Initiation and processing in vitro of the primary translation products of guinea-pig caseins

Abstract

1. Guinea-pig caseins synthesized in a mRNA-directed wheat-germ cell-free protein-synthesizing system represent the primary translation products, even though they appear to be of lower molecular weight when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in parallel with caseins isolated from guinea-pig milk. 2. Identification of the N-terminal dipeptide of the primary translational product of caseins A, B and C and alpha-lactalbumin showed that all shared a common sequence, which was identified as either Met-Arg or Met-Lys. 3. Procedures utilizing methionyl-tRNAfMet or methionyl-tRNAMet in the presence or absence of microsomal membranes during translation provide a rapid method of distinguishing between N-terminal processing of peptides synthesized in vitro and other post-translational modifications (glycosylation, phosphorylation), which also result in a change in mobility of peptides when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. The results demonstrate that guinea-pig caseins, in common with most other secretory proteins, are synthesized with transient N-terminal 'signal'-peptide extensions, which are cleaved during synthesis in the presence of microsomal membranes.

Characterisation of a Membrane‐Bound Serine‐Specific Casein Kinase Isolated from Lactating Guinea‐Pig Mammary Gland

“…[21] (see also [12]), the position of the proteins determined by autoradiography, and the individual proteins eluted. The recovered proteins were then each digested with trypsin and oxidised with performic acid as described previously [12].…”

“…The tissue was then removed from the medium, homogenised in 50 p1 of 15 mM sodium citrate, 150 mM NaCl, then incubated for 1 h in the presencc of 40 pg RNAse A ml-', and finally pooled with the original incubation medium. The caseins were then separated by electrophoresis on 10% polyacrylamide gels in the presence of sodium dodecylsulphate (0.1 w/v) and the individual caseins recovered from the gel as described previously [12].…”

“…The recovered proteins were then each digested with trypsin and oxidised with performic acid as described previously [12]. The resulting peptides were separated in two dimensions o n cellulose-coated flexible poly(ethy1enc terephthalate) supports (10 x 20 cm) (see [XI), using electrophoresis at pH 1.9 (glacial acetic acid/98 % formic acid/water, X7:25: 897) for 2.5 h at 300 V and 4 "C for the first dimension, and ascending chromatography in isobutyric acid/0.5 M N H 4 0 H ( 5 : 3) in the second dimension.…”

Extensive destruction of newly synthesized casein in mammary explants in organ culture

“…202 and into the alveolar lumen. This major secretory protein of the lactating mammary gland is synthesized on membrane-bound polyribosomes (Houdebine & Gaye, 1975;Harrison et al, 1976) with a signal peptide that undergoes co-translational proteolytic cleavage (Craig et al, 1979;Mercier & Gaye, 1980). Little or no degradation of radiolabelled casein was seen in mammary explants cultured in the presence of hormones when measured 24 and 48h after an initial 1h pulse of radioactivity (Wilde et al, 1980 by Forsyth & Myers (1971), and cultured for 18h at 370C in Medium 199 in the presence of insulin (5,ug/ml), prolactin (l,ug/ml) and cortisol (lpg/ml) as described by Al-Sarraj et al (1979).…”

“…It is well established that casein is synthesized, like many other secretory proteins, as a preprotein and that a signal peptide is cleaved as a co-translational event during processing of the proteins (Craig et al, 1979;Mercier & Gaye, 1980). The signal peptide of rabbit Jl-casein contains 15 amino acid residues, of which five are leucine.…”