The effect of amino acids on protein metabolism as measured in long‐term experiments in immature brain explants
Authors: Schneider Joseph F. , de Martini Joy E. , Toth Jeno , Lajtha Abel 
Published: January 2005

In a study of a system suitable for investigating long‐term effects on brain protein metabolism, we measured amino‐acid incorporation into isolated immature brain explants incubated under sterile conditions up to ten days.

Measurements of changes in total proteins, total DNA, cell number during the experiments, and 14C‐thymidine incorporation measurements indicated no significant net growth; new cell formation was below 5% in a 5‐day period; therefore, amino‐acid incorporation was mainly due to protein turnover.

The rate of incorporation in our immature brain preparation was similar to that of the adult brain in vivo: by ten days about one‐half of the tissue protein turned over. The label incorporated was released in subsequent incubations with cold amino acids. Such release occurred in all subcellular fractions examined.

Incorporation was fairly stable; at temperatures below 30°C it rapidly declined, but it was not affected when phenylalanine or the branched chain amino acids (leucine, isoleucine, valine) were elevated in the incubation medium. Brief exposure to low amino‐acid media had no effect; longer exposure resulted in tissue damage.

Our model system indicates that overall brain protein turnover is not sensitive to such variations in the level of most amino acids, which may occur under various conditions.

Protein metabolism of the nervous system occurs at a high rate. A recent long‐term labeling method (Lajtha, Latzkovits, and Toth, 1976) gave a best fit to incorporation curves by assuming two compartments for adult brain proteins, one of which (about 6%) has a half‐life of 15 hr and the other (94%) has a half‐life of ten days. The disappearance of protein‐bound label with time under conditions in which all proteins were previously labeled indicated that most, possibly all, proteins in brain are in a dynamic state (Lajtha and Toth, 1966). Incorporation of amino acids was found in all proteins and structures that have been studied to date; myelin proteins previously thought less active are also metabolized at a significant rate (Sabri, Bone, and Davison, 1974; Lajtha, Toth, Fujimoto, and Agrawal, 1977). We have fairly extensive information available in addition to turnover studies about the mechanisms of protein synthesis in brain (Roberts, 1971); protein breakdown was also studied in some detail (Marks and Lajtha, 1971).

In contrast to our knowledge about protein metabolism under physiological equilibrium conditions, our information about alterations during functional demands or pathological conditions is scanty. Although a significant amount of work has been reported, largely because of technical difficulties the results are difficult to interpret unequivocally. The present report represents our effort to address some of the obstacles: to develop a system in which influences on long‐term incorporation can be studied. Our system meets requirements that are difficult to meet in most other systems: (a) if needed, the specific activity of the precursor amino acid can be maintained at constant levels in the tissue for a number of days, and (b) the alterations of equilibrium (such as altered amino‐acid levels) can also be maintained within narrow limits throughout the experiment. Our system, isolated explanted segments from immature brain, showed stability in rates of incorporation during these experiments and thus should be a useful test system for protein metabolism studies. To our knowledge this is the first such study on brain explants; as a first approach we focussed our efforts on measuring protein turnover rates in this preparation and observing the effect of changes in amino‐acid levels on incorporation.