and amino acids are partly used as substrates for energy production when there is reduced pyruvate availability due to hypercapnia. It is proposed that amino acid carbon is made available for oxidation via transamination (aspartate aminotransferase reaction) and deamination (glutamate dehydrogenase reaction) and that citric acid cycle intermediates are metabolized following a reversal of reactions usually leading to CO₂ fixation.     

在生长、功能和萎退各期,大鼠乳腺磷酸酶的研究

前言虽然乳腺在生长、功能和萎退过程中形态上的变化是很大的(姚曾序、顾国彦,1957),可是和形态变化相伴随的化学和生物化学变化却研究得少。关于碱性磷酸酶(以下简称 AKP)方面,Kay(1925),Folley 和 Kay(1935)最早证明牛、羊和豚鼠乳腺含有相当量的这种酶。以后 Folley 和 Greenbaum(1947)进一步用生化方法研究了大鼠在怀     

STUDIES ON AMINO ACID LEVELS AND PROTEIN METABOLISM IN THE BRAINS OF GALACTOSE-INTOXICATED CHICKS1

Abstract— Levels of free amino acids, profiles of polyribosomes, and rates of protein synthesis and degradation were examined in the brains of chicks fed toxic levels of galactose. The content of a number of amino acids were altered; alanine and leucine were most strikingly depressed, whereas levels of aspartate were elevated. Polyribosomal profiles were unaltered. There appeared to be no detrimental effect on protein synthesis as judged by in vivo incorporation of L-[U-¹⁴C]leucine and L-[guanidino-¹⁴C]arginine. Likewise, the half-lives of proteins, measured by the loss of L-[guanidino-¹⁴C]arginine, were similar in experimental and control groups. In contrast, initial rates of incorporation of [³H]glucosamine into glycoproteins were enhanced. The effect was greatest in the microsomal fraction and typically 50 per cent greater than controls. Levels of free glucosamine and protein-bound hexosamine were essentially unaltered in the galactose-fed chicks.     

THE NATURE OF THE AMINO ACID POOL USED FOR PROTEIN SYNTHESIS IN RAT BRAIN SLICES

The incorporation into brain slice protein of externally provided [1-¹⁴C]valine was measured at varying levels of valine in the medium, under conditions of constant protein synthesis and equilibration of intracellular valine specific activity. The results indicate that the valine pool used for protein synthesis is not identical to the pool of total free valine. Neither does the incorporation solely occur from an extracellular pool which is in equilibrium with the incubation medium. The data are compatible with a two-site activation model in which aminoacylation of tRNA occurs at both an internal site utilizing amino acid from the intracellular pool and an external (possibly membranous) site converting extracellular valine directly to valyl-tRNA. A good fit to the experimental observations is also provided by a compartmented intracellular valine pool model.     

CORRELATION OF PLASMA AND BRAIN AMINO ACID AND PUTATIVE NEUROTRANSMITTER ALTERATIONS DURING ACUTE HEPATIC COMA IN THE RAT

During acute hepatic coma following two-stage hepatic devascularization in the rat, profound changes occurred in plasma and whole-brain amino acids and putative neurotransmitters. Brain ammonia, glutamine and GABA were increased, aspartate was decreased, while glutamate was unchanged. An increase in brain tryptophan was accompanied by a similar increase in plasma unbound tryptophan but decreased plasma total tryptophan. These changes occurred in the presence of high plasma levels of the other neutral amino acids, including the branched chain amino acids. Plasma insulin was unchanged while glucagon levels rose, resulting in a decreased insulin to glucagon ratio. These results suggest that while plasma unbound tryptophan may influence brain tryptophan levels, altered plasma concentrations of neutral amino acids which compete with tryptophan for transport into the brain do not contribute to the increase in brain tryptophan observed during acute hepatic coma.     

EFFECTS OF DIETARY PROTEINS ON FETAL BRAIN PROTEIN AND GLUTAMIC ACID METABOLISM IN RAT

Abstract— The effects of feeding dietary wheat and Bengal gram proteins to pregnant rats on brain protein and glutamic acid metabolism in 15-, 17- and 19-day fetuses were investigated. Wheat and Bengal gram diets resulted in loss of brain weight with decreased DNA, RNA, protein, free x amino N and deficits in the activities of brain glutamine synthetase, glutaminase I. glutaminase II and glutamate decarboxylase at all the gestational ages studied without any change in glutamine transferase activity. The concentrations of the amino acids alanine, glutamic acid, glutamine and GABA were found to be significantly lower on wheat and Bengal gram diets than the control on a 10% casein diet. The wheat with lysine and Bengal gram with methionine, cystine and tryptophan resulted in similar mean values of all the characteristics studied to the mean values observed in rats on the control diet. However, glutaminase I activity remained significantly low on lysine fortified wheat diet, and aspartic acid content was found to increase on both fortified and unfortified wheat and Bengal gram diets. A 20% casein diet showed increased brain weight, DNA. RNA. protein and free x amino N concentrations as compared with the 10% casein diet, while the other parameters remained unchanged.     

