ISOLATION OF HYDROPHOBIC PROTEINS BINDING AMINO ACIDS: γ-AMINOBUTYRIC ACID BINDING IN THE RAT CEREBRAL CORTEX

—The binding of [¹⁴C]GABA to nerve-ending membranes isolated from rat cerebral cortex follows a hyperbolic curve saturating at 0·4pmol/μg protein. This binding is about 60% inhibited by chloropromazine, and about 40%, inhibited by bicuculline. A hydrophobic protein fraction binding [¹⁴C]GABA was separated from the total. lipid extract of nerve-ending membranes. The binding follows a hyperbolic curve that saturates at 10·5 pmol of [¹⁴C]GABA/μg of protein, with an apparent K_d= 30 μm . The binding is competitively inhibited by bicuculline with a K_i= 273 μm . These results are compared with those previously obtained on a GABA binding protein from crustacean muscle.     

ISOLATION OF HYDROPHOBIC PROTEINS BINDING AMINO ACIDS. STEREOSELECTIVITY OF THE BINDING OF L-[14C]GLUTAMIC ACID IN CEREBRAL CORTEX

From the total lipid extract of ncrve-ending membranes or the homogenate of cerebral cortex a hydrophobic protein fraction binding L-[¹⁴C]glutamic acid was separated by chromatography on Sephadex LH20. This protein could only be partially separated from the [¹⁴C]GABA-binding protein and from the lipids that are present in the fraction; however, it was demonstrated that both amino acids bind to different sites. The saturation of the binding showed a high (Kd₁= 0.3μM), a medium (Kd, = 5 μM) and a low (Kd, = 55 μM) affinity binding site. The high affinity binding site had a binding capacity of 0.53 nmol/mg of protein and was highly stereoselective for the L-enantiomer. The binding of L-[¹⁴C]glutamic acid was not inhibited by GABA, was slightly inhibited by glycine and glutamine and was strongly inhibited in a progressive order by DL-a-methylglutamic acid, L-nuciferine, L-aspartic acid and L-glutamic acid diethyl ester. These results are compared with those previously obtained with the L-glutamic acid-binding protein isolated from crustacean muscle. The stereoselectivity of the binding and the possible role of this protein in synaptic transmission are discussed.     

ISOLATION OF HYDROPHOBIC PROTEINS BINDING NEUROTRANSMITTER AMINO ACIDS: γ-AMINOBUTYRIC ACID RECEPTOR OF THE SHRIMP MUSCLE

Abstract— The total lipid extract of shrimp muscle ( Artemisia longinaris ) was precipitated with ether. The supernatant containing 95 per cent of the phospholipids and 50 per cent of the protein showed binding for L-[¹⁴C]glutamatc in the first peak of protein. The sediment, redissolved in chloroform-methanol was chromatographed on a Sephadex LH-20 column. A single peak was eluted in the chloroform (20-40 ml) having no lipid phosphorous and high affinity binding for [¹⁴C]GABA. The saturation was achieved at 1 mole per 80.000 g protein and the curve revealed a single type of binding site. The purification achieved was of about 4000-fold. There was no binding of L-[¹⁴C]glulamate to the ether precipitate. The specificity of the binding of [¹⁴C]GABA was further supported by competition experiments with bicuculline. picro-toxin and muscimol. It is suggested that the hydrophobic protein isolated by us represents the GABA receptor. The findings presented in the two papers of the series suggest that the excitatory and inhibitory receptor from crustacean muscle can be separated as two different proteins.     

DEVELOPMENTAL TRANSITIONS IN UPTAKE OF AMINO ACIDS BY SYNAPTOSOMAL FRACTIONS ISOLATED FROM RAT CEREBRAL CORTEX

Developmental changes in mechanisms of synaptosomal amino acid transport have been studied in rat cerebral cortex. Well-defined changes over an age continuum could be observed in both the rates of amino acid accumulation and the effects of Na⁺ on the accumulation. The uptakes of five amino acids (threonine, serine and valine in Na⁺-free medium, aspartic acid and proline in Na⁺-containing medium) increased progressively with the age of the animal, whereas the uptakes of leucine and arginine (in Na⁺-free medium) decreased steadily. The uptake of serine or threonine by synaptosomal fractions prepared from newborn rats was markedly dependent on the presence of Na⁺in the incubation media. Na⁺exerted progressively less effect on the accumulation process with continuing postnatal development and to some extent inhibited uptake by fractions obtained from rats older than about 15 days. Na⁺significantly enhanced the accumulation of glycine in fractions from newborn and adult rats, but had only a slight effect in fractions prepared from 12 to 17-day old rats. A detailed study of the accumulation of glycine indicated that the synaptosomal transport of this amino acid proceeded by two independent systems, one of which was totally dependent on external Na⁺and the and adult animals than in fractions from 12 to 17-day-old rats, wheras the Na⁺-independent system was most active during this latter period of development. The decline in the Na⁺-independent accumulation of glycine from about the 15th day to adulthood was characterized by a decrease in the V_max. and an increase in the K_m.     

