DEGRADATION OF PYRENE-LABELED PHOSPHOLIPIDS BY LYSOSOMAL PHOSPHOLIPASES IN-VITRO - DEPENDENCE OF DEGRADATION ON THE LENGTH AND POSITION OF THE LABELED AND UNLABELED ACYL CHAINS

The hydrolysis of pyrenylacyl phosphatidylcholines (Pyr(n)PCs) (n indi cates the number of aliphatic carbons in the pyrene-chain) by crude ly sosomal phospholipases in vitro was investigated. Pyr(n)PCs consist of several sets in which the length of the pyrene-labelled or the unlabe lled acyl chain, linked to the sn-1 or sn-2 position, was systematical ly varied. Lysophosphatidylcholine and fatty acid were the only fluore scent breakdown products detected, thus indicating that Pyr(n)PCs were degraded by A-type phospholipases and lysophospholipases. Of these, m ainly A(1)-type phospholipases appear to be involved, as determined fr om the relative amounts of labelled fatty acid and lysolipid released from the positional isomers. Based on the effects of the length and po sition of the pyrene-labelled and unlabelled chains it is suggested th at (1) the lysosomal A-type phospholipases acting on Pyr(n)PCs recogni ze the carboxy-terminal part of the lipid acyl chains and (2) the rele vant part of the binding site is relatively narrow. Thus phospholipids with added bulk in the corresponding region, such as those that are p eroxidized and polymerized, may not be good substrates for the lysosom al phospholipases mentioned. The impaired hydrolysis of the most hydro phobic Pyr(n)PCs indicates that lysosomal phospholipases may not be ab le to penetrate significantly into the substrate interphase, but upwar d movement of the lipid may be required for efficient hydrolysis. Fina lly, the rate of hydrolysis of many pyrenyl derivatives was found to b e comparable to that of a natural phosphatidylcholine species, both in micelles and in lipoprotein particles, indicating that these derivati ves can be used as faithful reporters of lysosomal degradation of natu ral lipids in vivo and in vitro.