Summary
We have used highly purified lysosomes to investigate three models of hydrolytic injury by lysosomal phospholipases. Lysosomes, enriched up to 70-fold in marker enzyme activities, can be isolated from homogenized hepatic tissue by differential centrifugation and subsequent free flow electrophoresis. These organelles remain latent and can also be utilized to obtain ‘lysosol’, the soluble fraction of the lysosomes tissue containing acid active phospholipases. The first model investigated the effect of lysosol on non-lysosomal membranes. When this soluble fraction was incubated with plasmalemma (sarcolemma) from cardiac cells, selective hydrolysis of the phospholipids was observed: phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin were the preferred substrates, and only lysophosphatidylcholine and lysophosphatidylethanolamine accumulated in significant amounts. Hydrolysis of sphingomyelin was enhanced significantly by Triton-X-100. In the second model, when intact lysosomes were incubated at acid pH, hydrolysis of phospholipids by the endogenous lipases was observed. Once again this lipolysis was specific for phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin: significant amounts of lysophospholipids also accumulated in this model. Concurrent with these lipid changes, an increase in lysosomal permeability also occurred and pH 5.0 was optimal for this lipolytic activity. However, no phospholipase activity was detected when lysosomes were incubated at pH ranges found in acidotic tissue (pH 6.0 or higher). In the third model, lysosomes were incubated at pH 6.0 in the presence of exogenously generated free radicals (dihydroxyfumarate-FeADP). A rapid loss of membrane phospholipids was observed, and most of this loss could be contributed to peroxidation of membrane phospholipids; the production of malondialdehyde preceded loss of N-acetylglucosaminidase from the lysosome. However, significant accumulation of lysophospholipids, from 2% at control time to 6.6 and 8.7% at 10 and 20 minutes, suggested that lysosomal phospholipase were hydrolyzing lysosomal phospholipids. Thus, we hypothesize that this ‘free radical-induced lipolysis’ is a result of peroxidized phospholipids serving as preferred substrate for phospholipases at pH 6.0.
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Dickens, B.F., Mak, T. & Weglicki, W.B. Lysosomal lipolytic enzymes, lipid peroxidation, and injury. Mol Cell Biochem 82, 119–123 (1988). https://doi.org/10.1007/BF00242526
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DOI: https://doi.org/10.1007/BF00242526