EFFLUX OF PHENYLALANINE FROM RAT CEREBRAL CORTEX SLICES AS INFLUENCED BY EXTRA- AND INTRACELLULAR AMINO ACIDS

Abstract— Effects of other amino acids on the efflux of l -[³H]phenylalanine from rat cerebral cortex slices were studied in a superfusion system. Extracellular large neutral amino acids caused a strong trans-stimulation of [³H]phenylalanine efflux. Some small neutral amino acids were less effective, whereas acidic and basic amino acids and the amino acids without an amino group in the α-position were ineffective. Any trans -inhibition was not detected. The stimulatory trans -effects of phenylalanine and tryptophan were additive, reversible and concentration-dependent. They were apparently mediated by the same mechanisms. The efflux of [³H]phenylalanine was much slower at 273 K than at 310 K, but the effects of unlabelled phenylalanine and tryptophan on it were qualitatively similar at both temperatures. Amino acids accumulated intracellularly at moderately high concentrations did not inhibit [³H]phenylalanine efflux, but phenylalanine, leucine, isoleucine and norleucine caused an enhancement. Spontaneous efflux of [³H]phenylalanine showed some similarities to physical diffusion, but its selective and specific modification by other amino acids strongly suggests the involvement of mediated processes.     

INCORPORATION OF AMINO ACIDS INTO PROTEINS IN NEURONAL AND NEUROPIL FRACTIONS OF RAT CEREBRAL CORTEX: PRESENCE OF A RAPIDLY LABELLING NEURONAL FRACTION

Abstract— The time course of incorporation of intraperitoneally injected [³H]lysine and [¹⁴C]phenylalanine into neuronal and neuropil proteins has been followed for up to 8 days. At short times after injection (<2 h) the specific activity of the neuronal fraction was higher than that of the neuropil. At longer time intervals, although the total brain specific activity continued to rise, neuronal perikaryal specific activity fell below that of neuropil. Thus the neuronal/neuropil incorporation ratio with [³H]lysine as substrate was 1·5 at 1 h, but by 4 h had fallen to 0·4, a ratio which was maintained for up to 8 days. A similar reversal occurred with phenylalanine as substrate. These changes were interpreted as evidence for the presence of a rapidly-labelling protein fraction in the neurons which is subsequently transported out. Subcellular fractionation showed that over the 4 h period the rapidly labelling fraction was not transported to the synaptosomes. Incubation of prelabelled cortex slices followed by cell fractionation showed that a differential transport of protein of higher than average specific activity from both neurons and neuropil fractions occurred; there is a tendency for preformed highly labelled protein to accumulate during the in vitro incubation in Fraction D, a pellet enriched in red cells, some large neuronal perikarya and cell nuclei. When cell fractions were prepared after in vitro incubation, the distribution of the material down the gradient differed from that when fresh tissue was fractionated, as demonstrated by microscopic examination and the distribution of β-galactosidase, a neuronal marker. Double-label experiments showed that this redistribution could not account for the preferential loss and accumulation of prelabelled protein. It was noted that in vivo incorporation into the rapidly labelling neuronal protein is suppressed under certain changed environmental conditions, such as dark rearing. This is interpreted as lending support to the concept of the state-dependence of neuronal and neuropil protein synthesis and their inter-relations.     

