Protein Synthesis in a Cell-free System Derived from the Rat Parotid Gland

Abstract

Abstract A cell-free amino acid-incorporating system has been prepared from rat parotid salivary glands and has been shown to possess activity comparable to that of a similarly constituted rat liver cell-free system. Administration in vivo of isoproterenol, prior to removal of the glands, reduced the secretory ribonuclease content, along with the other exportable proteins, to sufficiently low levels to permit the preparation of intact ribonucleoprotein particle and pH 5 enzyme fractions. The pH 5 enzyme fraction was tested separately, and shown to be as active as a corresponding rat liver fraction in the formation of amino acyl transfer RNA. Ultracentrifugal analyses of the ribonucleoprotein particle fraction, derived from rat parotid tissue, revealed a number of ribonuclease-sensitive peaks with sedimentation coefficients consistent with polyribosomal species. The optimal conditions for amino acid incorporation were established revealing an optimal Mg++ ion concentration of 10 to 14 mm as contrasted with a lower optimum for the liver system, and a less prolonged time course of incorporation than in the rat liver system. The addition of polyuridylic acid to the rat parotid system stimulated the incorporation of phenylalanine into protein by approximately 5-fold with no shift in the optimal Mg++ ion concentration. This was in contrast to the 2.5 fold stimulation, requiring an upward shift in the Mg++ ion concentration, seen in the rat liver system. These findings represent the first report of a cell-free protein synthesis system derived from a mammalian salivary gland, and also demonstrate a new technique for reducing damage to subcellular fractions in exocrine glands by their endogenous secretory enzymes.