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. 1978 May;75(5):2338–2342. doi: 10.1073/pnas.75.5.2338

Coupled cell-free synthesis, segregation, and core glycosylation of a secretory protein

Vishwanath R Lingappa *, Jaisri R Lingappa *, Rajani Prasad †, Kurt E Ebner †, Günter Blobel *
PMCID: PMC392548  PMID: 276877

Abstract

mRNA from rat mammary glands 13-15 days post partum was translated in a wheat germ cell-free system either in the absence or in the presence of ribosome-denuded membranes prepared from isolated rough microsomes of dog pancreas. Newly synthesized α-lactalbumin was identified by immunoprecipitation with a monospecific rabbit antiserum against rat α-lactalbumin and was characterized by partial amino-terminal sequence determination and by lectin affinity chromatography. In the absence of membranes a presumably unglycosylated form of α-lactalbumin was synthesized that bound neither to concanavalin A-Sepharose nor to Ricinus communis lectin-agarose and that contained an amino-terminal signal peptide region comprising 19 amino acid residues. In the presence of membranes a processed form was synthesized that lacked the signal peptide portion and that had an amino-terminal sequence identical to that of mature α-lactalbumin. Furthermore, this processed form was found to be segregated, presumably within the microsomal vesicles, because it was resistant to post-translational proteolysis. It was also found to be glycosylated, and because it bound to concanavalin A-Sepharose, from which it could be eluted specifically by α-methyl mannoside, but not to R. communis lectin-agarose, it was presumably core-glycosylated. Processing, segregation, and core glycosylation were observed to proceed only when membranes were present during translation and not when they were added after translation.

Keywords: rat mammary mRNA, immunoprecipitation of α-lactalbumin, dog pancreas microsomal membranes, lectin chromatography, radiosequencing

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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