THYMIDINE METABOLISM AND DEOXYRIBONUCLEIC ACID SYNTHESIS IN THE DEVELOPING RAT BRAIN

—In growing rat brain, the specific activity of DNA at 12 h after the subcutaneous injection of [³H]thymidine underwent a sharp rise during the first 6 days of life, dropping just as precipitously by 15 days, thereafter continuing to decrease with increasing age. When [³H]thymidine was given to 6-day-old rats, a considerable amount was taken up immediately into the brain. Thymidine taken up into the acid-soluble fraction was readily phosphorylated to its nucleotides, thymidine mono-, di-, and triphosphate (TMP, TDP and TTP) within only 30 min following injection. The highest specific activity was found in TTP. The incorporation of of [3H]thymidine into DNA took place over a longer period of time after injection.     

AMINO ACID METABOLISM AND AMMONIA FORMATION IN BRAIN SLICES

The formation of ammonia and changes in the contents of free amino acids have been investigated in slices of guinea pig cerebral cortex incubated under the following conditions: (1) aerobically in glucose-free saline; (2) aerobically in glucose-free saline containing 10 mM-bromofuroic acid, an inhibitor of glutamate dehydrogenase (EC 1.4.1.2); (3) aerobically in saline containing 11-1 mM-glucose and (4) anaerobically in glucose-free saline. Ammonia was formed at a steady rate aerobically in glucose-free medium. The formation of ammonia was largely suppressed in the absence of oxygen or in the presence of glucose whereas the inhibitor of glutamate dehydrogenase produced about 50 per cent inhibition. Other inhibitors of glutamate dehydrogenase exerted a similar effect. Ammonia formation was also inhibited by some inhibitors of aminotransferases but not by others. Inhibition was generally more pronounced during the second and third hour of incubation. With the exception of glutamine which decreased slightly, the contents of all amino acids increased markedly during the anaerobic incubation. During aerobic incubation in a glucose-free medium, there was an almost complete disappearance of glutamic acid and GABA. Glutamine also decreased, but to a relatively smaller extent. The content of all other amino acids increased during aerobic incubation in glucose-free medium, although to a lesser extent than under anaerobic conditions. The greater increase of amino acids appearing anaerobically in comparison to the increase or decrease occurring under aerobic conditions corresponded closely to the greater amount of ammonia formed aerobically over that formed anaerobically. This finding is interpreted as indicating a similar degree of proteolysis under anaerobic and aerobic conditions; aerobically, the amino acids are partly metabolized with the concomitant liberation of ammonia. In glucose-supplemented medium, the content of glutamine was markedly increased. The content of glutamate and aspartate remained unchanged, whereas that of some other amino acids increased but to a lesser extent than in the absence of glucose. Proteolysis in the presence of glucose was estimated at about 65 per cent of that in its absence. In the presence of bromofuroate the rate of disappearance of glutamate was unchanged, but there was a larger increase in the content of aspartate and a smaller decrease of GABA and glutamine. Other changes did not differ significantly from those observed in the absence of bromofuroate. We conclude that the metabolism of amino acids in general and of glutamic acid in particular differs according to whether they are already present within the brain slice or are added to the incubation medium. Only the endogenous amino acids appear to be able to serve as precursors of ammonia and as substrates for energy production.     

THE RELATIONSHIP BETWEEN AMINO ACID INCORPORATION INTO PROTEIN IN ISOLATED NEOCORTEX SLICES AND THE TISSUE CONTENT OF FREE AMINO ACID

Abstract— The uptake of radioactive leucine by incubated neocortex slices was found to be increased by electrical stimulation, yielding a higher content of radioactive amino acid per g fresh weight of tissue which was maintained for prolonged periods of stimulation. The increased tissue content may be associated with tissue swelling found on electrical stimulation, but the additional amino acid uptake was by an active process rather than by passive diffusion. Additions of valine (2.5–10 m m ) or tryptophan (1 m m ) to the incubation medium was found to depress the tissue leucine content. Decreasing the tissue free leucine content by incubating slices in medium containing 5 m m -valine was found to decrease the incorporation of leucine and lysine into tissue protein, indicating that under these conditions tissue free amino acid becomes rate limiting for amino acid incorporation into protein. By analogy with the properties of cerebral tissue in oitro it is suggested that electrical activity in vivo may cause localized increases in free amino acid concentration which may serve to regulate protein synthesis in conditions where the concentration of free amino acids are rate limiting.     

