THE ACTIVATION OF THIAMINE DIPHOSPHATASE BY ATP IN RAT BRAIN

• 1 Thiamine diphosphatase (TDPase) of brain was activated by a low concentration of ATP.
• 2 In thiamine-deficient rats TDPase activity in the brain increased significantly relative to that in pair-fed animals, while in liver it decreased by the same amount as in the pair-fed controls.
• 3 Liver TDPase was localized almost entirely in the soluble fraction, but in the brain it was bound to insoluble protein. On treatment with Triton X-100 brain TDPase activity increased.
• 4 The ATP content of the brain of deficient rats, but not the ADP or AMP content, was significantly higher than in the control group. The level of inorganic phosphate in the brain and spinal cord of deficient animals was elevated markedly, while that of P-creatine was unchanged.
• 5 The possible roles of brain TDPase in relation to nerve conduction and the blood-brain barrier are discussed.

     

Postnatal Development of Thiamine Metabolism in Rat Brain

The activities of thiamine diphosphatase (TDPase), thiamine triphosphatase (TTPase), and thiamine pyrophosphokinase and the contents of thiamine and its phosphate esters were determined in rat brain cortex, cerebellum, and liver from birth to adulthood. Microsomal TTPase activity in the cerebral cortex and cerebellum increased from birth to 3 weeks, whereas that in the liver did not change during postnatal development. Microsomal TDPase activity in the cerebral cortex showed a transient increase at 1-2 weeks, but that in the cerebellum did not change during development. In contrast to the activity of the brain enzyme, that of liver microsomal TDPase increased stepwise after birth. Thiamine pyrophosphokinase activity in the cerebellum increased from birth to 3 weeks and then decreased, whereas that in the cerebral cortex and liver showed less change during development. TDP and thiamine monophosphate (TMP) levels increased after birth and plateaued at 3 weeks whereas TTP and thiamine levels showed little change during development in the cerebral cortex and cerebellum. The contents of thiamine and its phosphate esters in the liver showed more complicated changes during development. It is concluded that thiamine metabolism in the brain changes during postnatal development in a different way from that in the liver and that the development of thiamine metabolism differs among brain regions.     

ENHANCEMENT OF BRAIN THIAMINE DIPHOSPHATASE ACTIVITY OF RATS BY INJECTION OF CHOLINERGIC DRUGS

Abstract— The effects of cholinergic drugs on thiamine diphosphatase (TDPase) in rat brain, liver and kidney were studied to clarify the role of the enzyme in the central nervous system.
Brain TDPase activity was markedly increased by intraperitoneal injection of a sub-lethal dose of physostigmine, ambenonium or pentetrazol. These drugs also increased the activity in the kidney, but not liver. Strychnine, atropine, and scopolamine did not affect the activity of brain TDPase, but decreased the enzyme activity of liver and kidney. Physostigmine also increased the activity of brain thiamine monophosphatase.
Brain TDPase activity reacheda maximum 30 minafterphysostigmine injection (1.0mg/kg). However, inhibition of brain acetylcholinesterase activity was greatest 45 min after physostigmine injection. The TDPase and AChE activities had both returned to normal values 60 min after the injection. The durations of these changes of TDPase and AChE activity corresponded to the duration of the tremor induced by physostigmine. The contents of total and phosphorylated thiamines in the brain but not in the liver or kidney were significantly reduced by physostigmine.
The relationship between ACh and activation of TDPase activity by cholinesterase inhibitors is discussed.     

