My scientific work since 1946 can be epitomized as a search for patterns. What usually began as a single-minded devotion to in-depth analysis of one or a small number of variables always led to questions of how the results might relate to the whole living unit, whether it be cell, tissue, or organism. In studies of nitrogen metabolism of normal and neoplastic tissues (Roberts and Simonsen, 1960) it appeared desirable to determine the composition of pools of non-protein amino acids and related substances. It was anticipated that patterns of steady-state concentrations of these constituents would reflect characteristics of the tissues in a way that might reveal key metabolic differences. Progress was slow and curiosity limited until methods became available that enabled a large number of determinations to be made in a reasonably short period of time. The development of two-dimensional paper chromatographic procedures allowed the detection of microgram quantities of substances for which other adequate microanalytical procedures were not available and made it feasible to survey rapidly the distribution of free or loosely bound amino acids and other ninhydrin-reactive substances. I hastened to apply these techniques for the first time to animal tissues soon after they were introduced in the United States by C.E. Dent. The paper chromatographic procedures furnished tools that were ideally suited for giving simultaneous information rapidly about the maximal number of ninhydrin-reactive constituents and, although often employed in a semi-quantitative fashion, could give valuable hints about the presence of new materials and indicate which substances should be studied further in particular biological situations. Column chromatographic methods already were being applied extensively, but the procedures, although quantitative, were more time-consuming and allowed far fewer samples to be examined. In addition, unknown substances are much easier to detect by the paper chromatographic procedures.
My earliest observation with the two-dimensional paper chromatographic method showed that, in a given species at a particular stage of development, each normal tissue has a distribution of easily extractable ninhydrin-reactive constituents that is characteristic for that tissue, whereas quite similar patterns were observed in transplanted and spontaneous tumors. The latter findings agreed with Greenstein’s generalization based on enzyme assays: “no matter how or from which tissues tumors arise, they more nearly resemble each other chemically than they do normal tissues or than normal tissues resemble each other.”
Working during the summer of 1949 at the Roscoe B. Jackson Memorial Laboratory in Bar Harbor, Maine, where an unusually good selection of transplantable mouse tumors carried in a number of inbred mouse strains was available for study, I analyzed the free amino acid content of the C1300 transplantable neuroblastoma, then available only in solid form. Several mouse brain extracts were chromatographed for comparison with the neuroblastoma. An unidentified and previously unobserved ninhydrin-reactive material was seen on the chromatograms of the brain extracts. At most, only traces of this material had appeared in a large number of extracts of many other normal and neoplastic tissues previously examined, or in samples of urine and blood. Upon returning to my laboratories at Washington University in St. Louis, the unknown material was isolated from suitably prepared paper chromatograms. A study of the properties of the substance revealed it to be γ-aminobutyric acid (GABA). Initial identification was based on the comigration of the unknown with GABA on paper chromatography in three different solvent systems. I gave some of the material to Sidney Udenfriend, at that time also on the staff of Washington University, who made an absolute identification of the GABA in our extracts by the isotope derivative method. Abstracts were submitted to the Federation meetings that year (March 1950; see Figure 1, Figure 2). An abstract reporting the presence of an “unidentified amino acid in brain only” appeared from the laboratory of Jorge Awapara, in the proceedings of the same meeting (Awapara, 1950; see Figure 3). Ambiguity has arisen in some derivative accounts in the assignment of priority to the first report of the presence of GABA in brain. To clarify the matter, the relevant three abstracts appear here to attest to Roberts’ primacy of the discovery (Scans of the original abstracts Figure 1 Figure 2 Figure 3, and transcripts, see below).
We were helped initially in the identification of GABA in brain extracts by the fact that in 1949 GABA had been found to be prominent among the soluble nitrogenous components detectable by two-dimensional paper chromatography in the potato tuber (Steward, 1949). This caused me to write semi-facetiously in my notebook, “This proves that the brain is like a potato!” Unfortunately, little has happened to the state of the world since that time to change my mind. GABA had been found in nature before. In 1910 Prof. Dr. Dankwart Ackermann found it to be produced in putrefying mixtures by the action of bacteria (Ackermann, 1910, Ackermann and Kutscher 1910), and, subsequently, many reports had been made about the occurrence of GABA and/or its formation in bacteria, fungi, and plants. I was thrilled to receive a letter from Ackermann with congratulations upon publishing the first report of the presence of GABA in brain.
The only authentic sample of GABA that could be located at that time was in the chemical stockroom of the Department of Chemistry at the University of Illinois. Subsequently, we were able to make our own GABA by simple hydrolysis of a free and generous supply of 2-pyrrolidinone obtained from Cliff’s Dow Chemical Company in Marquette, Michigan. In order to demonstrate conclusively that the precursor for GABA was glutamic acid in crude brain preparations, it was necessary to employ 14C-labeled glutamic acid (3H-labeled substances not yet being available). No commercial sources were available. A sample of uniformly labeled L-glutamic acid isolated from a hydrolysate of algae grown in 14CO2 was kindly furnished by Konrad Bloch, then on the staff of the University of Chicago.
