Flotation through a 5.6% Percoll gradient of pinosomes from Chinese hamster ovary (CHO) cells labelled during a 10 min internalization period with horseradish peroxidase (HRP), a solute, revealed two pinosomal populations, the expected low-buoyancy population and an unexpected buoyant population. The buoyant pinosomes that sedimented similarly to plasma membrane were not an artifact of HRP trapping during homogenization or of cell surface-adherent HRP. No trapping or cell surface adherence of HRP could be detected by biochemical or cytochemical assays, even after internalization periods as short as 15 s to 1 min. With short uptake times, the buoyant pinosome population was the major HRP positive vesicle population, suggesting a precursor-to-product relationship between the two populations. In pulse-chase experiments, the buoyant pinosome population was shown to be highly exocytic and the precursor to later pinosomes. By electron-microscope cytochemistry, rapidly labelled, HRP positive pinosomes (15 s to 1 min uptake) were typically smooth vesicles with a median diameter of approximately equal to 0.30 micron and a size range from approximately equal to 0.10 micron to greater than 1.0 micron in diameter. We suggest that these rapidly labelled structures are a very early stage in the intracellular processing of pinocytic vesicles.

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