THE INCORPORATION OF14C-THREONINE AND14C-GLUCOSAMINE INTO SUBCELLULAR FRACTIONS AND INTO BOVINE SUBMAXILLARY MUCIN BY SLICES OF BOVINE SUBMAXILLARY GLAND

Publisher Website
Google Scholar
Abstract
Bovine submaxillary gland slices were used to investigate the subcellular site of biosynthesis of bovine submaxillary mucin (BSM). Slices were incubated with14C-threonine and14C-glucosamine in Krebs medium III at pH 7.4 and 37 °C for varying periods of time up to 180 minutes. Methods of subcellular fractionation based on those developed for liver proved unsatisfactory for use with submaxillary gland, probably because of the presence of large amounts of highly viscous mucin. A method of subcellular fractionation was developed which yielded five fractions: P (total soluble protein) and C (soluble BSM, purified by Cetavlon precipitation) were prepared from the 16,000 × g supernatant; and R (ribosomes), S (deoxycholate-soluble membrane-bound protein), and a residual fraction D were prepared from the 16,000 × g pellet. The incorporation of both tracers into these fractions was time-dependent and was inhibited by preincubation of the slices for 15 minutes with puromycin. The highest specific activities were recorded in the R and S fractions, implicating them as possible sites of glycoprotein biosynthesis. The time course of14C-threonine incorporation suggested that protein is made on ribosomes, is transferred to the endoplasmic reticulum, and is then ready for storage or secretion. The time course of14C-glucosamine incorporation suggested that carbohydrate is linked to the protein both while the protein is still associated with the ribosomes and after it has been released from them. The involvement of ribosomes in carbohydrate attachment to polypeptide received additional support from zone centrifugation analysis on sucrose-density gradients of ribosomes labeled with14C-glucosamine.