AGENTS ON AMINO ACIDS IN MAMMALIAN BRAIN 503 
full agreement with our results using thiosemicarbazide in rats" (Table I, Figs. 6—10). 
In the course of our studies with carbonyl-trapping agents we tested two which 
had remarkable specificity. Both hydroxylamine (NH,OH)” and aminooxyacetic 
acid!3 appear to be specific inhibitors of y-aminobutyric—a-ketoglutaric acid trans- 
aminase (GABA-T) in brain and /-alanine—a-ketoglutaric acid transaminase in liver. 
Present evidence favors the concept that these two enzymes in mammalian tissues 
may be identical”. 
Chromatographic evidence for the specificity of NH,OH in brain and liver are 
shown in Figs. 11-16. Administration of NH,OH resulted in increased levels of GABA 
in brain and f-alanine in liver. No significant changes were found in the level of 
other ninhydrin-reactive constituents in the tissues which were tested (Figs. 11-22). 
Although GABA is synthesized and degraded by enzymes requiring PyP, the in- 
TABLE II 
EFFECT OF NH,OH UPON GABA, GABA-T AND GAD 7m vivo 

GA BA-T activity, § GAD activity, §§ 


. (wmnoles GA BA|g pomoles GA BA|g 
Linial Woof Treatment** cee Brain area GA BA***, § tissue/h) tissue/h 
No. rats* (nin) (mg%) —§ = 
No + roo ug No + 50 ug 
addn. PyP/tube addn. PyP/tube 
I 2 Control o— Whole brain§§§ 22.1 29.5 — a 27a 
2 NEGOH 75 Whole brain 35-7 Wie — — 25-5 
NH,OH 210 Whole brain 47-5 15.1 = — 24.4 
2 3. Control —- Cortex 21.0 43-9 31.0 2e2 27.5 
3) Nid OH go Cortex 460.9 18.1 16.7 13.8 28.4 
3 2 Control — Cortex 48.7 39.9 
2 Control _- Cerebellum 68.7 50.7 
2 NET OE 90 Cortex 19.9 IEG feat 
Pe NIE OE go Cerebellum 30.9 20.2 
4 2 Control a Cortex 20.8 38.7 2 OAT 
2 Control -—— Cerebellum 18.5 53-1 B37 
2 NEC OE 100 Cortex 43.1 19.5 16.4 
2 NH,OH 100 Cerebellum 28.5 27.6 23.6 
5 2 Control 90 Cortex 16.1 27.8 
2 NIE OE 90 Cortex 13.4 278 

* All rats were Sprague Dawley females 180—210 g in weight. 
** NH,OH-HCI was adjusted to pH 6 before injecting at dosage of 75 mg/kg body wt. 
*** For additional confirmation see ref. 12. 
§ GABA-T was determined by a method described previously!® for acetone powder prepara- 
tions. In these assays 60 mg equivalent of brain homogenate was incubated at 37° C for 1h. GABA 
was determined enzymatically. GAD was determined by incubating tissue homogenate and 
glutamic acid substrate for 15 or 20 min at 37°C with shaking. The incubation system consisted 
of 0.2 ml phosphate buffer (0.1 M, pH 6.3), 0.15 ml glutamic acid (0.5 M neutralized to pH 6.3), 
the equivalent of 30 mg of brain tissue homogenate in 0.2 ml buffer and 0.05 ml pyridoxal phos- 
phate (1 mg/ml) or the equivalent amount of additional buffer. The reaction was terminated 
by the addition of 2.5 ml of 95% ethyl alcohol. The GABA formed, in excess of that present in 
the tissue at zero time, was determined enzymatically”. 
§§ These results have been confirmed measuring GAD activity by “CO, evolution from 
uniformly labeled 14C-glutamic acid substrate”. 
§§§ Whole brain less cerebellum, pons and medulla. 
References p. 508 