Since L-glutamic decarboxylase (GAD) from other sources was known to require pyridoxal phosphate as a coenzyme, it was necessary to test this substance for its effect on the decarboxylation of glutamic acid in brain preparations. The only source of pyridoxal phosphate available to us, after much searching, was in the possession of Wayne W. Umbright then at the Merck Institute for Therapeutic Research. He gave us a generous supply of this cofactor.
At the time of the discovery of GABA in brain, I was immediately faced with a serious conflict. I was working in the Wernse Laboratories of Cancer Research in Washington University School of Medicine under E.V. Cowdry, a great scientist, and a fine human being. I desperately wanted to work on the metabolism and function of GABA, but my obligations lay in the field of cancer research. Nonetheless, for almost three years thereafter, most of my research efforts were devoted to the study of GABA in brain. During that period I received much encouragement from Cowdry. Never once was I criticized or reprimanded for diverting my efforts from the main thrust of his program. I am most grateful to this gentleman for his support and forbearance during the period I remained in his laboratory and for his friendship in the subsequent years before his death.
Beyond the discovery
For several years, the unique presence of relatively large amounts of GABA in the tissue of the central nervous system (CNS) of various species remained a puzzle. The great neurochemist Heinrich Waelsch once discouragingly remarked that GABA was probably a metabolic wastebasket. My continuing efforts to convince some of the eminent neurophysiologists working at Washington University at that time to test GABA in various nerve preparations at the end of their planned experiments met with no cooperation, even though I brought GABA solutions personally to their laboratories in the hope of persuading them to test it.
In the first review on the subject in 1956 (Roberts, 1956), written after I had moved to my present position at City of Hope Medical Center, I concluded in desperation, “Perhaps the most difficult question to answer would be whether the presence in the gray matter of the CNS of uniquely high concentrations of γ-aminobutyric acid and the enzyme which forms it from glutamic acid has a direct or indirect connection to conduction of the nerve impulse in this tissue.” However, later that year, the first suggestion that GABA might have an inhibitory function in the vertebrate nervous system came from studies in which it was found that topically applied solutions of GABA exerted inhibitory effects on electrical activity in the brain (Hayashi and Nagai, 1956, Hayashi and Suhara, 1956). In 1957, evidence for an inhibitory function for GABA came from studies that established GABA as the major factor in brain extracts responsible for the inhibitory action of these extracts on the crayfish stretch receptor system (Bazemore et al., 1957). Within a brief period, the activity in this field increased greatly so that the research being carried out ranged from the study of the effects of GABA on ionic movements in single neurons to clinical evaluation of the role of the GABA system in, for example, epilepsy, schizophrenia, and various types of mental retardation. This warranted the convocation of a memorable interdisciplinary conference in 1959 at the City of Hope Research Institute attended by most of the individuals who had a role in opening up this exciting field and who presented summaries of their work (Roberts et al., 1960).
This first GABA conference was the greatest learning experience of my life. Having spent most of my scientific career in the rather narrow confines of classical biochemistry and the bare beginnings of molecular biology, I was thrust into the world of EEG, membranes, electrodes, voltage clamps, neuroanatomy, clinical seizures, neuroembryology and animal behavior. I had the privilege of meeting a number of the world’s leading neuroscientists among the participants, some of whom became close friends. What a mind-boggling intellectual feast! The meeting, itself, was over-whelming to a number of us. The sense of excitement was pervasive because we all sensed that a new era was beginning. The subject of neural inhibition finally had returned to front stage and center after many years of languishing in the wings. It was obvious that much of the future progress in the field would depend on interdisciplinary efforts and that we all would have to begin to learn each other’s language and ways of thinking. At times the proceedings resembled what one imagines might have taken place at the Tower of Babel. However, we all shared the optimistic feeling that we could help each other learn enough so that effective communication soon would take place. For some of us this turned out to be true, and many students in the laboratories of the participants reaped the benefit of the “new enlightenment.” It was a particularly heartening social occasion because individuals from Australia, Canada, England, France, Hungary, Japan, United States, and the Soviet Union met in enthusiastic amity and forged long-lasting scientific and personal links.
Abstracts reporting the discovery of GABA
Three abstracts related to GABA were submitted to the conference of the American Society of Biological Chemists, and published in the proceedings.
\(\gamma\)-Aminobutyric acid in brain. EUGENE ROBERTS and SAM FRANKEL (introduced by C. CARRUTHERS). Division of Cancer Research, Washington Univ., St. Louis, Mo.
