SOME PROPERTIES OF GLUTAMATE DECARBOXYLASE AND THE CONTENT OF PYRIDOXAL PHOSPHATE IN BRAINS OF THREE VERTEBRATE SPECIES

—Some properties of glutamate decarboxylase (GAD) were studied in the brain of the carp (Carassius auratus), the pigeon (Columbia livia) and the mouse (Mus musculus). The optimum pH for GAD in the three species was 6·3-6·5. In the three species studied, GAD activity of brain homogenates in water was higher than that of homogenates in buffer. The supernatant from homogenates in Triton-X-100 gave an enzyme preparation which showed greater activation by pyridoxal phosphate than those obtained from complete water or buffer homogenates or from the supernatant of Water homogenates. In the absence of pyridoxal phosphate, the activity of carp GAD was considerably lower than that of mouse or pigeon GAD. The addition of pyridoxal phosphate resulted in a much greater activation of carp GAD than that of pigeon or mouse GAD. Pyridoxal phosphate content was also measured in brains of the species studied. The difference between coenzyme levels in carp and mouse was very small in comparison to the difference in GAD activity in the absence of exogenous coenzyme. The pyridoxal phosphate content of pigeon brain was higher than that of the other two species.     

STUDIES ON THE REGULATION OF GABA SYNTHESIS: THE INTERACTION OF ADENINE NUCLEOTIDES AND GLUTAMATE WITH BRAIN GLUTAMATE DECARBOXYLASE

The effects of adenine nucleotides and glutamate on glutamate decarboxylase were studied in a dialyzed, high-speed supernatant of rat brain. When incubated with 10 μm -pyridoxal-P the enzyme was strongly inhibited by ATP, ADP and their Mg²⁺ complexes at concentrations which were well below tissue levels. The enzyme was not significantly inhibited by 15 mm -AMP or by 100 μM-3′-5’cyclic AMP or 3′-5’cyclic GMP. Inhibition by the nucleotides cannot be described in conventional steady-state kinetic terms. Addition of ATP in the presence of pyridoxal-P resulted in a slow, progressive decrease in the reaction rate which was similar to the inactivation observed when the enzyme was incubated in the absence of pyridoxal-P. The progressive inactivation in the presence of ATP was minimal at concentrations of glutamate which were well below K_m and became much more pronounced at higher glutamate concentrations. Addition of suprasaturating amounts of pyridoxal-P late in the incubation when the enzyme was almost completely inactivated resulted in an immediate and complete reactivation of the enzyme. Inhibition by ATP could be prevented by addition of saturating amounts of pyridoxal-P at the start of the reaction and was also relieved by addition of potassium phosphate buffer. The results suggest that inhibition by the nucleotides involves the prior formation of the inactive apoenzyme which results from the glutamate-promoted dissociation of pyridoxal-P. In the absence of the nucleotides, the enzyme is normally reactivated by the added pyridoxal-P. The nucleotides act to block this reassociation of pyridoxal-P with the apoenzyme thereby producing a progressive inactivation of the enzyme. The implications of these results for the regulation of GABA synthesis are discussed.     

KINETICS OF BRAIN GLUTAMATE DECARBOXYLASE. INTERACTIONS WITH GLUTAMATE,PYRIDOXAL 5-PHOSPHATE AND GLUTAMATE-PYRIDOXAL 5-PHOSPHATE SCHIFF BASE1

Abstract— The kinetic behavior of glutamate decarboxylase from mouse brain was analyzed in a wide range of glutamate and pyridoxal 5′-phosphate concentrations, approaching three limit conditions: (I) in the absence of glutamate-pyridoxal phosphate Schiff base; (II) when all glutamate is trapped in the form of Schiff base; (III) when all pyridoxal phosphate is trapped in the form of Schiff base. The experimental results in limit condition (I) are consistent with the existence of two different enzyme activities, one dependent and the other independent of free pyridoxal phosphate. The results obtained in limit conditions (II) and (III) give further support to this postulation. These data show that the free pyridoxal phosphate-dependent activity can be abolished when either all substrate or all cofactor are in the form of Schiff base. The free pyridoxal phosphate-independent activity is also abolished when all substrate is trapped as Schiff base, but it is not affected by the conversion of free pyridoxal phosphate into the Schiff base. A kinetic and mechanistic model for brain glutamate decarboxylase activity, which accounts for these observations as well as for the results of previous dead end-inhibition studies, is postulated. Computer simulations of this model, using the experimentally obtained kinetic constants, reproduced all the observed features of the enzyme behavior. The possible implications of the kinetic model for the regulation of the enzyme activity are discussed.     