Effects of thiamine antagonists on nerve conduction. II. Voltage clamp experiments with antimetabolites

Thiamine antimetabolites were externally applied to voltage clamped squid giant axons to investigate the possible role of thiamine in nerve conduction. Phenylthiazinothiamine, in concentrations as low as 250 m̈M, reduced peak early current and steady-state current, with the depression of the former being two to five times greater than that of the latter. Peak transient and steady-state conductances were about equally depressed by thiamine tert-butyl disulfide (2 mM) and L-586944-00P07 (5–10 mM). None of the antimetabolites produced an appreciable change in the kinetics of Na⁺ activation, K⁺ activation, or Na⁺ inactivation. Thiamine itself, applied externally up to 30 mM, had no appreciable effect on either the magnitude or time course of the ionic currents. Although these data are consistent with the hypothesis that thiamine may be involved in nerve conduction, they probably reflect a nonspecific stabilizing interaction of this class of compound with the axon membrane. Taken in this light, the hypothesis that thiamine plays a direct role in Na⁺ channel permeability changes must be reevaluated.     

PROPERTIES OF THIAMINE DI- AND TRIPHOSPHATASES IN RAT BRAIN MICROSOMES: EFFECTS OF CHLORPROMAZINE

Abstract— The mechanism of the action of chlorpromazine on rat brain thiamine phosphatases were studied to clarify the properties of these enzymes in the CNS. Chlorpromazine at concentrations of 0.25-1.0 m m caused marked decrease of microsomal and soluble thiamine triphosphatase (TTPase) activities and marked increase of microsomal thiamine diphosphatase (TDPase) activity. Imipramine and desipramine also inhibited TTPase but did not cause any marked change in TDPase activities. Addition of chlorpromazine (0.5 m m ) decreased the V_max of microsomal TTPase by about one-half, increased that of TDPase about 3-fold, and lowered the K _m value for TDP but not for TTP.
Acetone treatment of the microsomal fraction lowered the TTPase activity and markedly enhanced the TDPase activity. In acetone-treated microsomes, chlorpromazine also inhibited TTPase activity but did not activate TDPase. Deoxycholate had similar effects to chlorpromazine on these enzyme activities.     

Occurrence of Sialyltransferase Activity in the Synaptosomal Membranes Prepared from Calf Brain Cortex

The possible occurrence of sialyltransferase activity in the plasma membranes surrounding nerve endings (synaptosomal membranes) was studied, using calf brain cortex. The synaptosomal membranes were prepared by an improved procedure which provided: (a) a ?nerve ending fraction” consisting of at least 85% well-preserved nerve endings and containing only small quantities of membranes of intracellular origin; (b) a ?synaptosomal membrane fraction” carrying high amounts of authentic plasma membrane markers (Na⁺-K⁺ ATPase, 5′-nucleotidase, sialidase, gangliosides) with values of specific activity four to fivefold higher than those in the ?nerve ending fraction” and very small amounts of cerebroside sulphotransferase, marker of the Golgi apparatus, and of other markers of intracellular membranes (rotenone-insensitive NADH and NADPH: cytochrome c reductases), the specific activities of which were, respectively, 0.5- and 0.7-fold that in the ?nerve ending fraction”. Thus the preparation of synaptosomal membranes used had the characteristics of plasma membranes and carried a negligible contamination of membranes of intracellular origin. The distribution of sialyltransferase activity in the main brain subcellular fractions (microsomes; P₂ fraction; nerve ending fraction; mitochondria) resembled most closely that of thiamine pyrophosphatase, the enzyme known to be linked to the Golgi apparatus and the plasma membranes and of acetylcholine esterase, the enzyme known to be linked to either intracellular or plasma membranes. The enrichment of sialyltransferase activity in the ?synaptosomal membrane fraction”, referred to the ?nerve ending fraction”, was practically the same as that exhibited by authentic plasma membrane markers. All this is consistent with the hypothesis that in calf brain cortex sialyltransferase has two different subcellular locations: one at the level of intracellular structures, most likely the Golgi apparatus (as described by other authors), the other in the synaptosomal plasma membranes. The basic properties (pH optimum, V/S, V/t and V/protein relationships) and detergent requirements of the synaptosomal membrane-bound sialyltransferase were established. The highest enzyme activities were recorded on exogenous acceptors, lactosylceramide and ds -fetuin. The K_m values for CMP-NeuNAc were different using lactosylceramide and ds -fetuin as acceptor substrates (0.57 and 0.135 mm , respectively); the thermal stability of the enzyme acting on glycolipid acceptor was higher than that on the glycoprotein acceptor; the effect of detergents was different when using glycoprotein from glycolipid acceptors; no competition was observed between lactosylceramide and ds -fetuin. Thus the synaptosomal membranes carry at least two different sialyltransferase activities: one acting on lactosylceramide (and glycolipid acceptors), the other working on ds -fetuin (and glycoprotein acceptors). Ganglioside GM₃ was recognized as the product of synaptosomal membrane-bound sialyltransferase activity working on lactosylceramide as acceptor substrate.     

