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Abstracts of the Meeting of the Histamine Club 1975
In vivo and in vitro Rat Brain Histidine Deearboxylase Coenzymic Requirements by J. M. PALACIOS, G. MENGOD and I. BLANCO Instituto de Biologia Fundamental, Centro Coordinado del C.S.I.C. Universidad Aut6noma de Barcelona, Avda. S. Antonio M a Claret, 171 Barcelona, Spain We have studied the participation of pyridoxal-5-phosphate (PLP) in the decarboxylation of histidine by brain tissue, by measuring histidine decarboxylase (HD) using a modification of the chromatographic-spectrofluorimetric procedure of SCHWARTZ et al. [1]. As a first approach to the problem, the effects of PLP and its classical antagonist were studied. Using increasing concentrations of PLP, the highest reaction velocity of the enzyme was reached at 10-4 M. Higher concentrations of PLP inhibited HD. The action of the reducting compound NaBH4 and of classical carbonyl reagents such as semicarbazide, phenylhydrazide, hydroxylamine and isonicotinic acid hydrazide was studied by preincubating the tissue with these agents. While NaBH4 produced an irreversible loss of activity the carbonyl reagents caused an inhibition which was 90% at 10.2 M phenylhydrazine and 50% at 10-a M isonicotinic acid hydrazide. The binding of PLP with apoenzyme was studied by several dialysis procedures. When brain homogenates were dialyzed against phosphate buffer a 50% loss of activity was found. The loss did not occur when 10-a M PLP was added to the buffer. Stronger dialysis conditions such as dialyzing for longer periods against a concentration gradient of phosphate buffer caused no further resolution of HD. In all cases the addition of PLP to the incubation mixtures caused a total recuperation of activity. Dialysis against a solution containing 10.5 M semicarbazide produced the total resolution of fetal rat HD. Dialysis against a solution containing cysteine and E D T A has been reported as a method to resolve 'tightly bound' PLP in several PLP-containing enzymes. In rat brain homogenates dialyzed against such media only 50% of H D was lost which was restored by PLP addition. All these results indicated that brain HD, although depending on a carbonyl group for its
action, has at least two distinctly bound groups, one dialyzable, and possibly PLP, and the other non dialyzable. Looking for an unequal subceUular distribution of these two groups we dialyzed the pellet and supernatant of centrifugated rat brain homogenate at 10a g, 1 hour. The dialysis caused a 50% lost of H D activity either in the pellet or in the supernatant. These results indicated that the 'dialyzable' and the 'non dialyzable' rat brain H D were similarly distributed subcellularly. In order to check the role of PLP in brain H D in vivo, rats were injected intraperitoneaUy with 250 mg/kg semicarbazide. H D was significantly lowered at 15 minutes after injection, reaching a 60% of inhibition at 90 minutes. Brain histamine levels were also lowered at this time. 100 ~zg of PLP administered intracranially rised the brain histamine levels, in agreement with the results of SHAVER [2]. 250 mg/kg of PLP administered intraperitoneally also rised the brain histamine levels 90 minutes after the administration of the coenzyme. Conclusion Results both in vivo and in vitro agree with the role of PLP as coenzyme of the histidine decarboxylating enzyme in brain. However, the way in which PLP-apoenzyme interacts seems to differ from the mode of binding exhibited by the 'specific' enzyme in other tissues [3]. Received 1 September 1975.
References [11 J.C. SCHWARTZ, C. LAMPARTand C. RosE, J. Neurochem. 17, 1527 (1970). [2] R.W. SCHAYERand M.A. REALLY,Agents and Actions
4/3, 133 (1974). [3] D. AURES,R. H~.KANSONand W.G. CLARK,Handbook of Neurochemistry, vol. 4 (Plenum Press, New York 1970), p. 165.
Abstracts of the Meeting of the Histamine Club 1975
In vivo and in vitro Rat Brain Histidine Deearboxylase Coenzymic Requirements by J. M. PALACIOS, G. MENGOD and I. BLANCO Instituto de Biologia Fundamental, Centro Coordinado del C.S.I.C. Universidad Aut6noma de Barcelona, Avda. S. Antonio M a Claret, 171 Barcelona, Spain We have studied the participation of pyridoxal-5-phosphate (PLP) in the decarboxylation of histidine by brain tissue, by measuring histidine decarboxylase (HD) using a modification of the chromatographic-spectrofluorimetric procedure of SCHWARTZ et al. [1]. As a first approach to the problem, the effects of PLP and its classical antagonist were studied. Using increasing concentrations of PLP, the highest reaction velocity of the enzyme was reached at 10-4 M. Higher concentrations of PLP inhibited HD. The action of the reducting compound NaBH4 and of classical carbonyl reagents such as semicarbazide, phenylhydrazide, hydroxylamine and isonicotinic acid hydrazide was studied by preincubating the tissue with these agents. While NaBH4 produced an irreversible loss of activity the carbonyl reagents caused an inhibition which was 90% at 10.2 M phenylhydrazine and 50% at 10-a M isonicotinic acid hydrazide. The binding of PLP with apoenzyme was studied by several dialysis procedures. When brain homogenates were dialyzed against phosphate buffer a 50% loss of activity was found. The loss did not occur when 10-a M PLP was added to the buffer. Stronger dialysis conditions such as dialyzing for longer periods against a concentration gradient of phosphate buffer caused no further resolution of HD. In all cases the addition of PLP to the incubation mixtures caused a total recuperation of activity. Dialysis against a solution containing 10.5 M semicarbazide produced the total resolution of fetal rat HD. Dialysis against a solution containing cysteine and E D T A has been reported as a method to resolve 'tightly bound' PLP in several PLP-containing enzymes. In rat brain homogenates dialyzed against such media only 50% of H D was lost which was restored by PLP addition. All these results indicated that brain HD, although depending on a carbonyl group for its
action, has at least two distinctly bound groups, one dialyzable, and possibly PLP, and the other non dialyzable. Looking for an unequal subceUular distribution of these two groups we dialyzed the pellet and supernatant of centrifugated rat brain homogenate at 10a g, 1 hour. The dialysis caused a 50% lost of H D activity either in the pellet or in the supernatant. These results indicated that the 'dialyzable' and the 'non dialyzable' rat brain H D were similarly distributed subcellularly. In order to check the role of PLP in brain H D in vivo, rats were injected intraperitoneaUy with 250 mg/kg semicarbazide. H D was significantly lowered at 15 minutes after injection, reaching a 60% of inhibition at 90 minutes. Brain histamine levels were also lowered at this time. 100 ~zg of PLP administered intracranially rised the brain histamine levels, in agreement with the results of SHAVER [2]. 250 mg/kg of PLP administered intraperitoneally also rised the brain histamine levels 90 minutes after the administration of the coenzyme. Conclusion Results both in vivo and in vitro agree with the role of PLP as coenzyme of the histidine decarboxylating enzyme in brain. However, the way in which PLP-apoenzyme interacts seems to differ from the mode of binding exhibited by the 'specific' enzyme in other tissues [3]. Received 1 September 1975.
References [11 J.C. SCHWARTZ, C. LAMPARTand C. RosE, J. Neurochem. 17, 1527 (1970). [2] R.W. SCHAYERand M.A. REALLY,Agents and Actions
4/3, 133 (1974). [3] D. AURES,R. H~.KANSONand W.G. CLARK,Handbook of Neurochemistry, vol. 4 (Plenum Press, New York 1970), p. 165.
