The biosynthesis of rat serum albumin. In vivo studies on the formation of the disulfide bonds.

In order to learn at what stage the disulfide bonds of albumin are formed during its biosynthesis, we perfused rat livers with iodoacetamide and then isolated the intracellular precursor, proalbumin, from organelles known to be in the pathway of albumin synthesis and secretion. The alkylated cysteines in proalbumin were determined as a measure of its thiol groups in vivo. Proalbumin of smooth microsomes was found to contain a single thiol, which is proposed to be the noncoupling cysteine occurring residue 34 in circulating albumin. Proalbumin in rough microsomes contained an average of two cysteines; the additional cysteine thiol was largely situated in the COOH-terminal region and disappeared rapidly after blocking albumin synthesis with cycloheximide. In nascent chains of proalbumin, about 45% of the cysteine + cystine was in the thiol form. From these findings we propose that disulfide bond formation begins while the nascent chain is still attached to the ribosome and proceeds in an NH2 to COOH direction. The disulfide bonding apparently is completed into the endoplasmic reticulum. Possible intermediates in the process such as mixed disulfide forms of proalbumin with glutathione or cystamine were not detected. We suggest that cysteine-34 does not participate in disulfide bonding because the NH2 terminus of proalbumin remains loosely bound to the membrane, attached by a hydrophobic segment of the chain at residues 21-27.