The effect of ferriprotoporphyrin IX and chloroquine on phospholipid monolayers and the possible implications to antimalarial activity

Ferriprotoporphyrin IX intercalates into phospholipid membranes, as evidenced from its effect on the surface pressure of monolayers composed of different phospholipids. Ferriprotoporphyrin intercalation is enhanced by membrane hydrophobicity and decreased by negative surface potential. Chloroquine enhances the effect of ferriprotoporphyrin in relatively hydrophobic membranes but reduces it in monolayers composed of highly unsaturated phospholipids. These results are consistent with the differential effect of chloroquine on ferriprotoporphyrin-induced lysis of erythrocytes and of malarial parasites, thus supporting the membrane-lesion hypothesis of antimalarial action.     

Interactions of metal ions with phosphatidylserine bilayer membranes: effect of hydrocarbon chain unsaturation

A combination of surface monolayer, scanning calorimetry, 31P NMR, and spin-label ESR techniques has been used to monitor the interactions of monovalent (NH4+, Na+, and Li+) and divalent (Ca2+) cations with phosphatidylserines (PS) differing in their levels of chain unsaturation. Comparisons are made between the disaturated dimyristoyl-, dipalmitoyl-, and dihexadecyl-PS (DMPS, DPPS, and DHPS), saturated cis-monounsaturated palmitoyloleoyl-PS (POPS) (and bovine brain PS), di-trans-monounsaturated dielaidoyl-PS (DEPS), and di-cis-monounsaturated dioleoyl-PS (DOPS). Na+ and NH4+ cations interact weakly with all PS monolayers and bilayers without significant changes in molecular conformation, chain packing, or headgroup dynamics and without dependence on chain composition. In contrast, considering these structural and dynamic parameters, Li+ shows a gradation in its interaction with PS (DMPS greater than POPS approximately bovine brain PS greater than DOPS), suggesting that Li+-PS interactions depend on the interfacial properties of the PS molecules (e.g., surface area). Finally, Ca2+ interacts strongly with all PS monolayers and bilayers, without obvious chain selectivity. Thus, ion binding to PS depends not only on the properties of the cation (Na+ vs Li+ vs Ca2+) but also on the molecular details of the PS membrane surface.     

Substitution of Ser61----Gly61 in human apolipoprotein C-II does not alter its activation of lipoprotein lipase

Lipoprotein lipase (LpL) activity is enhanced by apolipoprotein C-II (apoC-II), a 79 amino acid residue peptide. The minimal apoC-II sequence required for activation of LpL resides between residues 56-79. To determine the possible role of an acyl-apoC-II intermediate involving Ser61 in enzyme catalysis, a synthetic peptide of apoC-II containing residues 56-79 was synthesized and compared to the corresponding peptide with serine at position 61 being substituted with glycine. With two different LpL assay systems, both peptides enhanced enzyme activity. Since glycine does not contain a hydroxyl group, these results rule out the possibility that an acyl-apoC-II intermediate with Ser61 is required for enzyme activation.     

Characterization of a Bacillus subtilis SecA mutant protein deficient in translocation ATPase and release from the membrane

SecA is the precursor protein binding subunit of the bacterial precursor protein translocase, which consists of the SecY/E protein as integral membrane domain. SecA is an ATPase, and couples the hydrolysis of ATP to the release of bound precursor proteins to allow their proton-motive-force-driven translocation across the cytoplasmic membrane. A putative ATP-binding motif can be predicted from the amino acid sequence of SecA with homology to the consensus Walker A-type motif. The role of this domain is not known. A lysine residue at position 106 at the end of the glycine-rich loop in the A motif of the Bacillus subtilis SecA was replaced by an asparagine through site-directed mutagenesis (K106N SecA). A similar replacement was introduced at an adjacent lysine residue at position 101 (K101N SecA). Wild-type and mutant SecA proteins were expressed to a high level and purified to homogeneity. The catalytic efficacy (k_cat/k_m) of the K106N SecA for lipid-stimulated ATP hydrolysis was only 1% of that of the wild-type and K101N SecA. K106N SecA retained the ability to bind ATP, but its ATPase activity was not stimulated by precursor proteins. Mutant and wild-type SecA bind with similar affinity to Escherichia coli inner membrane vesicles and insert into a phospholipid mono-layer, in contrast to the wild type, membrane insertion of the K106N SecA was not prevented by ATP. K106N SecA blocks the ATP and proton-motive-force-dependent chase of a translocation intermediate to fully translocated proOmpA. It is concluded that the GKT motif in the amino-terminal domain of SecA is part of the catalytic ATP-binding site. This site may be involved in the ATP-driven protein recycling function of SecA which allows the release of SecA from its association with precursor proteins, and the phospholipid bilayer.     

Microsomal and cytosolic cytochrome P450 mediated benzo(a)pyrene hydroxylation in Pleurotus pulmonarius

Summary Microsomal and soluble fractions of Pleurotus pulmonarius exhibited a reduced carbon monoxide difference spectrum with P450 maxima at 448nm and 450–452nm respectively. Substrate induced Type I spectra were observed on addition of benzo(a)pyrene to both fractions. Benzo(a)pyrene hydroxylation was measured using the aryl hydrocarbon hydroxylase assay and was observed to be P450 dependent as indicated by carbon monoxide inhibition together with the substrate binding characteristics. The activity of the fractions were observed to give K_m of 200mM and 660mM and V_max of 1.25 nmol/min/nmol P450 and 0.57 nmol/min/nmol P450 for the microsomal and cytosolic fractions respectively.