292 E. ROBERTS AND D.G. SIMONSEN 
to the brain sediment but not to that of other tissues or to brain acetone powder. 
The radioactivity in the sediment from heart homogenate was washed out by re- 
suspension in the medium but most of that in the original sediment from brain re- 
mained through two washings. Similar experiments employing equilibrium dialysis 
have shown that glutamic acid and possibly other amino acids may also be bound 
by brain preparations. Attempts to solubilize the binding material in brain so far 
have failed. Work is in progress to delineate further the properties of the binding 
material and to purify the factor or factors involved. Thus, at least in the case of 
brain, there is some evidence for the existence of substances in tissues with specific 
binding capacities for small molecules, but it cannot be determined at present 
whether or not any considerable proportion of the total intracellular content of 
any of the easily extractable amino constituents actually exist in this form, nor can 
it be said that such binding, where found, has any relationship to the physiological 
effects which some of the constituents may exert in various biological test systems. 
ARE THE FREE OR EASILY EXTRACTABLE AMINO ACIDS ARTIFACTS RESULTING 
FROM AUTOLYTIC CHANGES? 
One of the questions which was raised early in the work on free ninhydrin-reactive 
substances was whether these easily extractable constituents actually were normal 
cell constituents or whether they represented autolytic artifacts, since the quantities 
found were very small by comparison with the amounts of protein-bound amino 
acids occurring in the tissue samples studied. In order to minimize this possibility 
in our laboratory tissues are fixed immediately after excision. A number of the 
substances which occur prominently in one tissue or another such as taurine, GABA, 
carnosine and anserine, ethanolamine phosphate and glutathione are not normal 
constituents of proteins. However, recently a new nucleotide-peptide has been 
isolated from bovine liver which contains /-alanine, cysteic acid and taurine as well 
as other amino acids!®. Patterns of amino acids observed after hydrolysis of whole 
homogenates of tissues were never found to resemble those obtained in the protein- 
free extracts (see example in ref. 20). Furthermore, no evidence for the extensive 
occurrence of peptides other than glutathione was found in the extracts (see also 
ref. 21), the presence of which would be expected to a considerable extent if catheptic 
activity had played a prominent role in the origin of the detectable constituents. 
Fig. 29 shows a typical pattern of a picric acid filtrate of the ventricle of dog heart 
and Fig. 30 shows the changes produced in this pattern upon hydrolysis with 6 N 
HCl in a sealed tube for 24h (ref. rr). The changes noted on hydrolysis were the 
complete disappearance of glutamine, glutathione and ethanolamine phosphate with 
concomitant increases in the concentrations of glutamic acid, glycine, cysteine 
(detected as cysteic acid) and ethanolamine. In addition, /-alanine and histidine, 
the products of hydrolysis of carnosine, were detected. The latter substance, which 
gives a yellow-brown ninhydrin color, was below the level of detection on the chro- 
matogram of the unhydrolyzed extract. It is noteworthy that the content of alanine 
and taurine remained essentially constant and that most of the other amino acids 
did not even become detectable after acid hydrolysis, indicating that, at most, 
only small amounts of diverse peptides may be present in these extracts. Similar 
results were obtained in many instances in which protein-free extracts of different 
References p. 348/349 