Thiamine deficiency in rats affects thiamine metabolism possibly through the formation of oxidized thiamine pyrophosphate

BackgroundThiamine deficiency (TD) has a number of features in common with the neurodegenerative diseases development and close relationship between TD and oxidative stress (OS) has been repeatedly reported in the literature. The aim of this study is to understand how alimentary TD, accompanied by OS, affects the expression and level of two thiamine metabolism proteins in rat brain, namely, thiamine transporter 1 (THTR1) and thiamine pyrophosphokinase (TPK1), and what factors are responsible for the observed changes.MethodsThe effects of OS caused by TD on the THTR1and TPK1 expression in rat cortex, cerebellum and hippocampus were examined. The levels of active and oxidized forms of ThDP (enzymatically measured) in the blood and brain, ROS and SH-groups in the brain were also analyzed.ResultsTD increased the expression of THTR1 and protein level in all studied regions. In contrast, expression of TPK1 was depressed. TD-induced OS led to the accumulation of ThDP oxidized inactive form (ThDP_ox) in the blood and brain. In vitro reduction of ThDP_ox by dithiothreitol regenerates active ThDP suggesting that ThDP_ox is in disulfide form. A single high-dose thiamine administration to TD animals had no effect on THTR1 expression, partly raised TPK1 mRNA and protein levels, but is unable to normalize TPK1 enzyme activity. Brain and blood ThDP levels were increased in these conditions, but ThDP_ox was not decreased.General significanceIt is likely, that the accumulation of ThDP_ox in tissue could be seen as a potential marker of neurocellular dysfunction and thiamine metabolic state.     

Thiamine phosphate metabolism and possible coenzyme-independent functions of thiamine in brain.

The effect of depolarization of rat brain cortex slices on the relative distribution of thiamine among its various phosphate esters and on the efflux of thiamine was studied as a probe of possible coenzyme-independent neurophysiological functions of thiamine. Electrical pulses for 30 min increased lactate production but did not affect the levels of thiamine esters. Depolarization with 41 mM-potassium decreased thiamine diphosphate by only 3 percent (P= 0.05). Thiamine triphosphate levels (TTP) were unaffected by depolarization but doubled during incubation for 1 h in which time efflux of 40 percent of the total thiamine from the slices as unesterified thiamine occurred. Depolarization by potassium released a small but highly variable portion of the thiamine content of superfused cortex slices above the basal rate of efflux. The basal efflux was partially sodium dependent. Thiamine efflux was unaffected by acetylcholine, ouabain, or tetrodotoxin, compounds previously reported to increase thiamine efflux. The incorporation of ³²P₁ into the endogenous thiamine phosphates of cortex slices was studied. Incorporation into thiamine diphosphate reached only 20 percent of the specific activity of its precursor, ATP, after 2h of incubation while the incorporation into TTP approached equilibrium with ATP in 15-30 min indicating that the TTP pool was the most rapidly turning over of the thiamine phosphates. The data suggest that only a small portion of the TDP pool undergoes rapid turnover and serves as a precursor for TTP. The rapid turnover of TTP phosphoryl groups is consistent with specific functions for this compound related to its potential for phosphorylation reactions. An analog of TTP with the β, γ oxygen bridge replaced by a methylene group decreased TDP levels and increased thiamine when incubated with cortex slices, but did not effect thiamine monophosphate or triphosphate levels indicating inhibition of thiamine pyrophosphokinase.     

