2H( gamma,p)n cross section between 20 and 440 MeV

Rate control in acetylcholinesterase (AChE) involves a single anionic site whose anionic center controls rate-related biochemical and conformational changes in the E (free enzyme) and EA (acylated enzyme) conformers. Change in conformer structure and biochemistry affect binding, acylation, and hydrolysis. It is significant that the anionic-esteratic intersite distance is not altered during conformer change as E is converted to EA. In this enzyme system, cationic acetylcholine and anionic AChE are true structural, functional, and biochemical counterparts. The anionic center in the E conformer lies at the bottom of a sterically restricted, hydrophobic cleft < 8 A wide at the top and > 3 A wide at the bottom, while the anionic center in the EA conformer is relatively open. It is characterized by a decrease in the relative binding of hydrophobic cations and by an ability to bind large organic cations. Binding of acetylcholine, H+, or organic cations at the anionic site controls k2(acylation) in the E conformer and k3(hydrolysis) in the EA conformer. Acetylcholine binding forms the ES complex in which the cation maximizes k2. In the EAS complex, the cation reduces k3 and provides allosteric control. Anionic site structure and biochemistry and the effect of pH on k2 and k3 differentiates AChE from butyrylcholinesterase. This comprehensive study of kinetic and thermodynamic processes in AChE was made possible by the synthesis and/or use of families of over 30 cationic and acylation probes of known stereochemistry. They act as rulers of the E and EA conformers of AChE and provide comparative data on kinetic-based and thermodynamic-based constants. Cationic inhibitors affect decarbamylation rates in AChE and provide an additional set of comparative data related to the mechanism of substrate hydrolysis by AChE. Acridine araphanes are unique neural receptor and cholinergic enzyme probes. Their parallel plane and coplanar conformations are related to bridge length. Two parallel plane acridine araphanes are pure uncompetitive inhibitors of AChE. Scatchard plots of the binding of methylacridinium and 9-aminoacridine with the E conformer and 9-aminoacridine with the EA conformer indicate binding at a single anionic site. No ternary complex (EII or EAII) from two-site binding was detected. In AChE, nonspecific, low-level binding at surface ionic and hydrophobic areas is ubiquitous. Binding affinity differences greater than two orders of magnitude distinguish binding at the anionic site from low level binding at surface moieties. Surface binding provides environmental and stability changes in the enzyme but does not modify the fundamental biochemistry of the E and EA conformers.     

T20 measurements for 1H(d

When cytochrome b5 is added to large unilamellar vesicles (LUVs) of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), it binds predominantly in a 'loose,' or transferable form. Prolonged incubation of 30 degrees C leads to insertion in the physiological 'tight,' nontransferable form, with a halftime for the loose --> tight conversion of approx. 9 days. In this study, the effect of cholesterol on the rate of tight insertion was determined. Tight binding was assayed by depleting the LUVs of loose cytochrome b5 with an excess of SUV acceptors and then separating the liposome populations by gel-filtration or velocity sedimentation. Incorporation of cholesterol into the LUVs was found to markedly increase the rate of tight insertion, even though cholesterol decreases the equilibrium binding constant and saturation level of protein binding. The effect is not a continuously increasing function of cholesterol content, but attains a maximum at 20-25% mol%, where the rate enhancement is approx. 10-fold over baseline. At higher cholesterol levels, the rate decreases, returning to baseline at 40 mol% cholesterol. These observations are highly unusual in that cholesterol generally decreases the membrane binding affinity and the permeability of solutes, and does so as a monotonic function of cholesterol concentration (above the liquid-crystalline phase transition of the phospholipids). It is suggested that tight insertion is enhanced by lipid-protein packing mismatches and by bilayer fluidity; the former increases monotonically with increasing cholesterol whereas the latter decreases monotonically. At 20-25 mol% cholesterol the optimum balance of these physical properties is obtained for tight insertion.     

Noncompetitive enzyme immunoassay (hetero-two-site enzyme immunoassay) for gamma 2-melanocyte-stimulating hormone (gamma 2-MSH) and measurement of immunoreactive gamma 2-MSH in plasma of healthy subjects

A noncompetitive enzyme immunoassay (hetero-two-site enzyme immunoassay) for gamma 2-melanocyte-stimulating hormone (gamma 2-MSH) was developed. gamma 2-MSH (1-12) was biotinylated, trapped onto an anti-gamma 2-MSH (1-12) IgG-coated polystyrene bead, eluted at pH 1 after washing to eliminate other biotinylated substances, and measured using two streptavidin-coated polystyrene beads and affinity-purified anti-gamma 2-MSH (1-12) Fab'-peroxidase conjugate. The detection limit of gamma 2-MSH (1-12) was 10-30 amol (16-48 fg)/assay and 130-400 fmol (210-630 pg)/L of plasma. There was little or only slight cross reaction with alpha-MSH, beta-MSH, and gamma 1-MSH. By this immunoassay, the concentration and molecular size of immunoreactive gamma 2-MSH in plasma of healthy subjects were examined, and the results were compared with those by competitive enzyme immunoassay. Immunoreactive gamma 2-MSH measured by competitive enzyme immunoassay was a mixture of substances with high molecular weights (100-500 kDa), and its concentration was calculated to be 50-60 pmol/L using gamma 2-MSH (1-12) as standard. Immunoreactive gamma 2-MSH detected by the noncompetitive enzyme immunoassay after removal of high molecular weight substances was not homogeneous and smaller than gamma 2-MSH (1-12), and its concentration was approximately 1 pmol/L. The exact nature of these immunoreactive gamma 2-MSHs remains to be elucidated. gamma 2-MSH (1-12) added to plasma was degraded rapidly, and the concentration of gamma 2-MSH (1-12) was very low, if any, in plasma of healthy subjects.