Studies on the brush border membrane of mouse duodenum. II. Membrane protein metabolism

Mouse duodenal microvillus membrane protein metabolism was measured using radioactive labelling techniques. Labelled amino acids were introduced into the lumen of ligatured duodena. Following exposure to label, brush border membranes were isolated and analyzed. Experiments measuring the specific activity of protein labelled with a single amino acid revealed that total membrane protein appeared to turnover in about 14 hr. Protein in the mucosal homogenate had a faster turnover rate. Turnover rates of individual proteins were measured with single and dual isotope experiments. Membrane protein was solubilized with sodium dodecyl sulphate (SDS) buffer. Single isotope experiments showed that all polypeptides separated on SDS-gels were maximally labelled at 6 hr after injection. Bands did not incorporate label linearly. Rates of loss (degradation) of label from membrane proteins in the seventeen bands appeared to be related to the estimated molecular size of the proteins. Rates were highest for larger polypeptides. A double isotope technique, in which proteins were allowed to incorporate the same amino acid in two isotopic forms, delivered with a set time interval intervening, revealed that the ratios of the second label to the first in the SDS-separated polypeptides were highest for larger proteins and lowest for smaller polypeptides. Certain assumptions were outlined and the ratios taken as measures of turnover of proteins. Loss of label due to cell sloughing is discussed. A mixture of labelled amino acids (excluding leucine) was used to show that differences in leucine contents of different proteins was not an explanation for the variation in level of leucine radioactivity in different bands. For specific activity measurements throughout, protein in gels was quantitated with reference to the uptake of Coomassie stain. The use of this stain was validated by the finding that, at low protein concentration, the amount of stain taken up was proportional to the amount of bovine serum albumin or membrane protein loaded.