Apolipoprotein J (apoJ)-containing high-density lipoproteins (HDL), isolated from human plasma by immunoaffinity chromatography, are associated with apoAI and a protein of approximately 44 kDa. In order to advance our understanding of apoJ's role in the vasculature, a comprehensive investigation was performed to identify and characterize this 44-kDa protein and to study its interaction with apoJ. The 44-kDa protein, a monomeric glycoyslated polypeptide, was identified by N-terminal sequencing as serum paraoxonase. Paraoxonase exists in two oxidation states: one contains all free cysteines while the other has one disulfide bond between Cys42 and Cys284. Northern analysis of eight human tissues shows paraoxonase message present only in the liver. The majority of apoJ/paraoxonase-HDL are 90-140 kDa; however, not all of the plasma paraoxonase is associated with apoJ. The specificity of the apoJ/paraoxonase interaction, inferred by the constant mole ratio of the two proteins in affinity-purified apoJ-HDL, is confirmed in direct binding assays. For purified proteins, there is more than a 5-fold increase in the apparent affinity of apoJ for immobilized paraoxonase as the paraoxonase coating concentration is increased from 0.5 to 2.0 micrograms/mL. Both oxidation states of paraoxonase bind to apoJ with equal affinity. Our data combined with other evidence suggest that the plasma link of apoJ with paraoxonase will be implicated as a predictor of vascular damage.

Cobalamin-dependent methionine synthase catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine, forming tetrahydrofolate and methionine. The Escherichia coli enzyme, like its mammalian homologue, is occasionally inactivated by oxidation of the cofactor to cob(II)alamin. To return to the catalytic cycle, the cob(II)alamin forms of both the bacterial and mammalian enzymes must be reductively remethylated. Reduced flavodoxin donates an electron for this reaction in E. coli, and S-adenosylmethionine serves as the methyl donor. In humans, the electron is thought to be provided by methionine synthase reductase, a protein containing a domain with a significant degree of homology to flavodoxin. Because of this homology, studies of the interactions between E. coli flavodoxin and methionine synthase provide a model for the mammalian system. To characterize the binding interface between E. coli flavodoxin and methionine synthase, we have employed site-directed mutagenesis and chemical cross-linking using carbodiimide and N-hydroxysuccinimide. Glutamate 61 of flavodoxin is identified as a cross-linked residue, and lysine 959 of the C-terminal activation domain of methionine synthase is assigned as its partner. The mutation of lysine 959 to threonine results in a diminished level of cross-linking, but has only a small effect on the affinity of methionine synthase for flavodoxin. Identification of these cross-linked residues provides evidence in support of a docking model that will be useful in predicting the effects of mutations observed in mammalian homologues of E. coli flavodoxin and methionine synthase.

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