Studies on the biosynthesis of protein by lactating guinea-pig mammary gland. Characteristics of the synthesis of alpha-lactalbumin and total protein by slices and cell-free systems.

1. Slices of lactating guinea-pig mammary gland were incubated with radioactive amino acids and the various subcellular fractions separated by centrifugation after disruption of the cells by mincing and homogenization. The most active fraction for protein synthesis appeared to be the ;mitochondrial'. 2. When the subcellular fractions were prepared without previous incubation of the cells and were then incubated with radioactive amino acid and an energy-generating system, the ;mitochondrial fraction' was at least as active for protein synthesis as the ;microsomal fraction'. 3. The ribosomes in the microsomal fraction are mainly unattached to membrane whereas those in the mitochondrial fraction are probably attached to fragments of the rough-surfaced endoplasmic reticulum. This latter fraction contains few mitochondria. 4. The combined mitochondrial and microsomal fractions incorporated radioactive amino acids into alpha-lactalbumin. 5. The radioactive leucine isolated from tryptic and chymotryptic peptides of alpha-lactalbumin synthesized in the cell-free system was not of uniform specific radioactivity. This was consistent with the polypeptide being assembled by the sequential addition of amino acids. 6. Evidence is presented for the polypeptide chain of alpha-lactalbumin being assembled from the N-terminus and for chain initiation in the cell-free system. 7. It is concluded that cell-free extracts of lactating mammary gland synthesize alpha-lactalbumin.