Action of thiamine on different synapses

Thiamine at a concentration of 1×10^–14 to 1×10^–4 M facilitated neuromuscular transmission at the glutaminergic synapse of the crayfish adapter, manifesting as increased amplitude and quantal content of excitatory postsynaptic potentials and raised frequency of miniature excitatory postsynaptic potentials. Thiamine augmented spontaneous electrical activity and the amplitude of synaptic potentials in the longitudinal muscle of guinea pig taenia coli. It was found from studying the effects of thiamine on the membrane potential of rat brain synaptosomes that its presynaptic action is brought about by depolarization of the nerve terminal membrane. Interaction between thiamine and the nerve endings was described by a Hill coefficient of 0.22–0.30, indicating that it has several binding sites within the structure of the receptor concerned.A. V. Palladin Institute of Biochemistry, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 5, pp. 621–629, September–October, 1986.     

In vitro transformation of dehydroepiandrosterone or its sulphate into androstenediol or its sulphate by rat brain and blood preparations

Abstract— –Enzymic transformation of [4-¹⁴C]dehydroepiandrosterone or [4-¹⁴C]dehydro-epiandrosterone sulphate to androstenediol or its sulphate occurred when incubated with a microsomal preparation of rat brain or a whole rat blood homogenate. The brain enzyme which appeared to cause this transformation had a pH optimum at 60, was NADPH₂-dependent, and had an apparent K_m of 4·6 × 10^?6m . When the subcellular fractions of rat brain were compared for transformation, microsomes had the highest specific activity, followed by the cytosol. The crude nuclear and mitochondrial fractions had no significant activity. The level of enzymic activity in the brain microsomes increased from that for rats sacrificed at 7 days of postnatal age to a maximum for rats sacrificed at 1 month of age; then the activity appeared to level off in rats older than 1 month. Microsomes obtained from the cerebellum had the highest specific activity in comparison to that obtained from the cerebral cortex, the diencephalon, and the brain stem. The incubated preparations of rat brain also converted dehydroepiandrosterone sulphate to androstenediol sulphate without hydrolysis. The enzyme in rat blood which was similar to that in the brain was also partially characterized. The blood enzyme had a pH optimum at 6–5, was nearly exclusively present in erythrocytes, was also NADPH₂-dependent, and had an apparent K_m of 2·7 × 10^?4m . The developmental pattern of the blood enzyme specific activity was similar to that of the rat brain enzyme. Upon haemolysis, most activity was recovered in the haemolysate.     

Characterization of a glucuronyltransferase: neolactotetraosylceramide glucuronyltransferase from rat brain

The properties of a rat brain glucuronyltransferase, which is presumed to be associated with the biosynthesis of the HNK-1 epitope on sulfoglucuronyl glycolipids, are described. The enzyme required divalent cations for reaction, with maximal activity at 10mm Mn²⁺, and exhibited a dual optimum at pH 4–5 and pH 6 depending upon the buffer used, with the highest activity at pH 4.5 in MES buffer. This enzyme strictly recognized the Gal1-4GlcNAc terminal structure, and was highly specific for neolacto (type 2) glycolipids as acceptor. The enzyme was localized specifically in the brain, and was barely detected in other issues, including the thymus, spleen, liver, kidney, lung, and sciatic nerve fibres. Phosphatidylinositol and phosphatidylserine increased the enzymatic reaction 4.4- and 2.3-fold, respectively, whereas phosphatidylcholine slightly decreased the rate.Abbreviations GlcA glucuronic acid - Lc-PA₁₄ lactotetraose-phenyl-C₁₄H₂₉ - nLc-PA₁₄ neolactotetraose-phenyl-C₁₄H₂₉ - nLcOse₄-Cer neolactotetraosylceramide - NP-40 Nonidet P-40 - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - SGGL sulfoglucuronyl glycolipid     

