LYSOSOME LIPID STORAGE DISORDER IN NCTR-BALB/C MICE .3. ISOLATION AND ANALYSIS OF STORAGE INCLUSIONS FROM LIVER

  • 1 January 1982
    • journal article
    • research article
    • Vol. 108  (2) , 160-170
Abstract
Livers of NCTR-BALB/c mice, affected by excessive accumulation of cholesterol and phospholipid, were fractionated by sucrose density gradient centrifugation. Lysosomes of very low density (.rho. = 1.05 - 1.08) were found, which by EM appeared identical to the storage inclusions seen in fixed tissues. These lysosomes could be purified .apprx. 10-fold over the original homogenate and represented 4% of the total protein and 30-40% of the liver acid hydrolase content. The preparations were nearly free of mitochondrial, endoplasmic reticulum and plasma membrane contamination. The lysosomes were laden with cholesterol and phospholipid. Cholesterol (> 97% unesterified) accounted for half of the total lipid, and sphingomyelin accounted for another 20%. Phosphatidylcholine and phosphatidylethanolamine were also present in substantial quantities. All of the excess cholesterol and sphingomyelin of liver could be attributed to the low density lysosomes. Lysosomal acid sphingomyelinase activity, measured with a synthetic substrate, was 10-60% of BALB/c mouse control levels in liver, spleen and cerebellum, while 2 other lysosomal enzymes, N-acetyl-.beta.-glucosaminidase and .beta.-glucuronidase, were increased 2- to 8-fold in the same tissues. These data and the morphologic observations of the preceding paper establish that the disorder affecting NCTR-BALB/c mice is a lysosome storage disease. Several possible mechanisms to explain the cholesterol and phospholipid overloading of lysosomes are proposed. The specific gene defect remains to be established.