Relatively large quantities of an unidentified ninhydrin-reactive material were found in numerous two-dimensional paper chromatograms of protein-free extracts of fresh mouse, rabbit, and frog brains. At most, only traces of this material were found in a large number of many other normal and neoplastic tissues and in urine and blood. The eluate from suitably chosen strips of one-dimensional phenol chromatograms of mouse brain extract contained the unknown substance and only traces of valine and an unidentified peptide material. A comparison of the properties of the unknown substance in the eluate with those of known compounds by chromatography in different solvent systems showed it to be identical with \(\gamma\)-aminobutyric acid. This conclusion was independently confirmed by the isotopic derivative method using the I-131- and S-35 labeled p-iodo-phenyl sulfonyl derivatives (S. Udenfriend). Experiments with brain homogenates showed a formation of \(\gamma\)-aminobutyric acid which appeared to be accelerated when glutamic acid was added. \(\gamma\)-Aminobutyric acid was also formed in homogenates of liver and muscle. Experiments are under way to characterize the precursors of \(\gamma\)-aminobutyric acid and the enzymes involved in its formation.
A micro technique for identification of organic compounds using isotopic indicators and paper chromatography. SIDNEY UDENFRIEND (introduced by MILDRED COHN). Dept. of Biological Chemistry, Washington Univ. Med. School, St. Louis, Mo.
Isotopic indicators and paper chromatography, as used in the isotopic derivative method of amino acid analysis, can be employed for rigorous identification of microgram quantities of compounds. A reagent is synthesized in two isotopic forms, with isotopes that can be determined accurately, one in the presence of the other. The unknown substance and the compound being used for comparison are converted to derivatives of the reagents, each with a different isotope. The two derivatives are then mixed and subjected to chromatography on paper. If the two derivatives are identical then one band results in which the proportions of the two isotopes remain constant throughout. This is ascertained by cutting the band into consecutive transverse strips and measuring the isotope proportions in each. If the substances are not identical then the isotope ratio will vary from one end of the band to the other. The technique has been applied to the identification of a substance isolated from paper chromatograms of tissue extracts, having an Rf similar to \(\gamma\)-aminobutyric acid. An authentic sample of S35 labelled pipsyl \(\gamma\)-aminobutyric acid was mixed with the I131 labelled pipsyl derivative of the unknown. One band resulted with ratios of I131 to S35 of 0.433, 0.449, 0,439, 0.433, in consecutive strips. A mixture of S35-pipsyl leucine with I131-pipsyl isoleucine yielded one band with ratios of 3.35, 2.23, 1.90, 1.63, 1.38, in consecutive strips. Similarly, S35-pipsyl valine and I131-pipsyl norvaline yielded one band with consecutive ratios of 0.078, 0.136, 0.253, 0.498, 1.07, 1.26, 3.17.
Detection and identification of metabolites in tissues by means of paper chromatography. JORGE AWAPARA (introduced by HARRY J. DEUEL, JR.). Univ. of Texas, Anderson Hospital for Cancer Research, Houston.
Paper chromatography has been used as a quantitative method for some amino acids which are completely resolved by this procedure. The appearance of several unidentified components in chromatograms from tissue extracts has led to a system of isolating those components by means of paper chromatography, in sufficient quantity to allow identification. Thus far, ethanolaminophosporic ester has been identified in nearly all organs of the rat and some human tumors. Presently, a peptide has been detected in many organs and in blood and an unidentified amino acid in brain only. The identification of the latter is under way.
- Ackermann D. Über ein neues, auf bakteriellem Wege gewinnbares, Aporrhegma. Hoppe-Seyler’s Zeitschrift für physiologische Chemie 69:273-281, 1910.
- Ackermann D. and Kutscher, Fr. Über die Aporrhegmen. Hoppe-Seyler’s Zeitschrift für physiologische Chemie. 69:265-272, 1910.
- Awapara, J. Detection and identification of metabolites in tissues by means of paper chromatography. Fed. Proc. 9:148, 1950.
- Bazemore, A.W., Elliott, K.A.C., Florey, E. Isolation of Factor I. J. Neurochem., 1:334-339, 1957.
- Hayashi, T. and Nagai, K. Action of ω-amino acids on the motor cortex of higher animals, especially γ-amino-β-oxybutric acid as the real inhibitory principle in brain. In: Abstracts of Reviews: Abstracts of Communications. Brussels: 20th International Physiological Congress, p. 410, 1956.
- Hayashi T. and Suhara, R. Substances which produce epileptic seizure when applied on the motor cortex of dogs and substances which inhibit the seizure directly. In: Abstracts of Reviews: Abstracts of Communications. Brussels: 20th International Physiological Congress, p. 410, 1956.
- Roberts, E., Baxter, C.F., Van Harreveld, A., Wiersma, C.A.G., Adey. W.R., and Killam, K.F., eds. Inhibition in the Nervous System and Gamma-aminobutyric Acid. Oxford: Pergamon Press, 1960.
- Roberts, E. and Simonsen D.G. Free amino acids and related substances in normal and neoplastic tissues. In: Amino Acids, Proteins and Cancer Biochemistry, J.T. Edsall, editor, New York: Academic Press, pp. 121-145, 1960.
- Steward, F.C. Thompson, J.F., and Dent, C.E. γ-Aminobutyric Acid: A Constituent of the Potato Tuber? Science, 110:439-440, 1949.