EFFECTS OF METABOLIC INHIBITORS ON AMINO ACID METABOLISM IN RAT RETINA: A COMPARISON OF AMINO-OXYACETIC ACID AND ETHANOLAMINE-O-SULPHATE

Abstract— The abilities of AOAA and EOS to modify the utilisation of radioactively labelled glucose, acetate, glutamine and GABA were studied in isolated rat retina. AOAA inhibited the activities of GAD and GABA-T, while EOS inhibited GABA-T but not GAD. AOAA lowered the free amino acid contents of incubated retinae and suppressed the outflow of amino acids into the incubation medium, while EOS had no effect on either parameter. AOAA strongly inhibited the incorporation of ¹⁴C from labelled glucose, acetate and glutamine into GABA, and also suppressed the labelling of glutamate, aspartate and glutamine. These effects were qualitatively similar but quantitatively smaller with EOS. Both compounds markedly decreased the syntheses of aspartate and glutamate from exogenous GABA, while the passage of carbon from GABA to glutamine was much less affected. It is suggested that AOAA and EOS may act predominantly on neurones. It appears that inhibition of GABA-T alone does not cause a profound disturbance of the metabolism of other amino acids. Other metabolic inhibitors such as ouabain, malonate and fluoroacetate did not greatly affect the metabolism of GABA in rat retina.     

CONCOMITANT SYNTHESIS OF MEMBRANE PROTEIN AND EXPORTABLE PROTEIN OF THE SECRETORY GRANULE IN RAT PAROTID GLAND

After enzyme secretion the membrane of the secretory granule, which had been fused to the cell membrane, was resorbed into the cell. Experiments were therefore carried out to test whether formation of new secretory granules involves reutilization of the resorbed membrane or synthesis of a new membrane, de novo, from amino acids. Incorporation of amino acids-¹⁴C into proteins of various cell fractions was measured in vivo, 30, 120, and. 300 min after labeling. At all times the specific radioactivity of the secretory granule membrane was about equal to that of the granule's exportable content. At 120 and 300 min the specific radioactivity of the granule membrane and of the granule content was much higher than that of any other subcellular fraction. It is therefore concluded that the protein of the membrane is synthesized de novo concomitantly with the exportable protein. The proteins of the granule membrane could be distinguished from those of the granule content by gel electrophoresis. All major bands were labeled proportionately to their staining intensity. The amino acid composition of the secretory granule membrane was markedly different from that of the granule's content and also from that of the mitochondrial membrane. The granule membrane showed a high proline content, 30 moles/100 moles amino acids. The analyses show that the radioactivity of the granule membrane is indeed inherent in its proteins and is not due to contamination by other fractions. The possibility is considered that the exportable protein leaves the endoplasmic reticulum already enveloped by the newly synthesized membrane.     

PROTEIN AND NUCLEIC ACID METABOLISM IN INSECT FAT BODY

1. The appearance of larval fat body as seen under the light or electron microscope depends on the nutritional state of the larva and on the stage of larval development at which the fat body is observed. 2. Early in the last larval instar the cells usually possess a well-developed endo-plasmic reticulum rich in ribosomes, numerous mitochondria, glycogen granules, a Golgi complex and fat droplets, while later in the instar the endoplasmic reticulum is much reduced and mitochondria are few, but glycogen and fat droplets are present in greater amount together with the appearance of large numbers of proteinaceous spheres. 3. Early in the last instar the fat body synthesizes proteins and exports them into the blood, while later in the instar proteins are sequestered from the blood into the fat body. 4. The rate of protein synthesis by the fat body is high in the early to mid part of the last instar, but then falls off rapidly to a low level, at which it remains until the larva pupates. In diapausing pupae, protein synthesis remains at this low level. 5. The similarity between the electrophoretic patterns of proteins from the fat body and those from the blood provides strong evidence that the fat body is the site of synthesis of many of the blood proteins. 6. Some of the blood proteins have been shown to possess enzymic properties, while others are thought to play a role in the transportation of various types of compounds. 7. Ecdysone and juvenile hormone both stimulate the rate of protein synthesis by larval fat body. Protein synthesis in fat body from diapausing pupae is stimulated after injury to the pupae. 8. The appearance of adult fat body and the amount of protein it contains is often closely linked with the nutritional and reproductive states of the insect. 9. An important role of the fat body in the adult female insect is the synthesis of yolk proteins, which are released into the blood and then taken up by the developing oocytes. This synthesis and uptake are under the control of hormones secreted by the corpora allata and by the median neurosecretory cells of the pars intercerebralis. 10. The RNA content of fat body in final-instar larvae is not constant throughout the instar. In some larvae it is at its highest level early in the instar, falling to a low level as the instar progresses, while in other larvae (e.g. Calliphora) the level of RNA in fat body does not decrease as the instar progresses. 11. In some dipterous insects the base composition of total RNA is DNA-like in that the guanine + cytosine content is low, accounting for 40 % of the bases. A similar composition is seen in rapidly labelled RNA isolated from insects of other orders (Coleoptera and Lepidoptera), but the base content of total RNA from these latter insects resembles ribosomal RNA from vertebrate tissues in that it has a high (ca. 60 %) guanine + cytosine content. 12. The RNA/DNA ratios in blowfly larval tissues are high compared with those found in any vertebrate tissue. 13. In larval fat body, RNA synthesis is low at the time of a moult, increases during the early and mid-instar period and subsequently falls during the latter part of the instar. During the pupal period, especially during pupal diapause, the rate of RNA synthesis is very low and then increases during the subsequent development of the pharate adult. Injury to diapausing pupae results in an increased rate of RNA synthesis in most of their tissues. 14. Ecdysone and juvenile hormone both stimulate RNA and DNA synthesis in larval and adult fat body and in other tissues, although there is evidence that in some tissues these two hormones may act antagonistically to each other. The insecticide DDT also has been shown to stimulate RNA synthesis in tissues of adult insects.     

