The goal of the present study is to explore the functional involvement of the N-terminal region in the biological activity of phospholipase A2 (PLA2) enzyme. Native PLA2 from the venoms of Naja naja atra and Bungarus multicinctus and N-terminally mutated N. naja atra PLA2, i.e. an additional Met before Asn-1(M-PLA2), substitution of Asn-1 with Met-1(PLA2(N1M)) and removal of N-terminal seven residues (PLA2(¡µN7)), were employed in this study. Mutations on the N-terminal region insignificantly perturbed the binding ability of PLA2 for Ca2+ and ANS, but the enzymatic activity of mutants drastically decreased. Moreover, an alteration in the secondary structure was observed as revealed by CD spectra. Compared to other mutants, the fine structure of Ca2+-binding site within PLA2(¡µN7)) changed. Additionally, removal of the N-terminal region caused significant alternation in the structures of active site and substrate-binding site as evidenced by the results of fluorescence measurement, chemical modification and denaturation with detergents. In all N-terminal mutants, substituting Ans-1 with Met-1 affected the NNA-PLA2 structure to a least extent. The membrane-damage activity of PLA2(N1M) and M-PLA2 was 89% and 34% that of NNA-PLA2, respectively. PLA2(¡µN7) did not exhibit the membrane-damage activity. Studies on the biological activities of chemically modified N. naja atra PLA2 reflected a dissociation of the enzymatic activity from membrane-damage activity, and suggested the involvement of Trp-18, Trp-61, Lys-65, Tyr-3 and Tyr-63 in membrane-damage activity. Collectively, our data indicate that the intact N-terminus was crucial for maintaining of the functional conformation of PLA2 in the manifestation of the enzymatic activity and membrane-damage activity, and the enzymatic activity of PLA2 is in aid of but not exclusively essential for the membrane-damage effect.

The goal of the present study is to elucidate the modulatory effect of lipid compositions on phospholipase A2 (PLA2) activity. Sphingomyelin (SM) incorporation inhibited catalytic activity and membrane-damaging activity of native and mutated PLA2 toward egg yolk phosphatidylcholine (EYPC) vesicles. The inhibitory effects were through the reduction of membrane fluidity and modulation of the mode of membrane binding of PLA2 at water/lipid interface. The modulated effect of SM depended on inherent structural elements of PLA2. Moreover, cholesterol (Chol) incorporation into EYPC/egg yolk sphingomyelin (EYSM) vesicles relieved the inhibitory effect of sphingomyelin on PLA2 activity via lipid domain formation by SM and Chol. The effects on the interactive mode of PLA2 with phospholipids induced by the physical state changes of membrane bilayers abolished the inhibition of SM on catalytic activity and membrane-damaging activity of PLA2. Additionally, quercetin incorporation increased PLA2 activity and membrane-damaging activity toward EYPC/SM vesicles via its raft-making effect. Quercetin incorporation reduced PLA2 activity and membrane-damaging activity toward EYPC/SM/Chol vesicles via its raft-breaking effect. Membrane-inserted quercetin affected on membrane structure and membrane-bound mode of PLA2 to modulate PLA2 interfacial activity and membrane-damaging activity. Finally, studies on the effects of phosphatidylserine (PS) content on the sensitivity of lipid vesicles mimicking inner and outer plasma membrane toward PLA2 activity revealed that the membrane-binding mode adopted by PLA2 depended on the lipid composition. The effects of PS content on the extent of lipid domain formation and the conformation of PLA2 adopted at water-lipid interface modulate PLA2 catalytic activity. Collectively, these results indicate that lipid composition modulates PLA2 activity via its effects on membrane structure and membrane-bound mode of PLA2

Androgen receptor (AR) gene CAG polymorphisms may be associated with body shape, and are associated with certain breast and prostate cancers. In addition, body shape is associated with risk for a variety of diseases, including heart disease, diabetes, and certain forms of cancer. The CAG repeat in exon l of the AR gene was quantified using Perkin Elmer Applied Biosystems GeneScan analysis software in 96 and 59 healthy Caucasian men and women, respectively, who were over the age of 50 years. All participants had body measurements taken and donated a blood sample. Waist measurements included circumferences at the 1) umbilicus (wstumb), 2) top of the iliac crest (wstili), and 3) midpoint between the lowest rib and the iliac crest (wstwst). Waist-hip ratio (Wl-IR) was calculated using each corresponding waist measurement, respectively (WHRUMB, WHRILI, WHRWST). Mean repeat length was significantly different (p < 0.01) between men (22 ± 0.3 repeats) and women (23 ± 0.3 repeats). There was a significant relationship (p < 0.05) between mean individual CAG repeat number and tertile of WHRUMB in women based on the mean number of CAG repeats for each woman. Waist measurements in women were significantly different for all pairwise comparisons (p < 0.05). In addition, the three measurements of WHR in women, WHRUMB, WHRILI, and WHRWST, were significantly different from each other (p < 0.05). Thus, lesser numbers of CAG repeats may indicate a more androgenic phenotype in women.

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