THE DIFFERENTIATION OF PHOSPHOLIPASE A1 AND A2 IN RAT AND HUMAN NERVOUS TISSUES

—Lipid-free extracts of rat and human brain have been prepared and shown to contain phospholipase A₁ and A₂ activities and a lysophospholipase. The phospholipase Aj activity has pH optima of 4·2 and 4·6 in rat and human brain, respectively; it can be partially purified and isolated in high yields by dialysing the extracts at low pH. The purified preparations hydrolyse the ester bond at the 1-position in lecithin, phosphatidyl-ethanolamine and phosphatidylserine, but have little or no action on triglyceride or cholesterol ester. An assay system for the enzyme is described. Phospholipase A₂ activity is optimal at pH 5·5 in rat brain extracts and at pH 5·0 in extracts of human brain. The phospholipase A₂ activity of human cerebral cortex is largely unaffected by heating extracts at 70°C for 5 min, whereas this treatment substantially inactivates phospholipase A₁ and completely destroys lysophospholipase. Phospholipase A₁ is widely distributed in both grey and white matter of human brain and is also present in peripheral nerve. Phospholipase A₂ activity is lower than A₁ in all regions of the CNS examined so far, and is absent from peripheral nerve. Neither enzyme appears to require Ca²⁺ but both are inhibited by di-isopropylfluorophosphate (DFP, 2 × 10^?6 m) and thus differ from phospholipase A of pancreas. These studies confirm that the phospholipase A₁ and A₂ activities in brain are due to separate enzymes.     

Thiamine Triphosphate and Membrane-Associated Thiamine Phosphatases in the Electric Organ of Electrophorus electricus

The main electric organ of Electrophorus electricus is particularly rich in thiamine triphosphate, which represents 87% of the total thiamine content in this tissue. The thiamine pyrophosphate concentration, however, is very low in the eel electric organ and skeletal muscle as compared with other eel or rat tissues. Furthermore, electroplax membranes contain a whole set of enzymes responsible for the dephosphorylation of thiamine tri-, pyro- and monophosphate. Thiamine triphosphatase has a pH optimum of 6.8 and is dependent on Mg2+. The real substrate of the enzyme is probably a 1:1 complex of Mg2+ and thiamine triphosphate. Thiamine pyrophosphatase is activated by Ca2+. The apparent Km for thiamine triphosphate and Vmax are found to be, respectively, 1.76 mM and 5.95 nmol/mg of protein/min. Thiamine triphosphatase activity is inhibited at physiological K+ concentrations (up to 90 mM) and increasing Na+ concentrations (50% inhibition at 300 mM). ZnCl2 (10 mM) inhibits 90% of the enzyme activity. ATP and ITP are also strongly inhibitory. No significant effect of neurotoxins is seen. Membrane-associated thiamine triphosphatase is affected differently by proteolytic enzymes and is partially inactivated by pretreatment with phospholipase C and neuraminidase. The physiological significance of thiamine triphosphatase is discussed in relation to a specific role of thiamine in the nervous system.     

Isotopic labeling of thiamine

Thiamine of high specific radioactivity has been prepared by catalytic incorporation of tritium using ³H₂O and platinum. Purification of a crude commercially available product is described and the radiolabeling pattern is determined by NMR analysis of deuterated thiamine.     

