Hydrolysis of a fluorescent phospholipid substrate by phospholipase A2 and lipoprotein lipase

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Abstract

The fluorescent phospholipid 1-acyl-2-[6-[(7-nitro-2,1,3benzoxadiazol-4-yl)amino]-caproyl]phosphatidylcholine (C6-NBD-PC) was used as a substrate for porcine pancreatic phospholipase A2 (PA2) and bovine milk lipoprotein lipase (LpL). Hydrolysis of C6-NBD-PC by either enzyme resulted in a greater than 50-fold fluorescence enhancement with no shift in the emission maximum at 540 nm; Ca++ was required for PA2 catalysis. Identification of the products of hydrolysis showed cleavage at the sn-1 and sn-2 positions for LpL and PA2, respectively. For PA2, but not for LpL, there was a marked enhancement of enzyme catalysis at lipid concentrations above the critical micellar concentration of the lipid. Furthermore, apolipoprotein C-II, the activator protein of LpL for long-chain fatty acyl substrates, did not enhance the rate of catalysis of the water-soluble fluorescent phospholipid for either enzyme.

References (9)

  • D. Quinn et al.

    Prog. Lipid Res

    (1983)
  • M. Hamosh et al.

    Mol. Aspects Med

    (1983)
  • M. Shinomiya et al.

    Biochem. Biophys. Res. Commun

    (1983)
  • G. Bengtsson et al.

    FEBS Lett

    (1982)
There are more references available in the full text version of this article.

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Present address: The Second Department of Internal Medicine, Chiba University School of Medicine, Chiba, Japan

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