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Further Studies on Brain y-Aminobutyric Acid Metabolism after Administration of Pyridoxine to Vitamin B-&Deficient Suckling Rats BRIONY ACKROYD and WILLIAM R. D. SMITH Courtauld Institute of Biochemistry, The Middlesex Hospital Medical School, London W l P 5PR, U.K. Convulsions due to vitamin B-6 deficiency in humans can be controlled by the administration of pyridoxine, and the symptoms subside rapidly (Coursin, 1954). If derangement of y-aminobutyric acid metabolism were responsible for these convulsions, it would be expected that a return to normal y-aminobutyric acid metabolism occurs very rapidly after the pyridoxine administration. In vitamin B-&deficient suckling rats (Bayoumi & Smith, 1972), the amount of glutamate decarboxylase activity was shown to be decreased to 40% of control values. After intraperitoneal administration of pyridoxine hydrochloride (lOmg), the activity recovered almost immediately. Within 2min the activity was four times that in the untreated deficient animals (Bayoumi et al., 1974). The concentration of y-aminobutyric acid would also be expected to rise, following the same pattern as the glutamate decarboxylase. However, a lag of 20min occurred before the y-aminobutyric acid concentration began to rise. In order to resolve this apparent discrepancy, vitamin B-6-deficient suckling rats were given pyridoxine hydrochloride (10mg) by intraperitoneal injection at timeintervals ranging from 1 to 60min before they were killed. The animals were also given an intraventricular injection of ~-[U-'~C]glutamicacid (0.14pmol), 15min before they were killed. The brains were then prepared as for amino acid (Bayoumi et al., 1974). The y-amin~['~C]butyricacid and total brain glutamate concentrations of the preparations were determined. The y-aminobutyric acid was separated by a two-dimensional process by using, first, paper chromatography (butan-1-ol-acetic acid-water, 12: 3 :5, by vol.) on Whatman no. 1 paper, followed by high-voltage electrophoresis at 3 kV, 60mA at 90" to the direction of chromatography, in pyridine-acetic acid buffer, pH5.3. The radioactive spots corresponding to y-aminobutyric acid were located with a Pullan spark-chamber radiochromatogram scanner, cut out and counted for radioactivity in toluene scintillator [0.3 % 2,5-diphenyloxazole, 0.03 % 1,4-bis(5-phenyloxazol-2-yl)benzene] in a Packard 3385 Tri-Carb liquid-scintillation counter. The glutamic acid was measured after one-dimensional paper electrophoresis under the same conditions as above. The spots were quantitatively stained with ninhydrin (1 % in acetone). After heating, the glutamic acid spots were cut out and eluted in 5ml of 80% (v/v) ethanol. In order to compare relative glutamate decarboxylase activities in vivo, calculations are made assuming that, since the injected [14C]glutamic acid is negligible in amount compared with the total endogenous glutamic acid, the proportion of endogenous glutamate converted into y-aminobutyric acid approximates to the proportion of injected ['4C]glutamic acid converted into y-amin~['~C]butyricacid. Hence, the glutamate decarboxylase activity in vivo is expressed here as y-aminobutyric acid (pmol) produced/h per brain. This is found from Total y-aminobutyric acid produced

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[14C]glutamicacid y-amin~['~C]butyricacid

It was found that the production of y-aminobutyric acid began to rise after 7.5 min, increased up to 40min, and then declined. This point of initial increase is intermediate between the increase in the glutamate decarboxylase activity in uitro, and the increase in whole-brain y-aminobutyric acid (Fig. 1). Thus it appears that the rise in glutamate decarboxylase activity as measured in uitro is due to an activation in vitro of the apoenzyme by exogenous pyridoxine, but that the measurable rise in whole-brain y-aminobutyric acid lags behind the initial increasein VOl.

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Time after intraperitonealinjection (min) Fig. 1. Values of whole-brain glutamate decarboxylase activity (measured in vitro and in vivo) and y-aminobutyric acid ajier the intraperitoneal injection of pyridoxine hydrochloride For the experiments 2-week-old vitamin B-6-deficient suckling rats were injected with pyridoxine hydrochloride as described in the text. Values are expressed as percentage of control value. 0 , Glutamate decarboxylase in vitro; 0 , glutamate decarboxylase in vivo; H,y-aminobutyric acid.