The relation of the soluble thiamine triphosphatase activity of various rat tissues to nonspecific phosphatases

Polyacrylamide gel electrophoresis was used to investigate the relation of the soluble thiamine triphosphatase activity of various rat tissues to other phosphatases. This technique separated the thiamine triphosphatase of rat brain, heart, kidney, liver, lung, muscle and spleen from alkaline phosphatase (EC 3.1.3.1), acid phosphatase (EC 3.1.3.2) and other nonspecific phosphatase activities. In contrast, the hydrolytic activity for thiamine triphosphate in rat intestine moved identically with alkaline phosphatase in gel electrophoresis. Thiamine triphosphatase from rat liver and brain was also separated from alkaline phosphatase and acid phosphatase by gel chromatography on Sephadex G-100. This gave an apparent molecular weight of about 30,000 and a Stokes radius of 2.5 nanometers for brain and liver thiamine triphosphatase. The intestinal thiamine triphosphatase activity of the rat was eluted from the Sephadex G-100 column as two separate peaks (with apparent molecular weights of over 200,000 and 123,000) which exactly corresponded to the peaks of alkaline phosphatase. The isoelectric point (pI) of the brain thiamine triphosphatase was 4.6 (4 degrees C). The partially purified thiamine triphosphatase from brain and liver was highly specific for thiamine triphosphate. The results suggest that, apart from the intestine, the rat tissues studied contain a specific enzyme, thiamine triphosphatase (EC 3.6.1.28). The specific enzyme is responsible for most of the thiamine triphosphatase activity in these tissues. Rat intestine contains a high thiamine triphosphatase activity but all of it appears to be due to alkaline phosphatase.     

Product inhibition of mammalian thiamine pyrophosphokinase is an important mechanism for maintaining thiamine diphosphate homeostasis

BackgroundThiamine diphosphate (ThDP), an indispensable cofactor for oxidative energy metabolism, is synthesized through the reaction thiamine + ATP ? ThDP + AMP, catalyzed by thiamine pyrophosphokinase 1 (TPK1), a cytosolic dimeric enzyme. It was claimed that the equilibrium of the reaction is in favor of the formation of thiamine and ATP, at odds with thermodynamic calculations. Here we show that this discrepancy is due to feedback inhibition by the product ThDP.MethodsWe used a purified recombinant mouse TPK1 to study reaction kinetics in the forward (physiological) and for the first time also in the reverse direction.ResultsK_eq values reported previously are strongly underestimated, due to the fact the reaction in the forward direction rapidly slows down and reaches a pseudo-equilibrium as ThDP accumulates. We found that ThDP is a potent non-competitive inhibitor (K_i ≈ 0.4 μM) of the forward reaction. In the reverse direction, a true equilibrium is reached with a K_eq of about 2 × 10^?5, strongly in favor of ThDP formation. In the reverse direction, we found a very low K_m for ThDP (0.05 μM), in agreement with a tight binding of ThDP to the enzyme.General significanceInhibition of TPK1 by ThDP explains why intracellular ThDP levels remain low after administration of even very high doses of thiamine. Understanding the consequences of this feedback inhibition is essential for developing reliable methods for measuring TPK activity in tissue extracts and for optimizing the therapeutic use of thiamine and its prodrugs with higher bioavailability under pathological conditions.     

Thiamine pyrophosphatase (nucleoside diphosphatase) in the Golgi apparatus is distinct from microsomal nucleoside diphosphatase

The properties of thiamine pyrophosphatase in the Golgi apparatus of rat liver were studied. Thiamine pyrophosphatase in an extract of the Golgi apparatus was separated into 6 bands of between pH 5.4 and 6.3 by isoelectric focusing on polyacrylamide gel. On the gels all these subforms catalyzed the hydrolyses of GDP, IDP, UDP, and CDP as well as that of thiamine pyrophosphate. The characteristics resembled those of Type B nucleoside diphosphatase of rat brain, though the enzyme did not have 3 subforms of Type B nucleoside diphosphatase in the higher pH region on isoelectric focusing. Thiamine pyrophosphatase of the Golgi apparatus was separated from microsomal nucleoside diphosphatase by DEAE-cellulose column chromatography. The properties of the enzyme were quite similar to those of Type B nucleoside diphosphatase with respect to its substrate specificity, optimum pH for activity, and inhibition by ATP. These findings suggest that thiamine pyrophosphatase in the Golgi apparatus is different from microsomal nucleoside diphosphatase and that it might be basically the same enzyme as Type B nucleoside diphosphatase except for different extents of modification.     