AMINO ACIDS IN SYNAPTIC VESICLES FROM MAMMALIAN CEREBRAL CORTEX: A REAPPRAISAL

Synaptic vesicles were prepared from rat cerebral cortex and separated by gel filtration from small molecular weight compounds contaminating this fraction. Electron microscopy of the vesicle suspension showed that vesicles were by far the most abundant morphological entities. The amino acid content of the purified vesicle fraction was examined and the two amino acids appearing in the most significant amounts were found to be taurine and glutamate. This amino acid pool was not osmotically sensitive as is the vesicular pool of ACh and remained attached to the vesicular protein after passage through Sephadex columns equilibrated in water. However, amino acids added to the vesicle fraction prior to passage through Sephadex did not become associated with this pool and this indicated that the vesicular pool was not likely to be an artifact due to the vesicular protein non-specifically adsorbing amino acids. The release of taurine from incubated synaptosome beds was studied and elevated medium K⁺ (56 mm ) was found to cause a small increase (36 per cent) in the amount of the taurine released to the medium. During the same experiments another physiologically active amino acid, glutamate, was released in more significant amounts, increasing in the medium by 186 per cent. The possible significance of the presence of taurine is discussed.     

ISOLATION OF HYDROPHOBIC PROTEINS BINDING NEUROTRANSMITTER AMINOACIDS. GLUTAMATE RECEPTOR OF THE SHRIMP MUSCLE

Abstract— –Muscle of shrimps ( Artemisia longinaris ) were extracted with chloroform-methanol (2:1, v/v) and the proteolipids were separated by column chromatography on Sephadex LH-20. Three peaks of protein were eluted with chloroform and one with chloroform-methanol (4:1, v/v). Only the first peak eluted between 16 and 26 ml of chloroform showed binding for l -(¹⁴C]-glutamate. The type of saturation curve obtained suggests the existence of single type of binding site. The saturation is reached at one mole of l -glutamate per 320,000 g protein and the purification achieved about 3200-fold. The protein binding-glutamate does not bind GABA, aspartate or glutamine. The binding of l -[¹⁴C]-glutamate was inhibited by dl -α -methyl glutamic acid and l -glutamic acid diethyl ester. The binding properties of this hydrophobic protein fraction suggest that it may represent the isolated glutamate receptor of the shrimp muscle.     

EFFECTS OF TETRODOTOXIN, CALCIUM AND MAGNESIUM ON THE RELEASE OF AMINO ACIDS FROM SLICES OF GUINEA-PIG CEREBRAL CORTEX

Abstract— Tetrodotoxin, Ca²⁺-deprivation and high-Mg²⁺ were used in an effort to identify the portion of the evoked release of endogenous amino acids, labelled via metabolism of [¹⁴C]-glucose, and several exogenous labelled amino acids, that came from nerve terminals when slices of guinea pig cerebral cortex were superfused with glucose-free solutions and stimulated electrically. With some exceptions, spontaneous release of labelled amino acids was decreased by 2 μm -tetrodotoxin but increased in Ca²⁺-free medium and in solutions containing an extra 24 mm -MgCl₂. Tetrodotoxin suppressed 85–90% of the stimulated release of almost all labelled amino acids, but had a smaller effect on the release of endogenous ¹⁴C-labelled threonine-serine-glutamine (unseparated). In Ca²⁺-free solution, the stimulated release of endogenous ¹⁴C-labelled glutamate, aspartate and GABA was suppressed by 80–90%, but that of endogenous ¹⁴C-labelled threonine-serine-glutamine was unaffected as was most of the release of the other labelled amino acids. In medium containing an extra 24mM-MgCl₂, the stimulated release of endogenous ¹⁴C-labelled glutamate, aspartate and GABA was suppressed by 75-85%, that of exogenous labelled aspartate and GABA by 50–65%, but the release of the other labelled amino acids was unaffected. The control stimulated releases of endogenous ¹⁴C-labelled glutamate, aspartate and GABA were much larger than those of other labelled amino acids but were reduced by tetrodotoxin, Ca²⁺-deprivation and high-Mg²⁺ to a level similar to that of the control stimulated releases of the other labelled amino acids. These results suggest that almost all of the stimulated release of endogenous ¹⁴C-labelled glutamate, aspartate and GABA came from nerve terminals while those of the other labelled amino acids came from other tissue elements. In addition, they are in accord with a transmitter role for glutamate, aspartate and GABA in cerebral cortex.     

EFFECT OF PHOSPHATIDYLSERINE ON ACETYLCHOLINE OUTPUT FROM THE CEREBRAL CORTEX OF THE RAT

The effect of sonicated suspensions of phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine injected intravenously on acetylcholine release from the cerebral cortex was investigated in urethane anaesthetized rats. The electroeorticogram was also recorded. Phosphatidylserine caused a dose dependent, calcium dependent increase in acetylcholine output with no electrocorticografic changes. The increase, 75% peak effect after 150 mg/kg, was abolished by septal lesions and pretreatment with pimozide. Phosphatidylserine had no effect on acetylcholine release from brain slices in vitro. Phosphatidylethanolamine was approximately half as active as phosphatidylserine and phosphatidylcholine had no effect on acetylcholine output in vivo. It is concluded that phosphatidylserine exerts an indirect stimulating action on a septio-cortical cholinergic pathway.     

