Kinetic analysis of new formation, internalization, and turnover of bristle coated pits of the myeloid sinuses.

The rate of new formation of bristle coated pits (BCP) of the myeloid sinuses, their internalization, and turnover was determined by labeling of BCP with bovine albumin adsorbed to colloidal gold (Au) at low temperatures (0 degrees C to 4 degrees C), followed by endothelial activation with balanced saline at 38 degrees C to 40 degrees C for graded periods of time. New formation of BCP was practically instantaneous, occurring within 28 seconds, a time span which includes the in situ assembly of the coating protein clathrin as well as of the material effecting the binding of the ligand. Almost the entire population of BCP was turned over in 6 minutes, a time period which encompasses the initial formation of the plasmalemmal bristle coated binding site to the internalization of BCP. The number of BCP varied within a narrow range indicating a substantial degree of physiological constancy resulting from a quantitative parallelism between internalization and new formation of bristle coated binding sites. The number of BCP is markedly reduced in the prolonged (greater than 12 minutes) absence of endocytosable material. The reduction in the number of BCP is reversible. The binding to BCP at low temperatures (0 degrees C to 4 degrees C) of a series of proteinaceous substances adsorbed to Au was examined for the following Au complexes: bovine albumin/Au, rat albumin/Au, IgG/Au, orosomucoid/Au, ovalbumin/Au, fetuin/Au, transferrin/Au, insulin/Au, low density lipoprotein/Au, alpha 2 macroglobulin/Au, and Au stabilized by polyethylene glycol. All these Au complexes bound to BCP although there were differences in the degree of affinity as judged by the number of BCP labeled and by the number of Au/complex particles present in the labeled BCP. Au stabilized by polyethylene glycol did not bind to BCP. Under the condition of these experiments, bovine albumin/Au had the highest degree of affinity for BCP and was therefore selected for the kinetic studies.