the synthesis of y-aminobutyric acid resulting from the activation in uivo of glutamate decarboxylase by some 10-1 5 min. Two other enzymes involved in y-aminobutyric acid metabolism, aspartate transaminase and y-aminobutyrate transaminase, were measured in whole-brain homogenates of the vitamin B-&deficient suckling rats at various time-intervals after pyridoxine injection. For these measurements, 0.25 % Triton X-100 and 0.02-~-mercaptoethanol were added to the homogenization medium. Aspartate transaminase activity was measured automatically in an LKB 8600 reaction rate analyser, by using a Boehringer automated analysis kit. Pyridoxal S’-phosphate (10pg/ml) was added to the buffer when required. y-Aminobutyric acid transaminase was measured as previously described (Bayoumi et al., 1972). It has been previously reported that in brains of the vitamin B-&deficient suckling rats, glutamate decarboxylase activity and y-aminobutyric acid concentration were decreased t o 4 0 x a n d 50%of control values respectively (Bayoumi et al., 1974). In these studies, y-aminobutyrate transaminase activity is decreased to 64% of control values and aspartate transaminase is decreased to 23 % of control values. The activities of these enzymes, however, over a 60min recovery period after intraperitoneal pyridoxine injection, do not change significantly. The y-aminobutyrate transaminase activity in the deficient animals is maintained at 9.7 5 0.6,umol/h per g wet wt., which, when compared with the maximal activity, measured with pyridoxal 5’-phosphate added to the incubation mixture (100,ug/ml), shows that the enzyme is 55% saturated with cofactor. The activity in the control animals was maintained at 14.7 & 0.7pmol/h per g wet wt.. and the enzyme was 92.5% saturated. The aspartate transaminase activities 1975

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showed a similar pattern to the y-aminobutyrate transaminase. The enzyme was 56% saturated in the deficient animals, and 100% saturated in the control animals, but the apoenzyme activity (as measured in the presence of pyridoxal 5’-phosphate) in the deficient animals remained at 77% of the value in control animals. Thus apparently, over the period of recovery of neuronal activity after pyridoxine deprivation, in the y-aminobutyric acid system, the only parameters to change significantly are y-aminobutyric acid concentrations and glutamate decarboxylase activity. y-Aminobutyrate transaminase does not appear to play a significant part, perhaps owing to the unavailability of the added pyridoxine, since the enzyme has a mitochondrial localization. However, these results give further evidence in favour of y-aminobutyric acid metabolism being involved in the recovery of neuronal activity on administration of pyridoxine after its deprivation. The inhibitory properties of the y-aminobutyric acid system appear to depend on there being a continuous synthesis of y-aminobutyric acid (no matter how small) rather than the total amount of endogenous y-aminobutyric acid. Bayoumi, R. A. & Smith, W. R. D. (1972)J. Neurocbem. 19,1883-1897 Bayoumi, R.A., Kirwan, J. A. & Smith, W. R. D. (1972)J. Neurocbem. 19,569-576 Bayoumi, R.A., Ackroyd, E.B. & Smith, W. R. D. (1974)Biocbem. SOC.7’ram. 2,9699 Coursin, D. B. (1954)J. Am. Med. Ass. 154,406-408

Studies on nee Calcium Inside Pigeon Erythrocyte ‘Ghosts’ by Using the Calcium-Activated Luminescent Protein, Obelin ANTHONY K. CAMPBELL and ROBERT L. DORMER Department of Medical Biochemistry, Welsh National School of Medicine, Heath Park, CardifCF4 4XN, U.K. Changes in the concentration of free Ca2+inside cells appear to play an important role in the action of a number of hormones (Rasmussen et al., 1972). A major problem in studying the role of CaZ+in the control of intracellular processes is the low concentration of free Caz+, probably in the range O.l-lOp~,compared with the relatively high total cell Caz+,in the region of 1mM. A number of workers have attempted to investigate this problem by studying radioactive and total Ca2+fluxes, and by studying the effects of manipulations of the extracellular medium, including the removal of Caz+and Mg2+, the addition of EGTA [ethanedioxybis(ethylamine)tetra-acetic acid], local anaesthetics and bivalent cationic ionophores. Unfortunately, none of these investigations have been able to show definitively whether a particular hormone raises or lowers free cytoplasmic Ca2+or what the time-course of any changes might be. Cytoplasmic free Caz+ has been measured in the giant single muscle fibres of the barnacle (Ashley & Ridgeway, 1970) and in the giant axon of the squid (Baker et al., 1971) after micro-injection of the Caz+-activated luminescent protein, aequorin. There have been no reports of similar studies in small cells. The aim of the present communication is to show that a Caz+-activated luminescent protein can be resealed inside pigeon erythrocyte ‘ghosts’ and that this system can be used to investigate the effect of substances on free CaZ+within the ‘ghosts’. Pigeon erythrocytes were used, since stimulation by adrenaline produces large changes in the concentration of adenosine 3‘: S’-cyclic monophosphate (Davoren & Sutherland, 1963; A. K. Campbell, unpublished work). There are no readily available sources of aequorin in the United Kingdom. However, it has recently been shown that a similar protein, obelin, can be extracted and purified from the hydroid Obelia geniculata (Campbell, 1974). Sufficient quantities of obelin can be obtained to carry out physiological experiments (Ashley et al., 1975). A number of workers have investigated conditions for preparing human erythrocyte Vol. 3