CALCIUM-DEPENDENT BINDING OF BRAIN GLUTAMATE DECARBOXYLASE TO PHOSPHOLIPID VESICLES

The binding of glutamate decarboxylase (GAD), to phospholipid vesicles (liposomes) in the absence and in the presence of several Ca²⁺ and Mg²⁺ concentrations was studied. Phosphatidylcho-line-phosphatidylserine (4:1) liposomes are capable of binding GAD in a Ca²⁺-dependent manner. The per cent of GAD bound increased from 5 to 65°., in a sigmoid shape with Ca²⁺ concentrations in the 0.2-4 mm range. Mg²⁺ also induces GAD binding but is less effective than Ca²⁺ The Ca²⁺ -dependent binding of GAD is not the result of unspecific association of protein, since Ca²⁺ did not promote any binding of choline acetyltransferase or lactate dehydrogenase. Furthermore, the relative specific activity (^o_o enzyme activity/% protein) of GAD associated to liposomes increases 4-fold from 0 to 2 mm Ca²⁺. The per cent of GAD bound attains a plateau at a ratio phospholipid/protein of about 1.5. and decreases when the pH increases from 6.5 or 6.8 to 7 or 7.25. Na⁺ or K⁺ at a 100mm concentration also induce binding of GAD to liposomes. Phosphatidylcholine liposomes (without phosphatidylserine) practically did not bind GAD at any Ca²⁺ concentration. The Ca²⁺-dependent association of GAD to phosphatidylcholine-phosphatidylserine liposomes is very similar to that previously reported using brain membranes, and it correlates also well with the reported Ca²⁺-dependent aggregation of phosphatidylserine molecules in phospholipid membranes of similar composition. It is concluded that phosphatidylserine is probably involved in the Ca²⁺-dependent binding of GAD to brain membranes. Phospholipid vesicles seem to be a useful experimental model for studying the mechanisms of this GAD association to membranes and the possible physiological implications of the GAD-Ca²⁺-membrane interaction regarding the release of newly synthesized GABA from nerve endings.     

维生素C衍生物对癌细胞浸润转移能力的抑制作用机理研究

前期研究表明Asc2P6P1m能够有效地抑制癌细胞的浸润转移。本文试图以Asc2P6P1m对人成纤维瘤细胞浸润转移作用探讨维生素C衍生物对癌细胞转移能力抑制的机理。对HT-1080细胞分别以50—300μmol/LAsc2P6P1m处理1h,随着Asc2P6P1m浓度的增大,细胞移动的数目明显减少,Asc2P6P1m对HT-1080细胞移动的抑制作用呈现出量效关系。Asc2P6P1m对ROS的清除作用,通过自旋捕集剂DMPO以电子自旋共振方法进行研究。HT-1080细胞经Asc2P6P1m处理后,细胞内的自由基水平与对照组相比有显著的降低。用F-actin的分子探针NBD研究表明,随处理时间延长,细胞内荧光强度与对照组相比显著降低。Western blots研究表明,细胞核内的RhoA蛋白量随Asc2P6P1m处理时间延长而逐渐增加。研究提示,Asc2P6P1m对癌细胞浸润转移能力的抑制作用是与抑制癌细胞内的ROS、提高细胞核内RhoA水平、降低细胞质内F-actin相关。     

THE SYNTHESIS OF GLUTAMATE BY RAT BRAIN MITOCHONDRIA

The synthesis of glutamate from 2-oxoglutarate generated by the citric acid cycle and ammonium acetate has been studied in brain mitochondria of synaptic or non synaptic origin. Non synaptic brain mitochondria synthesise glutamate at twice the rate (1.3 nmol. min^?1. mg protein^?1) of synaptic mitochondria (0.65 nmol. min^?1. mg protein^?1) when pyruvate is the precursor for 2-oxoglutarate, but at a similar rate (0.9 and 0.7 nmol. min^?1, mg protein^?1) when 3 hydroxybutyrate is the precursor. Glutamate synthesis from ammonium acetate and extramitochondrially addcd 2-oxoglutarate (5 mM) by both synaptic and nonsynaptic mitochondria was 5-fold higher (5-6nmol. min^?1. mg protein^?1) than glutamate synthesis from endogenously produced 2-oxoglutarate. In the uncoupled state (or un-coupler + oligomycin) the rate was reduced by half. (2.5-3 nmol. min^?1. mg protein^?1) as compared to mitochondria synthesising glutamate in states 3 or 4 (± oligomycin). The changes in brain mitochondrial nicotinamide nucleotide redox state have been monitored by fluorimetric, spectrophotometric and enzymatic techniques during glutamate synthesis and compared with liver mitochondria under similar conditions. On the instigation of glutamate synthesis by NH⁺₄ addition a significant NAD(P)H oxidation occurs with liver mitochondria but no detectable change occurs with brain mitochondria. Leucine (2 mM) causes a doubling of glutamate synthesis by both synaptic and non synaptic brain mitochondria with no detectable change in the NAD(P)H redox state. The results are discussed with respect to the control of glutamate synthesis by mitochondrial redox potential and the possible intramitochondrial compartmentation of this process.     

