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A complex profile of metabolites of [14C]arachidonic acid are formed in NADPH-dependent reactions in liver microsomes from phenobarbital-treated rats. The products were resolved by HPLC and analyzed initially with an on-line diode array detector set to monitor 205, 220, 235, and 270 nm. In addition to the epoxyeicosatrienoic acids and their diol hydrolysis products, prominent hydroxyeicosatetraenoic acids (HETE) metabolites were identified as omega-hydroxy derivatives and three bis-allylic products, 7-HETE, 10-HETE, and 13-HETE. The formation of 13-HETE was reported by Oliw et al. (Arch. Biochem. Biophys. 300, 434-439 (1993)). Structures of the new products were established by GC-MS, and for 10-HETE, by comparison to authentic synthetic material. Chiral column analysis indicated that 7-HETE and 10-HETE were essentially racemic. The 13-HETE resolved into a 40:60 ratio of 13S:13R. The bis-allylic HETEs readily undergo rearrangement to conjugated diene-containing HETEs in mild acidic conditions (1% acetic acid). Using [18O]13-HETE we observed 69% retention of the labeled oxygen in the 11- and 15-HETE rearrangement products. Furthermore, steric analysis of products from acid-treated 10-HETE and 13-HETE enantiomers revealed partial retention of chirality (80:20 ratio of enantiomers for methyl esters, and 60:40 ratio for free acids) in the course of mild acid-catalyzed rearrangement. This represents a novel reaction of this class of arachidonic acid metabolites of cytochromes P450.

Dansyl-phosphatidylserine (D-PS) was used as a fluorescence probe to study interactions between protein kinase C (PKC) with phospholipid vesicles. It was found that D-PS fluorescence (520 nm) was enhanced by PKC (excited at 285 nm). The fluorescence energy transfer, indicative of a close association of PKC with D-PS vesicles, was differentially modulated by various phospholipids, depending upon their effects on PKC activation state and the manners in which they were present. PKC inhibitors (e.g. polymyxin B and ether lipids) potently inhibited the PKC-enhanced D-PS fluorescence. It is suggested that certain spatial arrangements between PKC and its phospholipid cofactor are essential for the enzyme activation and that D-PS would be a useful probe to study fluorimetrically such interactions.