An improved procedure for the isolation of lamellar bodies from human lung. Lamellar bodies free of lysosomes contain a spectrum of lysosomal-type hydrolases

Abstract

We have recently shown that lamellar body fractions purified from human lung contain a distinct acid α-glucosidase distinguishable from lysosomal acid α-glucosidase in that it does not cross-react with antibodies raised against the lysosomal enzyme and does not bind to concanavalin A. In order to study the relationship between the non-concanavalin A-binding α-glueosidase and lamellar bodies more closely a method was developed for the further purification of the organelles. A purified lamelar body preparation isolated from human lung homogenate by discontinuous sucrose density centrifugation was subjected to gel filtration with Sepharose 4B followed by Percoli density gradient centrifugation, which yielded a lamellar body preparation with a phospholipid phosphorus/protein ratio of 12.57 ± 0.38 (µmol/mg) (n = 3) as compared to a ratio of 3.34 ± 0.16 (µmol/mg) (n = 3) in the sucrose density gradient preparation. Concomitantly there was a 3.3 ± 0.1 (n = 3) -fold enrichment in the content of total acid α-glutosidase and a 3.2 ± 0.1 (n = 3) -fold enrichment of non-concanavalin A-binding acid α-glucosidase. The new purification method removes adhering proteins without changing the phospholipid composition. During the successive purification steps the concanavalin A-sensitive and -insensitive α-glucosidases remained fully lamellar body fraction associated. Differences between a lysosome-enriched fraction and a lamellar body preparation at varying stages of purification with respect to the ratio between soluble acid hydrdases and the membrane-associate lysosomal enzyme glucocerebrosidase indicate that the purified lamellar bodies were not contaminated with lysosomes. The absence of lysosomes in the purified lamellar body fraction was confirmed by experiments with the weak base glycyl-L-phenylalanine-β-naphthylamide, which is an artificial substrate for the lysosomal enzyme cathepsin C and brings about lysis of lysosomes. Morphological examination by electron microscopy endorses the absence of contaminating vesicles and organelles and showed a structural integrity of the lamellar bodies in the final preparation. The improved isolation procedure strongly suggests that the concanavalin A-insensitive acid α-glucosidase is endogenous to lamellar bodies and supports our earlier idea that it can be used as a lamellar body-specific marker enzyme. In addition, the experiments show that lamellar bodies free of lysosomes contain a spectrum of lysosomal-type enzymes.