INHIBITION OF AMINO ACID UPTAKE BY THE ABSENCE OF Na+ IN SLICES OF BRAIN

—The Na+ requirement of amino acid transport was measured in brain slices. The tissue was first washed free of Na+ and then Na+ was replaced by one of the following: choline, Li+, Rb+, or mannose. Amino acid uptake was measured at different times (5–120 min) and at low (10^-7–10^-5m ) and high (10^-3m ) concentrations. Most of the Na+ could be washed out of the tissue; this also decreased K+ levels despite increased K+ in the medium. K+ tissue levels were partially restored when Na+ was added. The absence of Na+ abolished the uptake of Glu, Asp, GABA, Gly, Tau and Pro. Most of the neutral amino acids (Ala, Val, Trp, His) were very strongly inhibited by the absence of Na+ under most experimental conditions. Basic amino acids (Arg, Lys) were not completely inhibited, in that 30 per cent of the equilibrium uptake remained and some of the basic amino acid influx was independent of the Na+ tissue level. The uptake of amines (tyramine, cadaverine, putrescine) did not require Na+, and often was greater in the absence of Na+. We conclude that amino acid uptake in brain slices is Na+ dependent, although the absence of Na+ may affect transport indirectly.     

BIOGENIC AMINE-MEDIATED ALTERATION OF PYRIDOXAL PHOSPHATE FORMATION IN RAT BRAIN

Abstract— DOPA, dopamine, norepinephrine, tyramine, serotonin, histamine and GABA inhibited pyridoxal kinase; whereas, tyrosine, 5-hydroxytryptophan, histidine, glutamic acid, hypotaurine and taurine were without inhibitory effects. Tetrahydroisoquinoline derivatives formed from Pictet-Spengler condensation between DOPA, dopamine and norepinephrine with pyridoxal and pyridoxal phosphate did not inhibit pyridoxal kinase. These results are interpreted to indicate that interaction of biogenic amines and pyridoxal kinase may alter the formation of pyridoxal phosphate which in turn may influence the activity of numerous pyridoxal phosphate dependent enzymatic reactions in brain.     

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.     

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.     

EFFECTS OF UNDER NUTRITION AND PROTEIN DEFICIENCY ON GLUTAMATE DEHYDROGENASE AND DECARBOXYLASE IN RAT BRAIN

Abstract— Studies were made on the effects of undernutrition at different ages during the neonatal period and of the comparative effects of postweaning protein and calorie deficiencies in neonatally undernourished or normally reared animals. Neonatal undernutrition resulted in deficits in body wt, brain wt and the activities of brain glutamate dehydrogenase and glutamate decarboxylase. Percentage deficits in brain wt were maximum in the first week of life but those in brain enzymes were greater in the second week. Rehabilitation of neonatally undernourished animals reversed the deficits in brain wt and brain enzymes. Post-weaning protein deficiency produced similar deficits in brain enzymes in both neonatally undernourished and normally reared animals. With post-weaning undernutrition, however, these deficits were found only in animals subjected to neonatal undernutrition as well.     

EFFECTS OF DIFFERENT LEVELS OF DIETARY PROTEIN ON BRAIN GLUTAMATE DEHYDROGENASE AND DECARBOXYLASE IN YOUNG ALBINO RATS

Abstract— Studies were carried out to identify the minimum levels of protein (casein) needed in the diet in order to prevent or reverse the deficits in brain enzymes previously found with protein deficiency. Groups of weanling albino rats were fed diets containing variable amounts of protein (5, 8, 10, 15 or 20 per cent in experiment I, and 5, 6, 7, 8 or 20 per cent in experiment II) for 5 or 10 weeks. Deficits in brain wt and brain glutamate dehydrogenase and decarboxylase were found to be prevented by a diet containing 8 per cent or more of protein, although for optimum growth 15 per cent protein in the diet was found to be necessary. Groups of rats were fed a 5 or 20% protein diet for 10 weeks after which the 5% protein animals were either continued on the diet for another 10 weeks or changed to one containing 8, 10, 15 or 20% protein. The brain enzyme deficits found with the 5% protein diet were found to be fully reversed by feeding a 10% protein diet during rehabilitation.     

KINETICS OF BRAIN GLUTAMATE DECARBOXYLASE. INHIBITION STUDIES WITH N-(5′-PHOSPHOPYRIDOXYL) AMINO ACIDS1

Abstract— Seven N-(5′-phosphopyridoxyl) amino acids, reduced analogs of the glutamate-pyridoxal phosphate Schiff base, were synthesized and purified. All of them inhibited mouse brain glutamate decarboxylase activity. The four most potent inhibitors were the aminooxyacetate, GABA, cysteinesul-finate and glutamate derivatives, and the effect of these compounds was studied kinetically. The inhibition produced was in all cases mixed function with respect to glutamate and competitive with respect to pyridoxal phosphate. The inhibition kinetics were non-linear. These results are interpreted in terms of an ordered binding of pyridoxal phosphate and glutamate to the enzyme. Furthermore, they are consistent with previous findings suggesting the existence of two kinds of glutamate decarboxylase activity differing in their dependence on free pyridoxal phosphate.     

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.