Renal protein absorption into sub-cellular particles. I. Studies with intact kidneys and fractionated homogenates

• 1.1. Intravenously injected bovine pancreatic ribonuclease A labeled with iodine 125 was rapidly cleared from the blood into the kidneys. Liver and gut absorbed little, and spleen and lung, none.
• 2.2. Most of the radioactivity in kidney homogenates sedimented upon centrifugation, showing it to be associated with particles. The labeled particles were partially resolved by centrifugation at high speed in a density gradient into two overlapping bands: fractions I and II.
• 3.3. Particles of fraction II were also isolated by centrifugation in 41.5% sucrose solution. In confirmation, the resuspended precipitate rebanded at a density near that characteristic of fraction II upon density gradient centrifugation.
• 4.4. The labeled particles were identified as pinocytic vesicles (fraction I) and lysosomes (fraction II) respectively by the following criteria:
  • 4.1.(a) Analyses performed at varying intervals after intravenous injection of ribonuclease showed that fraction I rapidly became labeled as the kidneys took up the ribonuclease; label in this fraction then diminished while fraction II continued to increase in radioactivity. Later, fraction II also lost activity, accounting for the concomitant decrease in kidney labeling. Taken together with the rapid removal of label from the blood, these facts indicate that fraction II acquired exogenous protein from fraction I, just as lysosomes have been observed to take up material from pinocytic vacuoles.
  • 4.2.(b) Suspensions predominantly of fraction II released phosphotungstic acid-soluble label into the supernatant, upon incubation at 37 °C and pH 5.5, indicating degradation of the exogenous ribonuclease by these particles. Such activity is characteristic of lysosomes. When fraction I predominated, no degradation occurred upon incubation.
• 5.5. Damaged particles were distinguished from intact ones by their release of protein bound radioactivity upon centrifugation in 0.9% saline-0.25 M sucrose. Centrifugation in isotonic sucrose alone did not necessarily lead to release of label. Intact particles were unaffected by centrifugation in saline sucrose. The method was used to detect osmotic damage caused by incubation in hypotonic medium, and damage from centrifugation and resuspension.
• 6.6. Labeled particles of either type were most stable at slightly acid pH.