Biomedical subjects

M L Shank-Retzlaff

Publications and source records attributed to M L Shank-Retzlaff.

2 recordsLinked to original sources

PubMed2000

Analyte gradient-surface plasmon resonance: a one-step method for determining kinetic rates and macromolecular binding affinities.

A novel one-step method for determining kinetic rates and equilibrium binding affinities, termed analyte gradient-surface plasmon resonance (AG-SPR) is described. A gradient maker or HPLC pump system is used to produce a gradient so that, under continuous-flow conditions, the concentration of analyte passing over the sensor surface increases linearly with time. The rate at which analyte binds to the immobilized receptors is measured by monitoring the change in the surface plasmon resonance minimum as the analyte concentration increases. Kinetic rates are determined by fitting the data to a modified version of the previously described two-compartment model (Schuck, P.; Minton, A. P. Anal. Biochem. 1996, 240, 262-272). Numerical simulations indicate that AG-SPR results in accurate estimates of both kinetic rates and equilibrium affinities regardless of the intrinsic kinetics of the interaction and can be used for systems under mass transport limitations. Simulations also indicate that AG-SPR can be used to characterize interactions that do not obey pseudo-first-order kinetics due to the presence of a heterogeneous receptor population. Experimentally, the interaction of cytochrome c with cytochrome b5 immobilized on a negatively charged monolayer has been characterized by AG-SPR, and both the specific and the nonspecific interactions were quantitatively analyzed. This new technique is advantageous over traditional SPR methods because it eliminates the need for surface regeneration and is significantly faster than traditional titration experiments.

Cytochrome c Group↗

PubMed1998

Membrane topology of cytochrome P450 2B4 in Langmuir-Blodgett monolayers.

Using Langmuir-Blodgett monolayers of both phosphatidylethanolamines and phosphatidylcholines as membrane mimics, we have examined the topology of cytochrome P450 2B4 anchoring. The interaction of wild-type P450 2B4 with phosphatidylethanolamine monolayers can be characterized as a biphasic reaction, with the initial fast phase explained by the specific insertion of membrane-spanning segments of the protein into the monolayer. Injection of cytochrome b5 (b5) beneath dipalmitoyl-phosphatidylcholine monolayers also resulted in biphasic kinetics. Regardless of the nature of the lipid employed, neither a truncated cytochrome P450 2B4 (P450 2B4 Delta2-27) lacking the amino-terminal hydrophobic residues widely believed to be the major transmembrane segment nor a soluble b5 fragment (Deltab5) lacking its carboxy terminus anchor exhibit the fast-phase behavior characteristic of specific insertion. To further characterize the membrane topology of P450 2B4, its insertion area in DPPE monolayers was measured and analyzed with use of the Gibbs equation for adsorption at an interface. The mean molecular insertion area derived from isotherms of P450 2B4 in a DPPE monolayer at a pressure of 19 mN/m, 680 +/- 95 A2 is large enough to accommodate two to four transmembrane helices. The large insertion area and the fact that the truncated cytochrome retains as much as 30% of its membrane localization when expressed in Escherichia coli (Pernecky, S. J., Larson, J. R., Philpot, R. M., and Coon, M. J. (1993) Proc. Natl. Acad. Sci. USA 90, 2651-2655) suggest that this cytochrome is not deeply embedded but that other regions, in addition to the amino-terminal 26 residues, may be involved in the interaction of cytochrome P450 with the membrane.

1,2-Dipalmitoylphosphatidylcholine↗