FREE AMINO ACIDS IN ANIMAL TISSUE 201 
which have been employed. Likewise, even if these small molecular-weight constit- 
uents were shown to be associated in loose linkage with a subcellular component 
or structure when procedures are employed which disrupt cell structure but do not 
degrade complex molecules and organelles, it could not be assumed that such an 
association exists 7m vivo. When the cell structure is destroyed myriad new oppor- 
tunities for interactions between cellular constituents may arise which do not exist 
in the living cell. We have recently obtained experimental evidence which indicates 
the possibility of the existence of a physical binding of GABA in brain which is 
TABLE II 
UPTAKE OF [1-MC]-y-AMINOBUTYRIC ACID INTO SEDIMENTS OF HOMOGENATES 
OF BRAIN AND OTHER TISSUES!8 


% of total % of total 
Expt. ees volume in counts in 
No. LESS sediment* sediment BjA 
(A) (B) 
I Brain 10.0 260.0 2.60 
Liver 9.0 5.9 0.05 
Heart 10.4 7-4 0.71 
Lung 7-4 5-9 0.80 
Spleen 8.6 5.7 0.66 
Kidney 9.9 7.9 0.80 
2 Brain: 
a. Original sediment 10.1 37.8 Bu 7A: 
b. t x washed sediment 29.9 2.96 
c. 2 washed sediment 32.3 3.20 
Heart: 
a. Original sediment 9.8 8.3 0.85 
b. 1 x washed sediment 1.0 0.10 
c. 2 * washed sediment 0.8 0.09 
3 Mouse brain acetone powder** 15.7 L257, 0.81 

* Derived from residue weight assuming a density of unity for both the sediment and sus- 
pending medium. The suspending medium had the following composition: 0.154 \/ NaCl, 10.4 
parts; 0.154 M MgSO,, 0.1; 0.25 M glucose, 0.3; 0.11 M sodium phosphate buffer (pH 7.2), 1.2. 
o.1 mg of {[1-MC)}]GABA (2.7 mC/mM) was employed in to ml of the above medium. The sedi- 
ments in Expt. 2 were resuspended in isotope-free medium. 
** Too mg of acetone powder containing high levels of L-glutamic acid decarboxylase and 
y-aminobutyrate—a-ketoglutarate transaminase activities was suspended in 2.5 ml of medium 
and incubated for 70 min. 
not enzymatic (Table II)!8. The experiments were performed at o-—4° under con- 
ditions which did not permit metabolism of the added |1-''C)GABA to take place. 
To weighed portions of freshly dissected rat brain, lung, heart, spleen, kidney and 
liver were added g vols. of incubation medium containing the labeled amino acid. 
Tissues were homogenized, incubated with shaking for 50 min at 0°, and were then 
centrifuged for 30 min at 23 000 « g at o°. The supernatant fluid was poured off, 
the residue weighed and resuspended in the original volume of fresh medium not 
containing isotope, and suitable aliquots of the original homogenate and resuspended 
residue were counted in the scintillation counter. Results show that GABA is bound 
References p. 348/349 
