The purpose of the present study was to investigate the following:
(1) Cellular and subcellular distribution of apolipoprotein after injection of ¹²⁵I-labeled canine Apo A-I into dogs.
(2) In vitro proteolysis of canine high density lipoprotein subfraction, HDL₃, by acid proteases in canine liver lysosomes.
(3) Binding, uptake and proteolytic degradation of rat high density lipoprotein subfraction, HDL3, by isolated rat liver parenchymal cells.
(1) Canine Apo A-I was purified by column chromatography of the delipidated canine high density lipoprotein subfraction, HDL₃, using Sephadex G-100. The purified Apo A-I showed only a single band on SDS polyacrylamide gel electrophoresis. The calculated molecular weight of canine Apo A-I was 28,000. The C- and N- terminal amino acid was alanine and aspartic acid, respectively. The purified canine Apo A-I was iodinated by the iodine monochloride method and injected intravenously into healthy male mongrel dogs. At one day after the injection of ¹²⁵I-Apo A-I, the liver took up 4.8 times more radioactive counts than the kidney and twenty times as many counts as the spleen. The highest radioactivity recovered in the subcellular fractions of the liver was found in the lysosomal fraction.
(2) Soluble lysosomal fraction had 23-fold greater proteolytic activity of HDL₃ than homogenate, while the mitochondrial and microsomal fractions showed almost no activity. Proteolysis of HDL₃ and bovine serum albumin by acid lysosomal proteases conformed to a hyperbolic courve. The straight line was obtained on Lineweaver-Burk plots. Apparent Km for HDL₃ was 46 micromolar and 374 micromolar for bovine serum albumin proteolysis. Iodoacetate inhibited HDL₃ proteolysis 100 percent at concentrations greater than 50 micromolar, while bovine serum albumin proteolysis was inhibited 68 percent by iodoacetate at a concentration of 100 micromolar. Maximum inhibition of HDL₃ and bovine serum albumin proteolysis by pepstatin was 45 percent and 70 percent, respectively. These observations suggest that the endopeptidase, cathepsin B, plays a more significant role in HDL₃ proteolysis.
(3) In the standard assay of ¹²⁵I-labeled HDL₃ binding, uptake and proteolytic degradation by rat liver parenchymal cells, approximately 10 million cells were incubated at 37°C? in 2.0ml of Krebs-Henseleit bicarbonate buffer, pH 7.4. The maximum rat HDL₃-binding capacity (B_max) and the dissociation constant (Kd) of the isolated rat liver cell was 31 nanograms/mg dry weight of cells and 60×80^-8M, based on M. W. 28,000 of Apo A-I, respectively. Radioactivity in cells reached a maximum at 30 minutes of incubation and this plateau persisted for at least 2 hours. There was a lag phase for 15 minutes and after that a constant rate of proteolytic degradation was observed up to 2 hours at 37°C. Proteolytic degradation of ¹²⁵I-labeled HDL₃ was inhibited by chloroquine. Inhibition of HDL₃ proteolytic degradation by chloroquine may be explained by an effect on cathepsin B in liver lysosomes.
These data suggegt that binding, uptake and proteolytic degradation of rat HDL₃ by isolated rat liver parenchymal cells are actively performed and linked in the sequence: binding, then uptake and finally proteolytic degradation. There may be a specific HDL₃ or lipoprotein A recognition sites on the plasma membrane. The important role of liver lysosomes in proteolytic degradation of HDL₃ was indicated.