Amino acid activation and formation of amino acyl s-RNA in bovine parotid gland.

Our previous report described the general characteristics of cell-free amino acid incorporating system consisting of bovine parotid gland microsome and cell sap [4]. The requirement of adenosine triphosphate (ATP) , guanosine triphosphate (GTP) and cell sap for the maximal incorporation in the cell-free system may indicate that the general pathway of the protein biosynthesis, amino acid activation, amino acyl soluble ribonucleic acid (amino acyl s-RNA) formation and transfer of amino acid to microsomes, may operate in this system. However, there has been no study on each step by which this protein biosynthesis takes place in parotid gland . In an attempt to elucidate in detail the each step in protein biosynthesis of parotid gland, the following experiments were carried out ; Experiments on the amino acid activation and amino acyl s-RNA formation in parotid gland cell-free system. P32-labeled pyrophosphate (PP32) was prepared by the method of KORNBERG and PRicER[7] and purified by the modified procedure of COHN and CARTER[1] . The preparation of microsome and soluble fraction from parotid gland was carried out as described in the previous paper[4] . Assay Method of Amino Acid Activating Activity One ml. of incubation mixture for the PP32-ATP exchange assay contained 3 μmoles of ATP, 3 ƒÊmoles of PP32(about 100 ,000 counts/minute by Geiger-Muller counter), 10 ƒÊmoles of MgCl2, 100 ƒÊmoles of tris-HCl buffer (pH 7 .8), each 3 ƒÊmoles of amino acid and the enzyme fraction (1 mg . of protein). The cell sap, pH 5.2 fraction , pH 4.5 fraction and pH 4.5 supernatant fraction prepared by the method of SATAKE