Figure 4.

Carrot cells (d 2) were incubated with [¹⁴C]ethanolamine (7.5 μCi/g fresh weight), [¹⁴C]myristic acid (25 μCi/g fresh weight), [³H]inositol (50 μCi/g fresh weight), or with [¹⁴C]linoleic acid 0.1 mCi/g fresh weight) for 2 d. Cells were placed into phosphate-free medium 30 min before the addition of [³²P]Pi (0.4 mCi/g fresh weight) and incubated with isotope for 16 h. Cells were harvested as described in Methods. A, Proteins were separated by 10% SDS-PAGE and visualized by Coomassie brilliant blue staining. Lanes 1 to 3, Partially purified eEF-1A fraction (2 μg of total protein); lanes 4 to 6, microsomal fraction (20 μg of total protein); and lanes 7 to 9, supernatant fraction (20 μg of total protein). In lanes 1, 4, and 7, cells were labeled with [¹⁴C]ethanolamine; in lanes 2, 5, and 8 with [¹⁴C]myristic acid; and in lanes 3, 6, and 9 with [³H]inositol. B, Image of gel shown in A using a phosphor imager tritium screen exposed for 13 d. C, Purified eEF-1A labeled with [³²P]Pi. Lane 1, 10% SDS-PAGE visualized by Coomassie brilliant blue staining (2 μg); lane 2, autoradiograph of [³²P]Pi eEF-1A exposed to film for 5 d. D, [¹⁴C]Linoleic acid-labeled cells: 40,000g supernatant protein and microsomes. Lanes 1 and 2, 10% SDS-PAGE visualized by Coomassie brilliant blue staining. Lane 1, Microsomes (20 μg of total protein); lane 2, soluble protein (20 μg of total protein); and lanes 3 and 4, autoradiograph of SDS-PAGE. Arrows indicate migration of 50-kD standard. Molecular mass standards are shown at left (in kD). In vivo-labeling experiments were repeated at least twice with duplicates.