No abstract

No abstract

The levels of casein mRNA, polysomal casein synthesis, and intracellular casein have been determined during mid-pregnancy in the rat mammary gland. Ovariectomy of the mid-pregnant rat and hormone replacement were used as a model for studying the regulation of casein synthesis. Following ovariectomy a twofold increase in both casein mRNA activity, determined in a wheat germ translation assay, and casein mRNA sequence concentration, measured using a selective cDNA hybridization probe, was observed. This effect on mRNA accumulation was first observed within 16 h following ovariectomy and was maximal between 24 and 48 h. Progesterone, but not estradiol or hydrocortisone, administered at the time of ovariectomy prevented the increase in casein m R N A . These changes in casein m R N A were compared with the mRNA levels normally observed during development of the rat mammary gland from virginity until lactation. A comparison of polysomes isolated from mid-pregnant and lactating mammary glands revealed a shift from small polysomes, containing predominantly monosomes, disomes, and trisomes E e v i o u s results from our laboratory have demonstrated that appreciable amounts of casein m R N A are present in the rat mammary gland during early and mid-pregnancy . Although secretion of casein does not occur at these times we found that at mid-pregnancy the level of casein mRNA per alveolar cell was 25 to 50% of the maximal levels reached during lactation. This increase in casein m R N A sequences, measured with a selective cDNA-hybridization probe, was coincident with an increase in casein m R N A activity, as measured by cell-free translation (Rosen and Barker, 1976). Thus the relationship between the levels of casein m R N A and the synthesis and secretion of casein are not understood, and the precise hormonal mechanisms regulating these processes remain unclear.The levels of casein mRNA observed during mid-pregnancy may rcflect both the positive and negative effects of several peptide and steroid hormones. Thus, the increase in casein mRNA activity seen during early and mid-pregnancy may be related to changes in serum prolactin and, especially in the rat, placental lactogen. These two polypeptide hormones have been shown to induce casein synthesis (Lockwood et al., 1966; Turkington, 1968) and casein mRNA (Houdebine and Gaye, 1975 both in vivo and in mammary gland organ culture. Furthermore, high levels of progesterone during pregnancy are thought to antagonize the lactogenic effects of these hormones (Davis et ai., 1972;Assairi et al., 1974). The present studies were therefore designed to investigate the relationship among casein m R N A levels, polysomal casein synthesis. and the intracellular concentrations of casein during mid-pregnancy in the rat.to larger polysomal aggregates, containing 7 to 1 1 ribosomes per mRNA. A similar shift was observed within 48 h following ovariectomy. Using specific immunoprecipitation, casein synthesis on isolated polysomes was measured in a cell-free translation assay. Casein comprised ...

(8, 13, 19. 20, 26, 27). The Bacillus subtilis, rat liver, and ovine glutamine synthetases have been similarly investigated (4-6, 14, 18, 21) although not as intensively.With respect to higher plants, the pea seed enzyme has been purified and partially characterized (23,25), as have the carrot (3) and rice (9) enzymes. An earlier paper (16) described the preparation of purified glutamine synthetase from pea leaves, which is perhaps distinct from the pea seed enzyme. In this paper, further information concerning its kinetic properties are reported. MATERIALS AND METHODSThe methods were the same as described in our earlier paper (16). However, in the present study an additional assay method was employed; namely, the transfer assay. In this assay the ability of the enzyme to catalyze the following reaction was measured:Pi, or AsO4 y-glutamyl hydroxamate +NH3 ,y-Glutamyl hydroxamate was determined colorimetrically by the procedure described previously (16). The components of the reaction mixture are given in Table VI.In all cases, the enzyme used in the present studies was at least 75% pure and usually homogenous as determined by disc gel electrophoresis. The enzyme preparations were stored for periods of up to 3 months. RESULTSExcept where noted, the results below were similar using either the modified Pi assay (16) or the glutamyl hydroxamate assay (16).Metal Ion Specificity. The purified enzyme had an absolute requirement for a divalent cation, and Mg`gave the optimal activity. Mn2+ and Co2+ were 45 to 60% and 30 to 45% as effective, respectively, but with Co2' and especially Mn'+, the ratio of concentration of divalent cation and ATP was critical; this ratio strongly influenced the pH optimum (16). Zinc and Fe2+ gave less than 5% of maximal activity, although no studies were made to determine condition (such as pH optimum) for optimal activity with these cations. (Fig. 1), when assayed at pH 7.8. At pH 6.2, however, the addition of 0.5 mM Mn'2 stimulated the reaction by at least 100% (ATP, 8 mm, MgSO4, 20 mM), but only at lower levels (10 mM) of L-glutamate. With 30 mm glutamate, the addition of 0.25 to 1.0 mm Mn2+ caused some inhibition, but much less than that which occurred at pH 7.8 under the same conditions. Part of the inhibition caused by Mn2+ at pH 7.8 is due to a change in pH optimum, from 7.8 to 8.0 (Mg2+ alone) to below 5.8 (Mg2+ plus Mn2+) (Fig. 2). This is similar to the optimum pH for Mn2+ alone (where [Mn'+] = [ATP]) which is about 5.2 (16).

No abstract