Horseradish peroxidase (HRP), an enzyme internalized by fluid phase pinocytosis, has been used to study the process by which pinosome contents are delivered to lysosomes in Chinese hamster ovary cells. Pinosome contents were labeled by allowing cells to internalize HRP for 3-5 min . Following various chase times, cells were either processed for HRP and acid phosphatase (AcPase) cytochemistry or homogenized and fractionated in Percoll gradients . In Percoll gradients, pinosomes labeled by a 3-5-min HRP pulse behaved as a vesicle population more dense than plasma membrane and less dense than lysosomes. In pulse-chase experiments, internalized HRP was chased rapidly (3-6-min chase) to a density position intermediate between the "initial" pinocytic vesicle population and lysosomes. With longer chase periods, a progressive accumulation of HRP in more dense vesicles was observed . Correspondence between the HRP distribution and lysosomal marker distribution was reached after a^-1-h chase. By electron microscope cytochemistry of intact cells, the predominant class of HRPpositive vesicles after pulse uptakes or a 3-min chase period was characterized by a peripheral rim of reaction product and was AcPase negative . After 10-120-min chase periods, the predominant class of HRP-positive vesicles was characterized by luminal deposits and HRP activity was frequently observed in multivesicular bodies . HRP-positive vesicles after a 10-or 30-min chase were AcPase-positive. No H RP activity was detected in Golgi apparatus . Together these observations indicate that progressive processing of vesicular components of the vacuolar apparatus occurs at both a prelysosomal and lysosomal stage .Prelysosomal endocytic compartments have recently been the subject of much interest in cell biology . These compartments, referred to variously as pinosomes, endosomes, intermediate vacuoles, or receptosomes (for reviews, see 6, 16, 31), have been described predominantly by electron microscopy and recently by cell fractionation . A major physiological function ofthese compartments appears to be ligand-receptor dissociation in response to vesicle acidification. In the present work, we have addressed the question of whether lysosomal as well as prelysosomal endocytic intermediates exist. Horseradish peroxidase (HRP'), an enzyme internalized by fluid phase pinocytosis in fibroblasts (1), has been chosen as the pinocytic marker because as a solute it should be included in all 'Abbreviations used in this paper: AcPase, acid phosphatase; CHO, Chinese hamster ovary; DAB, diaminobenzidine ; FC10, 10% fetal calf serum ; HRP, horseradish peroxidase ; aMEM, Eagle's minimum essential medium, alpha modification ; MIT, monoiodotyrosine . 108 pinocytic vesicles irrespective of their origin and because it can be readily assayed spectrophotometrically and localized cytochemically. A pulse-chase approach combined with cell fractionation and cytochemistry has been taken . The chief result of this work has been to demonstrate a rapid and progressive process...

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Pinocytic vesicles (pinosomes) and lysosomes from suspension cultured, Chinese hamster ovary (CHO-S) cells have been resolved as two non-overlapping organelle populations by analytical centrifugation in Percoll gradients. Pinosomes were labeled with either horseradish peroxidase (HRP), a fluid phase content marker, or by radioiodination by pinocytosed lactoperoxidase (LPO). CHO-S cell lysosomes followed by three different marker enzymes and electron microscopy behaved as a single, dense organelle population. Pinosomes were partially resolved from plasma membrane, a less dense organelle population.