Pig tissues express a catalytically inefficient 25-kDa thiamine triphosphatase: Insight in the catalytic mechanisms of this enzyme

Thiamine triphosphate (ThTP) is found in most organisms and may be an intracellular signal molecule produced in response to stress. We have recently cloned the cDNA coding for a highly specific mammalian 25-kDa thiamine triphosphatase. The enzyme was active in all mammalian species studied except pig, although the corresponding mRNA was present. In order to determine whether the very low ThTPase activity in pig tissues is due to the absence of the protein or to a lack of catalytic efficiency, we expressed human and pig ThTPase in E. coli as GST fusion proteins. The purified recombinant pig GST-ThTPase was found to be 2–3 orders of magnitude less active than human GST-ThTPase. Using site-directed mutagenesis, we show that, in particular, the change of Glu85 to lysine is responsible for decreased solubility and catalytic activity of the pig enzyme. Immunohistochemical studies revealed a distribution of the protein in pig brain very similar to the one reported in rodent brain. Thus, our results suggest that a 25-kDa protein homologous to hThTPase but practically devoid of enzyme activity is expressed in pig tissues. This raises the possibility that this protein may play a physiological role other than ThTP hydrolysis.     

l-Histidine Decarboxylase in the Human Brain: Properties and Localization

The properties of the histamine-forming enzyme in human brain samples were studied utilizing a radiochromatographic procedure. The influence of postmortem conditions was checked with rat brains, and the results indicated that the enzyme activity is not altered in situ for a delay not exceeding 4 h at ambient temperature. Moreover, tissue blocks or homogenates can be stored at low temperatures for up to 3 months with a good preservation of the enzyme activity. The data indicate that histamine synthesis in the human brain involves the ?specific” histidine decarboxylase (HD, EC 4.1.1.22) and not the aromatic l -amino acid decarboxylase; (1) the optimum pH is 7.4 at 10^-6m-l -histidine; (2) the apparent K_m is about 3.10^-5m ; (3) it is inhibited by α-hydrazino histidine and brocresine but not affected by α-methyl DOPA. Moreover, a major portion of the enzyme is localized in a subcellular fraction containing nerve terminals and it shows an uneven regional distribution which parallels that observed in the brain of other mammalian species. Taken together these data strongly suggest that histamine could play a neurotransmitter role in the human brain.     

Rat brain aspartate β-decarboxylase. A comparative study with the liver enzyme

Aspartate β-decarboxylase (AspD), which catalyses the β-decarboxylation of aspartate (Asp) to alanine (Ala), was found in significant quantities only in the brain, kidney and liver. This enzyme has an optimum pH at 7.4. Addition of exogenous pyridoxal 5′-phosphate did not increase enzyme activity presumably because of firmly bound cofactor. However, aminooxyacetic acid is a potent inhibitor.There is an apparent 8-fold variation in AspD in the seven brain regions studied, with the highest activities in the cortex and the lowest in the striatum and hippocampus. In the presence of α-ketoglutarate, the production of ¹⁴CO₂ from [¹⁴C]Asp may no longer represent AspD activity due to active transamination of Asp, presumably by aspartate aminotransferase, to oxaloacetate. Under such conditions, comparable AspD activities were observed in all seven brain regions.Kinetic analysis showed that the liver and kidney enzymes have identical affinity for Asp (K_m = 3.5 mM) while the brain enzyme has a higher affinit (K_m = 1.3 mM). The V_max values obtained indicated that the enzyme populations in liver, kidney and brain are in the ratio 18:4:1. Various amino acids were found to inhibit both brain and liver AspD. Serine, however, activated the liver enzyme but inhibited competitively the kidney and brain enzymes. These results indicate that AspD may exist as two or more isozymes.