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ATP-binding cassette protein G1 (ABCG1) plays an important role in eliminating excess cholesterol from macrophages and in the formation of high-density lipoprotein (HDL), which contributes to the prevention and regression of atherosclerosis. The post-translational regulation of ABCG1 remains elusive, although phosphorylation by protein kinase A destabilizes ABCG1 proteins. We examined the phosphorylation of ABCG1 using HEK293 and Raw264.7 cells. ABCG1 phosphorylation was enhanced by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator. PKC activation by TPA increased ABCG1 protein levels and promoted ABCG1-dependent cholesterol efflux to HDL. This activity was suppressed by Go6976, a PKCα/βI inhibitor, suggesting that PKC activation stabilizes ABCG1. To confirm this, the degradation rate of ABCG1 was analysed; ABCG1 degradation was suppressed upon PKC activation, suggesting that PKC phosphorylation regulates ABCG1 levels. To confirm this involvement, we co-expressed ABCG1 and a constitutively active form of PKCα in HEK cells. ABCG1 was increased upon co-expression. These results suggest that PKC-mediated phosphorylation, probably PKCα, stabilizes ABCG1, consequently increasing ABCG1-mediated cholesterol efflux, by suppressing ABCG1 degradation. PKC activation could thus be a therapeutic target to suppress the development of atherosclerosis.

The chloroplastic isoform of monodehydroascorbate (MDA) radical reductase was purified from spinach chloroplasts and leaves. The cDNA of chloroplastic MDA reductase was cloned, and its deduced amino acid sequence, consisting of 497 residues, showed high homology with those of putative organellar MDA reductases deduced from cDNAs of several plants. The amino acid sequence of the amino terminal of the purified enzyme suggested that the chloroplastic enzyme has a transit peptide consisting of 53 residues. A southern blot analysis suggested the occurrence of a gene encoding another isoform homologous to the chloroplastic isoform in spinach. The recombinant enzyme was highly expressed in Eschericia coli using the cDNA, and purified to a homogeneous state with high specific activity. The enzyme properties of the chloroplastic isoform are presented in comparison with those of the cytosolic form.

Activity staining with NADPH-nitroblue tetrazolium after native-PAGE of membrane proteins of Synechocystis PCC6803, solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS), revealed four NAD(P)H dehydrogenase (NDH) activities; an NDH complex of the respiratory chain, a ferredoxin NADP + reductase (FNR), a drgA product which oxidized both NADH and NADPH, and an uncharacterized NADH-specific enzyme. The NDH complex was purified with anion exchange and gel filtration chromatographies. The purified complex had a molecular mass of 376 kDa and was composed of 9 subunits. Western analysis showed that the complex contained the NDH-H subunit, but not NDH-A or B. The enzyme reduced ferricyanide much faster than plastoquinone and used NADPH as its prefered electron donor rather than NADH. The enzymatic activity was inhibited by diphenyleneiodonium chloride and salicylhydroxamic acid, but not by rotenone, p-chloromercuribenzoate, TV-ethylmaleimide, flavon, dicumarol, or antimycin A. These results suggest that the purified complex is a hydrophilic subcomplex which contains an NADPH binding site and flavin, and is dissociated from a hydrophobic subcomplex, which contains quinone binding site.