Abstract
Iron binding proteins obtained from rat intestinal mucosa were analysed in order to clarify their roles in iron absorption. The upper intestinal mucosa of rats which were administered a 59Fe solution through a stomach tube was homogenized with a Dounce homogenizer. The homogenate was centrifuged at 20, 000. g and then the precipitate was extracted with Triton X-100. Two iron binding proteins (Peak 1 and Peak 2) were isolated by gel filtration on Sepharose 6 B from the supernate and Triton extract. Since Peak 2 has been identified as mucosal ferritin, Peak 1 was analysed.
Peak 1 was a large molecular membrane protein with an isoelectric point of 2.5, whose apparent molecular weight was over 106 as estimated by gel filtration. In the time course experiment, the amount of 59Fe incorporated into Peak 1 decreased from the maximal value found at one hour, while that into ferritin onthe contrary increased up to five hours. This finding suggested that 59Fe of Peak 1 was transferred to ferritin.
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of Peak 1 from the supernate and Triton extract revealed four bands whose apparent molecular weights were in the range of 145, 000 to 78, 000. They were stained by both Coomassie brilliant blue and PAS stain. Although Peak 1 reacted weakly against anti-human lactoferrin antiserum by immunoprecipitin reaction, the nature of these protein bands separated by SDS-PAGE could not be further characterized.
To determine the subcellular localization of Peak 1, the brush border membrane from the upper intestinal mucosal homogenate of rats orally administered the 59Fe solution was prepared by the CaC12 precipitation method. Two 59Fe peaks were isolated by gel filtration on Sepharose 6 B from both the Triton extract of the brush border membrane and the CaC12 precipitate. Peak 1 found in the CaC12 precipitate was shown to be a contaminant of Peak 1 in the brush border, because 59Fe per total sucrase activity of Peak 1 from the brush border was coincident with that from the CaCl2 precipitate in a similar experiment. Therefore, it was suggested that Peak 1 was localized on the brush border membrane.
These results show that Peak 1, a large molecular iron binding protein, participates in the initial step of iron absorption.
