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  • Title: Synthesis of prodan-phosphatidylcholine, a new fluorescent probe, and its interactions with pancreatic and snake venom phospholipases A2.
    Author: Hendrickson HS, Dumdei EJ, Batchelder AG, Carlson GL.
    Journal: Biochemistry; 1987 Jun 16; 26(12):3697-703. PubMed ID: 3651404.
    Abstract:
    A new fluorescent probe, prodan-PC, was synthesized by incubating thio-PC, a thiol ester analogue of phosphatidylcholine [1,2-bis(decanoylthio)-1,2-dideoxy-sn-glycero-3-phosphocholine], with acrylodan, a fluorescent thiol-reactive reagent [6-acryloyl-2-(dimethylamino)naphthalene], in the presence of phospholipase A2, which served to generate lysothio-PC in situ. Prodan-PC (PPC) showed maximum absorption in ethanol at 370 nm. The fluorescence emission spectrum showed maximum emission at 530 nm in water and at 498 nm in ethanol. In the presence of a saturating amount of phospholipase A2, the emission maximum shifted to about 470 nm. PPC showed a critical micellar concentration around 5 microM, with evidence of premicellar aggregation above 1 microM. Binding of PPC to Crotalus adamanteus phospholipase A2 was evidenced by an increase in emission at 480 nm and an increase in fluorescence anisotropy. An apparent dissociation constant of 0.323 microM was calculated for this enzyme complex. Binding was dependent on the presence of calcium ion and was abolished by blocking the active site with p-bromophenacyl bromide. Binding was also followed by energy transfer from tryptophan in the enzyme to PPC. Apparent dissociation constants for PPC complexes with phospholipases A2 from Naja naja naja and porcine pancreas and the prophospholipase A2 from porcine pancreas were 0.509, 0.107, and 0.114 microM, respectively. PPC was shown to inhibit the activity of pancreatic phospholipase A2 in thio-PC-sodium cholate mixed micelles. Inhibition studies were complicated because PPC can also serve as an activator of the snake venom enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)
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