水稻温敏失绿突变体的Rubisco活化酶活性、蛋白质与氨基酸组分的变化

在水稻温敏失绿突变性状表达过程中,对其Rubsico 含量、Rubsico 活化酶活性,全叶蛋白及游离氨基酸组分变化进行测定。结果表明:突变体的Rubisco 结构和含量与野生型一样,保持相对稳定;而其Rubisco 活化酶活性则随一个分子量为56.2kD(PI=4.5)的特异蛋白质的存在与消失发生明显改变。当突变性状表达时,分子量为56.2kD(PT=4.5)的特异蛋白消失,其Rubisco 活化酶活性下降;当叶片失绿区域复绿时,56.2kD(PI=4.5)特异蛋白出现,则Rubisco 活化酶活性上升。这一密切地相关关系表明,突变体的Rubisco 活化酶活性变化在光合作用过程中,除与自身结构和含量有关外,还与叶片中这一特异蛋白的存在密切相关,它可能是Rubisco 活化酶活性的调节蛋白。这种调节具体表现在氨基酸代谢上,是对上游氨基酸的阻遏调控,从而使叶绿体的结构物质合成受阻,最终导致类囊体膜的退化。     

GLUCOSE AND AMINO ACID METABOLISM IN OCTOPUS OPTIC AND VERTICAL LOBES IN VITRO

(1) The metabolism of glucose and amino acids in vitro was compared in the rat cerebral cortex and the optic and vertical lobes of the octopus brain. (2) Specific activities and pool sizes of the five amino acids, glutamate, aspartate, glutamine, alanine and γ-aminobutyric acid (GABA), were determined in octopus and rat brain slices after 2 hr incubation with 10 mm -[U-¹⁴C]glucose, 10 mm -L-[U-¹⁴C]glutamate, and 10mm -^L-[U-¹⁴C]glutamate with added 10 mM-glucose. Amino acid pool sizes were similar in rat and octopus brain, with the exception of alanine, which was higher in the octopus. Generally specific activities were from four- to 20-fold higher in rat brain. With [U-¹⁴C]glucose as substrate, specific activities of GABA and glutamate were highest in rat; those of alanine and glutamine highest in octopus brain. With ^L-[U-¹⁴C]glutamate the specific activities of GABA and aspartate were highest in rat, that of aspartate highest and GABA lowest in octopus. The addition of glucose to ^L-[U-¹⁴C]glutamate as substrate had little effect on the specific activities of any of the amino acids. (3) The uptake of some amino acids was determined by incubation with [U-¹⁴C]amino acids for 2 hr, and ¹⁴CO₂ formation was also measured. The amount of label taken up by octopus was uniformly 20-25 per cent of that found for rat brain. The amount of ¹⁴CO₂, however, differed according to the amino acid. Four times as much ¹⁴CO₂ was generated from alanine by octopus optic lobe and twice as much by the vertical lobe than rat cortex, but from glutamate, only 24 per cent in the optic and 15 per cent in the vertical lobe. No ¹⁴CO₂ was generated from [U-¹⁴C]GABA in the octopus, by contrast with the rat. (4) Activity of some of the enzymes involved in amino acid metabolism was determined in homogenates of rat cortex and octopus optic and vertical lobes, with and without activation by Triton X-100. Enzymic activities in the octopus, with the exception of alanine aminotransferase, were lower than in the rat, and glutamate decarboxylase could not be detected in octopus brain, in the absence of detergent.