FREE AMINO ACIDS IN ANIMAL TISSUE 289 
known. Relatively gentle procedures such as homogenization in cold 80%, alcohol, 
heat coagulation and dialysis, or deproteinization with cold trichloroacetic and per- 
chloric acids yield extracts which give virtually identical chromatograms. Similar pat- 
terns of free amino acids were found in whole cells, ground cytoplasm, and cell nuclei 
prepared from livers of fed Wistar rats by the BEHRENS technique, which utilizes 
organic solvents!’. All of the numerous attempts in our laboratory to analyze free 
amino acids in cell particulates of liver and tumor cells prepared in aqueous media 
TABLE I 
CONCENTRATIONS OF FREE AMINO ACIDS IN VARIOUS AREAS 
OF I9-YEAR OLD HUMAN BRAIN!3* 
Values expressed in mg/1oo g wet wt. 





Constituent Bo COTE etic ee ihiatannis yeah) Tareas 
Glycerophosphoethanol- 
amine 22.5 23.4 24.9 16.6 23.0 24.8 50.7 
Phosphoethanolamine 10.5 — 14.6 4.6 10.8 5-4 2.8 
Taurine 18.1 11.9 15.1 14.1 9.2 14.9 13.5 
Aspartic acid 38.2 10.7 26.8 3377) 38.8 39.2 16.2 
Threonine 638 6.4 9.1 18.0 12.4 9.9 10.0 
Serine 27) axe) 232 18.9 18.3 15-7 22.9 
Glutamic acid 154.2 61.5 180.5 146.2 135.8 103.5 94.0 
Glycine and alanine 34.3 22.6 39.2 49.8 42.7 30.4 54-4 
Cystathionine 197 9.5 2.3 — — 353 5.3 
Isoleucine 2.4 3.6 4.0 — — 1.8 7.8 
Leucine 7-3 5.2 It Teer 9.4 9.3 11.6 15.6 
y-Aminobutyric acid 21.0 jez 27-4 70.5 39-5 48.0 22.0 

* Method of MooRE AND STEIN}. 
ordinarily considered suitable for the metabolic study of cell fractions have resulted 
in the virtually complete liberation of free amino acids into the suspending medium. 
Typical results are shown in Figs. 23-28. Livers were removed from male rats which 
had been fasted for 24h. The livers were homogenized either in 0.25 M sucrose 
alone or in a medium containing 0.25 M sucrose, 0.1 M potassium phosphate buffer, 
and 0.002 M versene at pH 7.38. All operations were performed at 0°. Smears made 
of the homogenates revealed that at least 99°, of the cells had been disrupted. The 
homogenates were centrifuged for 30 min at 105 000 x g in the Spinco Model L 
ultracentrifuge. The supernatant fluid was removed and the precipitates were ex- 
tracted with 80° alcohol in the usual fashion for chromatography. Chromatograms 
prepared from the original fresh homogenate and from the residue which contained 
all the particulate matter (microsomes, mitochondria, nuclei and cell debris) re- 
vealed that virtually all of the free or easily extractable amino acids had been re- 
moved into the suspending medium. Similar experiments were performed in which 
the suspending medium contained 10% polyvinylpyrrolidone and 20%, sucrose. 
The latter experiments were performed both at 0° and 25°. In each instance the free 
amino acids were found largely in the supernatant medium. 
References p. 348/349 
