Abstract
Fractionation techniques, based on zonal centrifugation in density gradients, were developed for examining the heterogeneity of lysosomal populations and also for the purification of lysosomes from normal rat liver. Small lysosomes of rat liver were observed to be relatively rich in acid phosphatase whilst larger lysosomes were richer in other enzymes, notably acid ribonuclease. Examination of acid hydrolase distributions amongst partially disrupted lysosomes indicated that this 'apparent' heterogeneity was due mainly to the membrane association of acid phosphatase. These observations were confirmed by morphology and by cytochemical staining for acid phosphatase. The heterogeneity could not be attributed to different populations of lysosomes originating from different cell types of rat liver. A large scale method for isolating 60-fold purified lysosomes (from up to 200 g of liver) was developed. Lysosomes purified by this method were utilised for raising anti-lysosomal antibodies. Examination of lysosomal proteins with the aid of these antibodies (by immunoelectrophoresis) and by SDS polyacrylamide electrophoresis indicated significant interchange between lysosomes and serum and bile but relatively little between lysosomes and related structures, such as the plasma membrane. Fractionation of 'microsomes' from isolated hepatocytes and non-hepatocytes showed that the plasma membrane (PM) of non-hepatocytes has a much lower banding density than the PM of hepatocytes. These observations partly explain the heterogeneity of PM vesicles recovered in the microsomal fraction of whole liver. Lysosomes of the rat kidney cortex were subfractionated in a systematic study, in order to examine and characterise the complete range of lysosomal populations with respect to their size, enzymic composition and possibly function. A rate zonal sedimentation method was developed to provide a 'profile' of lysosomes present in the classical 'ML' fraction. Small lysosomes, which were similar in size to those of liver, were extremely heterogeneous in terms of enzyme distributions. The characteristic large lysosomes (protein droplets) of the kidney tubular cells were well resolved from the small lysosomes and contained a distinct complement of lysosomal enzymes. Subfractionation of kidney 'microsomes. ' revealed that this fraction contained a high proportion of small lysosomes which were also very heterogeneous. All populations of kidney lysosomes appeared to be involved in the uptake and degradation of a typical low molecular weight protein which is reabsorbed from the glomerular filtrate into the proximal tubule cells.