Differences in the efficiency and pattern of incorporation of [ 3H]leucine and [ 3H]proline into proteins of adult cat brain

Previous EM autoradiographic studies have shown that injection of [ 3H]proline ([ 3H]Pro) into the cat dorsal column nuclei (DCN) results in heavy labeling of microglia but negligible labeling of DCN neurons. [ 3H]Leucine ([ 3H]Leu), in contrast, was extensively incorporated into both neurons and glia. We now report preliminary assessment of differences in molecular labeling patterns produced by the two amino acid precursors in DCN injection sites (24 h following injection; equal amounts of [ 3H]Pro and [ 3H]Leu of equal specific radioactivity). [ 3H]Pro, despite its lack of incorporation into neurons, labeled DCN to a significantly greater extent than [ 3H]Leu (30.0 dpm/μg protein/μCi for [ 3H]Pro vs 11.7 dpm/μg protein/μCi for [ 3H]Leu). Greater than 90% of the radioactivity from both precursors was recovered in protein as opposed to TCA soluble or ethanol soluble molecules. Of the [ 3H]Pro- or [ 3H]Leu-derived radioactivity recovered in protein, greater than 94% was found to remain in the original precursor form. Fluorographic analysis of SDS acrylamide gels showed labeling of a wide variety of individual proteins with either amino acid. However, particular molecular weight classes were relatively more heavily labeled with either [ 3H]Leu (47, 63, 77 kDa) or [ 3H]Pro (22, 45, 50, 66, 80 kDa). Overall the results indicate that the difference in cellular distribution of incorporated [ 3H]Pro and [ 3H]Leu, as observed by EM autoradiography is a reflection of the extent of labeling and specific labeling pattern of proteins isolated from the tissue.