Guinea-pig milk-protein synthesis. Isolation and characterization of messenger ribonucleic acids from lactating mammary gland and identification of caseins and pre-α-lactalbumin as translation products in heterologous cell-free systems

Abstract

1. The major milk proteins synthesized by the lactating mammary gland of the guinea pig were identified and designated as caseins A, B and C and alpha-lactalbumin, with estimated mol.wts. of 28000, 25500, 20500 and 14500 respectively. 2. Antisera to the total casein fraction and to alpha-lactalbumin were prepared from rabbits. The milk proteins were also iodinated with either 131I or 125I. 3. A poly(A)-rich RNA fraction was isolated from lactating guinea-pig mammary glands. Isolation was by affinity chromatography on oligo(dT)-cellulose. 4. Examination of this RNA fraction by electrophoresis on polyacrylamide gels containing formamide indicated three major species 1, 2 and 3, with estimated wol.wts. of 5.4 X 10(5) and 3.3 X 10(5), and the apparent absence of rRNA species. 5. The poly(A)-rich RNA stimulated protein synthesis in heterologous cell-free systems based on wheat germ, Krebs II ascites-tumour cells, and the latter supplemented with an initiation factor-3 fraction from rabbit reticulocyte ribosomes. 6. Between 80 and 90% of the protein synthesis directed by the mRNA was for milk proteins. 7. Analysis of the proteins immunoprecipitated by the alpha-lactalbumin antiserum showed in the wheat-germ system that the product was a protein with a molecular weight greater than that of alpha-lactalbumin, whereas in the ascites-tumour-cell systems both this protein and alpha-lactalbumin were found. When the larger protein was treated with CNBr and the resulting peptides were examined, it was shown that the extra peptide was at the N-terminus. This and other evidence is adduced for the initial translation product of alpha-lactalbumin being a precursor with an addition of about ten amino acids at the N-terminus. 8. Similar analysis of the casein immlnospecific proteins produced under the direction of mRNA indicated that the products had a molecular weight that was apparently a littel smaller than that of the caseins synthesized in vivo. This was not consistent with higher-molecular weight casein precursors. 9. Possible explanations for the results obtained are discussed, especially in terms of the physiological significance of the pre-alpha-lactalbumin as a secretory protein.

Pre-β-lactoglobulin Synthesis by mRNA from Bovine Mammary Gland

“…[2][3][4][5][6][7][8] This result, together with the fact that p-Iactoglobulin is synthesized on membrane-bound polysomes, 9) strongly supports that the biosynthesis and transport of p-Iactoglobulin follow signal hypothesis. OUf results, with the previous identification of prea-Iactalbumin 13 ) and precaseins H ) confirm that signal hypothesis is applicable for the biosynthesis and processing of all of major milk proteins.…”

Structure and expression of c-fgr protooncogene mRNA in Epstein-Barr virus converted cell lines

“…Polyadenylated RNA was isolated by affinity chromatography on oligo-dT cellulose (Collaborative Research Ltd.) according to Craig et al (1976).…”

Differential tissue expression of the glutathione transferase multigene family

“…of absolute ethanol. Separation of poly(A)containing RNA was then performed as previously described (Craig et al, 1976).…”

“…Immunoprecipitation procedures using antibodies raised against GSH transferase subunits 1, 2 and 3 ( Fig. lb) were as previously described (Craig et al, 1976).…”