大鼠大脑皮质星形胶质细胞条件培养液促进小脑皮质神经元的生长

本研究从大鼠大脑皮质分离、纯化星形胶质细胞,再经培养后收集星形胶质细胞的无血清条件培养液。用盖玻片培养法与快速自动比色微量分析法研究了星形胶质细胞条件培养液对小脑皮质神经元生存以及神经元活力的影响。发现星形胶质细胞条件培养液能够明显提高小脑皮质神经元的体外存活率,增强神经元的活力。表明星形胶质细胞具有神经营养性作用。     

NONRIBOSOMAL INCORPORATION OF AMINO ACIDS INTO THE TROPONIN-LIKE PROTEIN FROM SYNAPTOSOMES

A preparation of synaptosomal cytoplasm was isolated from forebrain of young rats and incubated with various amino acids in vitro. Incorporation of amino acids into protein was observed. This incorporation did not occur by ribosomal protein synthesis. The amino acid incorporating system was not stimulated by ATP and was inhibited by calcium. The system incorporated amino acids enzymatically. An electrophoretic analysis of the synaptosomal preparation, following incubation in the presence of radioactive amino acids, showed only three labelled protein species (molecular weights 37,000, 26,000 and 20,000). This incorporation of amino acids was found to have a high degree of specificity for three protein species. Migration of the three protein species was found to be nearly identical to that of rabbit muscle troponin. The proteins incorporating amino acids were also found to have other characteristics of the troponin subunits. A possible role of troponin modification is discussed.     

亲和层析法分离腺苷结合蛋白质

本文报告一种新的腺苷亲和层析凝胶的合成方法。利用这种凝胶可从大鼠心脏、肝脏及小牛主动脉平滑肌的水溶部份分离出几种腺苷结合蛋白质,其亚基分子量(据SDS-PAGE)分别为35,000、37,000、46,000、43,000及15,300Dal。现已证明,35,000Dal蛋白质是乳酸脱氢酶及苹果酸脱氢酶,43,000Dal蛋白质是腺苷激酶,46,000Dal蛋白质可能是S-腺苷同型半胱氨酸水解酶。15,000Dal蛋白质前人未有报道。它对腺苷具有高度特导性和亲和力,推测是腺苷的细胞内受体和/或载体。测定了这种低分子量腺苷结合蛋白质的氨基酸组成及某些物理常数:pI=6.5;沉降系数2.42S,微分比容0.727cm~3/g,与腺苷复合物的解离常数K_D=2.3μM。     

STUDIES ON SOLUBLE PROTEINS RELEASED FROM THE SYNAPTIC VESICLES OF RAT BRAIN CORTEX

Abstract— The soluble proteins released from the synaptic vesicles of rat cerebral cortex were studied. One fraction (D₄) of these proteins was released in parallel with release of acetylcholine when synaptic vesicles were incubated at 37°C for 10 min in isotonic medium. Another fraction (Dj) was liberated from synaptic vesicles when their membranes were ruptured by mild treatment under hyposmotic conditions and freeze-thawing after release of D₁ fraction. Fractions D₁ and D₂ contained 12 and 9 per cent, respectively, of the total protein in the synaptic vesicles. Some properties of these fractions were investigated by zone electrophoresis and ultracentrifugation, and by measuring their binding capacities for [¹⁴C]acetylcholine and various enzyme activities related to acetylcholine metabolism.     

THE ABSENCE OF ''SURPLUS'' ACETYLCHOLINE FORMATION IN PRISMS PREPARED FROM RAT CEREBRAL CORTEX

Abstract— Prisms of rat cerebral cortex incubated without cholinesterase inhibitor showed an increase in ACh content during the first hour of incubation. The effects of adding cholinesterase inhibitors during the second hour depended on the potassium concentration. At 6 mM-K⁺ there was only a small rise in ACh but when K⁺ was raised to 25 mM a large increase in ACh content was observed. It is proposed that the increase in ACh is due to the reuptake of released ACh.