Human lysosomal acid lipase/cholesteryl ester hydrolase. Purification and properties of the form secreted by fibroblasts in microcarrier culture.

G N Sando,L M Rosenbaum

doi:10.1016/s0021-9258(18)95720-0

Abstract

Lysosomal acid lipase was purified to near homogeneity in a yield of 25-30% from secretions of human fibroblasts grown on microcarriers in spinner culture. Ammonium chloride was added to the serum-free medium to stimulate production of extracellular enzyme and minimize modifications, including proteolytic processing and destruction of the mannose 6-phosphate recognition marker, that have been associated with packaging and maturation of acid hydrolases in lysosomes. Chromatography of secretions by decyl-agarose, hydroxylapatite, phenylboronate-agarose, and gel filtration resulted in greater than 1500-fold purification of the lipase, representing a 10,000-fold increase above the specific activity of intracellular enzyme. The apparent molecular weight of approximately 49,000, estimated for the lipase by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, was similar to that determined for the native enzyme by gel filtration (Mr approximately 47,000). By contrast, a smaller molecular weight (Mr approximately 41,000) was estimated for the intracellular enzyme. The purified enzyme was susceptible to hydrolysis by endo-beta-N-acetylglucosaminidase H, which resulted in at least two new forms, reduced in apparent molecular weight by approximately 4,000-6,000. Treatment with the endoglycosidase did not alter the catalytic activity or heat stability of the acid lipase. However, the treated enzyme was no longer internalized by fibroblasts via the mannose 6-phosphate receptor and thereby had lost the capacity to correct cholesteryl ester accumulation in cultured lipase-deficient cells. Acid fatty acyl hydrolase activity for cholesteryl oleate, triolein, and methylumbelliferyl oleate co-purified. All three esters were hydrolyzed optimally at pH 4.0, but the pH profile was altered by addition of salts or albumin to the phospholipid-bile salt substrate mixtures. In a series of saturated fatty acyl esters of 4-methylumbelliferone, a derivative with an intermediate chain length (9 carbons) was the best substrate and was hydrolyzed at a rate comparable to that of the oleate ester at pH 4. The optimal pH for hydrolysis of the intermediate and shorter chain length esters was higher by about 2 pH units than that for the longer chain esters (pH approximately 4). The activity of the purified lipase was stimulated by several different proteins. The relationship of this effect to the possible requirement for a natural activator substance has not been determined.(ABSTRACT TRUNCATED AT 400 WORDS)

Highlights

A comparison of the gel filtration profile of the highly purified secreted fibroblast lipase with that of the intracellular form indicated that the average molecular weight of the intracellular enzyme was lower byabout 6000 (Fig.6)
The purified lipase did not hydrolyze fatty acyl esters of phosphatidylcholine under conditions in which acid phospholipase A activity was detected in sonicates of normal and cholesteryl ester hydrolase-deficient cells, and in a preparation of the secreted M U 0 hydrolase that was less retarded by decyl-agarose
PH optima were used to distinguish acid lipase were identified with M, = 30,600 and 102,500 [9]. among lipases, an approach that may not be valid, since the Imanaka et al [50] recently published a value of 42,000 for lysosomalacid lipase/cholesteryl ester hydrolase is active over the molecular weight of the acid lipase isolated from rabbit a broad pH range that depends on substrate preparation and liver

Summary

Introduction

Purification of Secreted Human Fibroblast Acid Lipase activity when individual fractions along the activity peak from the next to last gel chromatography step (2nd AcA 44) were analyzed by SDS-polyacrylamide gel electrophoresis (Fig. 5, lanes E-H). A comparison of the gel filtration profile of the highly purified secreted fibroblast lipase with that of the intracellular form indicated that the average molecular weight of the intracellular enzyme was lower byabout 6000 (Fig.6).

Results

Conclusion