Rapid incorporation of functional rhodopsin into nanoscale apolipoprotein bound bilayer (NABB) particles.

Human apolipoprotein A-I (apo A-I) and its engineered constructs form discoidal lipid bilayers upon interaction with lipids in vitro. We now report the cloning, expression, and purification of apo A-I derived from zebrafish (Danio rerio), which combines with phospholipids to form similar discoidal bilayers and may prove to be superior to human apo A-I constructs for rapid reconstitution of seven-transmembrane helix receptors into nanoscale apolipoprotein bound bilayers (NABBs). We characterized NABBs by gel-filtration chromatography, native polyacrylamide gradient gel electrophoresis, UV-visible photobleaching difference spectroscopy, and fluorescence spectroscopy. We used electron microscopy to determine the stoichiometry and orientation of rhodopsin (rho)-containing NABBs prepared under various conditions and correlated stability and signaling efficiency of rho in NABBs with either one or two receptors. We discovered that the specific activity of G protein coupling for single rhos sequestered in individual NABBs was nearly identical with that of two rhos per NABB under conditions where stoichiometry and orientation could be inferred by electron microscopy imaging. Thermal stability of rho in NABBs was superior to that of rho in various commonly used detergents. We conclude that the NABB system using engineered zebrafish apo A-I is a native-like membrane mimetic system for G-protein-coupled receptors and discuss strategies for rapid incorporation of expressed membrane proteins into NABBs.

Ventricular hypertrophy plus neurohumoral activation is necessary to alter the cardiac beta-adrenoceptor system in experimental heart failure.

Treatment of rats with monocrotaline (MCT) leads to pulmonary hypertension, right ventricular (RV) hypertrophy, and finally to RV heart failure. This is associated with characteristic changes in right ventricular beta-adrenoceptors (beta-AR), neuronal noradrenaline transporter (NAT) density and activity (uptake1), and G protein-coupled receptor kinase (GRK) activity. This study aimed to find out factors that determine beta-AR, uptake1, and GRK changes. Thus, 6-week-old rats were treated with 50 mg/kg MCT subcutaneous or 0.9% saline. Within 13 to 19 days after MCT application (group A), RV weight (222+/-6 versus 147+/-5 mg) and RV/left ventricular (LV) weight ratio (0.42+/-0.01 versus 0.29+/-0.01) were significantly increased, whereas plasma noradrenaline, RV beta-AR density, RV NAT density and activity, and RV GRK activity were not significantly altered. Twenty-one to twenty-eight days after MCT (group B), however, not only RV weight (316+/-4 versus 148+/-2 mg) and RV/LV weight ratio (0.61+/-0.01 versus 0.3+/-0.01) were markedly increased but also plasma noradrenaline (645+/-63 versus 278+/-18 pg/mL); now, RV beta-AR density (13.4+/-1.3 versus 26.5+/-1.1 fmol/mg protein), RV NAT density (50.9+/-11.3 versus 79.6+/-2.9 fmol/mg protein), and RV NAT activity (65.4+/-7.4 versus 111.8+/-15.9 pmol [3H]-NA/mg tissue slices/15 min) were significantly decreased and RV-membrane GRK activity (100+/-15 versus 67+/-6 [32P]-rhodopsin in cpm) significantly increased. LV parameters of MCT-treated rats were only marginally different from control LV. We conclude that in MCT-treated rats ventricular hypertrophy per se is not sufficient to cause characteristic alterations in the myocardial beta-AR system often seen in heart failure; only if ventricular hypertrophy is associated with neurohumoral activation beta-ARs are downregulated and GRK activity is increased.

Rod outer segment structure influences the apparent kinetic parameters of cyclic GMP phosphodiesterase.

Cyclic GMP hydrolysis by the phosphodiesterase (PDE) of retinal rod outer segments (ROS) is a key amplification step in phototransduction. Definitive estimates of the turnover number, kcat, and of the Km are crucial to quantifying the amplification contributed by the PDE. Published estimates for these kinetic parameters vary widely; moreover, light-dependent changes in the Km of PDE have been reported. The experiments and analyses reported here account for most observed variations in apparent Km, and they lead to definitive estimates of the intrinsic kinetic parameters in amphibian rods. We first obtained a new and highly accurate estimate of the ratio of holo-PDE to rhodopsin in the amphibian ROS, 1:270. We then estimated the apparent kinetic parameters of light-activated PDE of suspensions of disrupted frog ROS whose structural integrity was systematically varied. In the most severely disrupted ROS preparation, we found Km = 95 microM and kcat = 4,400 cGMP.s-1. In suspensions of disc-stack fragments of greater integrity, the apparent Km increased to approximately 600 microM, though kcat remained unchanged. In contrast, the Km for cAMP was not shifted in the disc stack preparations. A theoretical analysis shows that the elevated apparent Km of suspensions of disc stacks can be explained as a consequence of diffusion with hydrolysis in the disc stack, which causes active PDEs nearer the center of the stack to be exposed to a lower concentration of cyclic GMP than PDEs at the disc stack rim. The analysis predicts our observation that the apparent Km for cGMP is elevated with no accompanying decrease in kcat. The analysis also predicts the lack of a Km shift for cAMP and the previously reported light dependence of the apparent Km for cGMP. We conclude that the intrinsic kinetic parameters of the PDE do not vary with light or structural integrity, and are those of the most severely disrupted disc stacks.