Assessment of arginine 97 and lysine 72 as determinants of substrate specificity in cytochrome P450 2C9 (CYP2C9)

CYP2C9 is distinguished by a preference for substrates bearing a negative charge at physiological pH. Previous studies have suggested that CYP2C9 residues R97 and K72 may play roles in determining preference for anionic substrates by interaction at the active site or in the access channel. The aim of the present study was to assess the role of these two residues in determining substrate selectivity. R97 and K72 were substituted with negative, uncharged polar and hydrophobic residues using a degenerate polymerase chain reaction-directed strategy. Wild-type and mutant enzymes were expressed in bicistronic format with human cytochrome P450 reductase in Escherichia coli. Mutation of R97 led to a loss of holoenzyme expression for R97A, R97V, R97L, R97T, and R97E mutants. Low levels of hemoprotein were detected for R97Q, R97K, R97I, and R97P mutants. Significant apoenzyme was observed, suggesting that heme insertion or protein stability was compromised in R97 mutants. These observations are consistent with a structural role for R97 in addition to any role in substrate binding. By contrast, all K72 mutants examined (K72E, K72Q, K72V, and K72L) could be expressed as hemoprotein at levels comparable to wild-type. Type I binding spectra were obtained with wildtype and K72 mutants using diclofenac and ibuprofen. Mutation of K72 had little or no effect on the interaction with these substrates, arguing against a critical role in determining substrate specificity. Thus, neither residue appears to play a role in determining substrate specificity, but a structural role for R97 can be proposed consistent with recently published crystallographic data for CYP2C9 and CYP2C5.

E-cadherin transport from the trans-Golgi network in tubulovesicular carriers is selectively regulated by Golgin-97

E-cadherin is a cell-cell adhesion protein that is trafficked and delivered to the basolateral cell surface. Membrane-bound carriers for the post-Golgi exocytosis of E-cadherin have not been characterized. Green fluorescent protein (GFP)-tagged E-cadherin (Ecad-GFP) is transported from the trans-Golgi network (TGN) to the recycling endosome on its way to the cell surface in tubulovesicular carriers that resemble TGN tubules labeled by members of the golgin family of tethering proteins. Here, we examine the association of golgins with tubular carriers containing E-cadherin as cargo. Fluorescent GRIP domains from golgin proteins replicate the membrane binding of the full-length proteins and were coexpressed with Ecad-GFP. The GRIP domains of p230/golgin-245 and golgin-97 had overlapping but nonidentical distributions on the TGN; both domains were on TGN-derived tubules but only the golgin-97 GRIP domain coincided with Ecad-GFP tubules in live cells. When the Arl1-binding endogenous golgins, p230/golgin-245 and golgin-97 were displaced from Golgi membranes by overexpression of the p230 GRIP domain, trafficking of Ecad-GFP was inhibited. siRNA knockdown of golgin-97 also inhibited trafficking of Ecad-GFP. Thus, the GRIP domains of p230/golgin-245 and golgin-97 bind discriminately to distinct membrane subdomains of the TGN. Golgin-97 is identified as a selective and essential component of the tubulovesicular carriers transporting E-cadherin out of the TGN.

A total electron content space weather study of the Nighttime Weddell Sea Anomaly of 1996/97 southern summer with TOPEX/Poseidon radar altimetry

This paper reports on a total electron content space weather study of the nighttime Weddell Sea Anomaly, overlooked by previously published TOPEX/Poseidon climate studies, and of the nighttime ionosphere during the 1996/1997 southern summer. To ascertain the morphology of spatial TEC distribution over the oceans in terms of hourly, geomagnetic, longitudinal and summer-winter variations, the TOPEX TEC, magnetic, and published neutral wind velocity data are utilized. To understand the underlying physical processes, the TEC results are combined with inclination and declination data plus global magnetic field-line maps. To investigate spatial and temporal TEC variations, geographic/magnetic latitudes and local times are computed. As results show, the nighttime Weddell Sea Anomaly is a large (∼1,600(°)2; ∼22 million km2 estimated for a steady ionosphere) space weather feature. Extending between 200°E and 300°E (geographic), it is an ionization enhancement peaking at 50°S–60°S/250°E–270°E and continuing beyond 66°S. It develops where the spacing between the magnetic field lines is wide/medium, easterly declination is large-medium (20°–50°), and inclination is optimum (∼55°S). Its development and hourly variations are closely correlated with wind speed variations. There is a noticeable (∼43%) reduction in its average area during the high magnetic activity period investigated. Southern summer nighttime TECs follow closely the variations of declination and field-line configuration and therefore introduce a longitudinal division of four (Indian, western/eastern Pacific, Atlantic). Northern winter nighttime TECs measured over a limited area are rather uniform longitudinally because of the small declination variation. TOPEX maps depict the expected strong asymmetry in TEC distribution about the magnetic dip equator.