The work proposed aims to determine whether during maturation, all immature mammalian red cells lose selected membrane components, and in particular, the transferrin receptor by the extrusion of membrane limited vesicles. The objective is to determine whether (and how) the protein and enzyme activities of these vesicles varies between species, and how (or if) this variation reflects the components disappearing from the red cell during maturation. The work involves the use of various species made anemic by phlebotomy or phenyl hydrazine treatment, separation of vesicles from the plasma or culture medium. The analysis of the protein content is by SDS-PAGE as well as with specific antibodies, such as those for the transferrin receptor and the clathrin uncoating ATPase and assays for specific enzymes and functions, such as nucleoside transport. The stage of the life cycle of the red cell at which vesicle formation starts is known. To address this question, immature, nucleated red blood cells from spleens of anemic mice will be isolated and cultured to determine whether vesicle formation is a postenucleation event. To address the question whether a single vesicle contains all the lost activities or ether these are segregated amongst different vesicles, the isolated vesicles from maturing sheep reticulocytes will be separated by isoelectric focusing or chromato-focusing to see if the specific functions can be segregated. Electron microscopic studies using gold labelled antibodies to detect specific functions will be used to confirm whether a single vesicle is a repository for several functions. Red cells lose many plasma membrane functions during maturation. It is not known whether all these functions are lost with a common time frame. It is proposed to address the time course for the loss of several selected functions and whether physiological significance can be attributed to the putative differences in rate of loss. The ultimate objective is to determine what signals are used to eliminate obsolete, but still functional membrane proteins, during maturation of the red cell. Circulating transferrin receptors have been detected in man during elevated reticulocyte formation and in some malignancies. Knowledge of the composition of the released vesicles and the type of proteins lost may provide additional approaches to diagnosis of malignancy and aberration in red cell production.
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