Polysomes of bovine anterior pituitary gland and their role in hormone and protein biosynthesis.

Abstract

This laboratory has shown that a cell-free ribosomal system from bovine anterior pituitary glands is capable of incorporating labeled amino acids not only into protein, but also into adrenocorticotropin, growth hormone, and prolactin fractions.', 2 An investigation of the physicochemical and biosynthetic characteristics of this system strongly suggested the presence of functional polysomes.3 In the present study, the pituitary polysomes were resolved into fractions of discrete particle size, and the various factors that affect this function and organization were examined. The polysomes were shown to promote the biosynthesis of prolactin and growth hormone, as well as mixed proteins. Attempts have been made to correlate the aggregate size of the polysomes with the chain length of the hormones synthesized. Materials and Methods.-(1) Radioactive compounds: 3,4-H3-L-proline (5 c/mmole), 4,5-L-H3-leucine (5 c/mmole), and gl-L-H3-phenylalanine (4.25 c/mmole) were all purchased from New England Nuclear Corp. H3-poly U (0.025 c/mmole) and nonlabeled poly U were products of Miles Chemical Co., and Sigma Chemical Co., respectively. (2) Biochemicals: puromycin was obtained from Nutritional Biochemical Corp., and cycloheximide was a gift of Upjohn Co. Crystalline pancreatic ribonuclease (RNase) was a product of the Worthington Biochemical Corp. The sources of the other biochemicals used were those described previously.1 (3) Hormone standards: bovine prolactin (NIH-P-B1) and growth hormone (NIH-GH-B10) preparations were provided by the Endocrine Study Section, National Institutes of Health. (4) Polysomes and other components of the pituitary cell-free system: these were prepared from fresh bovine anterior pituitary glands.' 3 Density gradient analysis of polysomes: All operations were conducted at 0-4?. The polysomal preparations were resolved into components of discrete particle size by sucrose density-gradient fractionation. Linear gradients (15-35% w/v in Medium M1 minus mercaptoethanol, 25 ml vol) were layered on cushions of 5 ml of 50% sucrose in Medium M, using I X 3 in. cellulose nitrate tubes. The polysomal preparation in 1-1.5 ml of Medium S1 was gently layered on top of the gradient. The tube was centrifuged for 270 min at 25,000 rpm in a Spinco model L ultracentrifuge equipped with an SW25.1 swinging-bucket rotor. Fractions of 0.7 ml were then collected using the Isco model 180 density gradient fractionator, Instrument Specialties Co. Ultraviolet (UV) absorbancies were either monitored continuously at 254 my with an Isco model UA-2 UV analyzer equipped with a recorder, or measured at 260 mA on each fraction (after dilution to 1 ml with water) in a Beckman DU spectrophotometer. Presumptive identification of the polysomal species separated on the gradient was made by direct electron microscopy examination, or by sedimentation analysis in a Spinco model E ultracentrifuge. H3-poly U binding to ribosomes: Measurements were made as previously described.3 Incorporation of H3-amino acids into protein and hormones: Previous methods3, 4 were used, except that prolactin was purified by column chromatography on O-(diethylaminoethyl) cellulose (DEAE-cellulose) rather than by the isoelectric precipitation step at pH 5.7. RNA and protein: These were determined as described in earlier papers.', 3 Electron microscopy: Polysome suspensions (suitably diluted) were deposited directly