HPLC analysis of free amino acids and amino acids of total proteins in cultured cells: An application to the study of rat sertoli cell protein metabolism

Abstract

A simple, rapid and, sensitive HPLC method, coupled with fluorometric detection, has been worked out and employed to determine the intracellular free amino acid concentrations and the amino acid composition of total proteins in rat Sertoli cell primary culture. Sertoli cells were isolated enzymatically from testes of 20- and 28-day-old rats and cultured at 32°C in Eagle's minimum essential medium. On the second day of culture, cell monolayers were quickly rinsed with ice-cold saline, immediately frozen in liquid nitrogen, accurately harvested, and homogenized in 10% trichloroacetic acid. Tissue free amino acids were determined in the acidic soluble fraction following neutralization, while the precipitate was hydrolyzed for the evaluation of the fractional content of amino acids into total proteins. Amino acid samples were derivatized with o-phthaldialdehyde/3-mercaptopropionic acid and resolved by a linear one-step acetonitrile gradient in 12.5 m m sodium phosphate buffer, pH 7.2, employing a 5-μm particle size reversed-phase column. Fluorescence was monitored with excitation at 330 nm and emission at 450 nm. Under these conditions all major physiological amino acids could be satisfactory separated, identified, and subsequently quantified with the aid of standards. The run time was about 50 min; the linearity was excellent over a large range of concentrations (1–800 pmol) and the lower limit of sensitivity appeared to be 0.5 pmol. This method permits us to demonstrate age-dependent modifications in the intracellular amino acid pool and to adequately evaluate the process of protein synthesis in cultured Sertoli cells. The precise evaluation of the specific activity in the amino acid precursor pool allows us the quantitative estimation of protein synthesis by incorporation of a radioactive amino acid; the fractional content of the amino acid precursor into total cell protein was used for the calculation of the rate of protein synthesis.