Charles Pidgeon

Intestinal peptide transport systems and oral drug availability

The intestinal peptide transport system has broad substrate specificities. In addition to its physiological function of absorbing di- and tripeptides resulting from the digestion of dietary proteins, this transport system also absorbs some orally administered peptidomimetic drugs, including β-lactam antibiotics, angiotensin converting enzyme inhibitors, renin inhibitors, bestatin, thrombin inhibitors, and thyrotropin-releasing hormone and its analogues. There have been several studies on the mechanism and substrate structure-affinity relationship for this transport system. Rapid progress has been made recently in studies on the molecular basis of the intestinal peptide transport system. A protein apparently involved in peptide transport has been isolated from rabbit small intestines, and genes for human intestinal peptide transporters have been cloned, sequenced and functionally expressed. This review summarizes these studies and addresses the pharmaceutical potential of the intestinal peptide transport system.

Immobilized artificial membrane chromatography : rapid purification of functional membrane proteins

A solid-phase membrane mimetic system, denoted as immobilized artificial membranes (IAM), has been developed and utilized as a novel high-performance liquid chromatography (HPLC) matrix for the first step in the rapid purification of functional membrane proteins. IAM phases consist of monolayers of amphiphilic membrane lipid molecules covalently bonded to a rigid silica particle. These monolayers of lipids have proved remarkably effective for the chromatography of biomolecules. Several cytochrome P450 isozymes, an extremely important family of hydrophobic membrane proteins with a labile heme catalytic center, have been partially purified in functional conformations from rat liver, kidney, and adrenal microsomes on IAM supports. Functionality of purified P450 and P450 reductase has been demonstrated by optical difference spectroscopy, by carbon monoxide binding, and by reconstitution of enzymatic activity in vitro. Other membrane proteins, including rat liver plasma membrane NADH oxidase and ferricyanide oxidoreductase have also been partially purified by IAM HPLC. The methods for purification of these proteins are described.

Magnetically induced orientation of phosphatidylcholine membranes

Lipid bilayers prepared from natural phospholipids orient in magnetic fields with the long axis of the lipid molecules perpendicular to the magnetic field. This magnetically induced orientation was studied at high (11.7 Tesla (T)), mid (9.36 T), and low (4.68 T) magnetic field strengths using lipid aggregates prepared from natural and synthetic phosphatidylcholine analogs. Phosphatidylcholine analogs containing saturated diacylated chains (12 to 16 carbons/chain) exhibited extensive orientation of the lipid when bilayer formation occurred by gentle hydration conditions. Gentle hydration involved incubating dried phosphatidylcholine C above the main phase transition (Tm); brief shaking or swirling by hand was occasionally needed to completely disperse the lipids. The method of bilayer formation significantly influenced the amount of lipid that orients in magnetic fields. Thus the supramolecular structures (and % orientation) above Tm in an 11.7 T field of dimyristoylphosphatidylcholine (DMPC) bilayers are SUV (0%), LUV (approximately 15%), SPLV (approximately 40%), vortexed-MLV (approximately 60%) and non-vortexed MLV (approximately 90%). Single layered vesicles prepared by the REV method exhibited orientation at 11.7 T similar to LUV prepared by freeze thaw cycles. Aqueous dispersions of eggPC prepared by gentle hydration exhibit approximately 40% orientation at 11.7 T which decreased to approximately 30% orientation if 30% cholesterol is added to the membrane. Magnetic orientation of bilayers thus appears to be a general phenomenon for both saturated and unsaturated natural phospholipids either with or without cholesterol in the membrane.

A simple and efficient method for preparation of unsymmetrical sulfides

Abstract A simple and high efficient method for preparation of unsymmetrical sulfides is described.

Fourier transform infrared assay of membrane lipids immobilized to silica: Leaching and stability of immobilized artificial membrane-bonded phases

A nondestructive, sensitive assay to monitor the hydrocarbon content of silica-based chromatography particles has been developed. The assay requires a microscope accessory interfaced with a Fourier transform infrared (FTIR) spectrometer. For determining hydrocarbon content, undiluted alkyl-silica-bonded phases were pressed into a thin wafer. Hydrocarbon content was quantitated using the integrated hydrocarbon band intensity between 2995 and 2825 cm-1 [i.e., band area C-H] and the integrated silica oxide band intensity between 1945 and 1780 cm-1 [i.e., band area Si-O]. Plotting the [band area C-H]/[band area Si-O] ratio vs the carbon content determined by elemental analysis gave a correlation coefficient of r = 0.997. The FTIR assay was validated on 5-, 7-, and 12-microns silica particles using three different immobilized artificial membrane (IAM) silica-bonded phases. The utility of the FTIR assay in determining hydrocarbon content was demonstrated by evaluating hydrocarbon leaching from IAM phases exposed to mobile-phase solvents. The ability of organic solvents to leach hydrocarbon from IAM phases containing phosphatidylcholine (PC) as the immobilized ligand was chloroform greater than ethanol approximately methanol greater than ethyl acetate greater than methylene chloride greater than acetonitrile greater than acetone. Acetone and acetonitrile cause very little hydrocarbon leaching from HPLC-IAM.PC columns. When challenged with different mobile phases, IAM.PC columns perfused with mobile phase are more stable than IAM.PC-bonded phases stirred in mobile phases. IAM.PC contains lecithin linked to silica by amide bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid purification of cotton seed membrane-bound N-acylphosphatidylethanolamine synthase by immobilized artificial membrane chromatography

N-Acylphosphatidylethanolamine synthase (NAPES) is a membrane-bound enzyme present in cotton seedlings at a concentration of < or = 0.02% of the total protein. NAPES was purified to electrophoretic homogeneity in a single chromatographic step using immobilized artificial membrane (IAM) chromatography. The IAM column used for NAPES purification was etherIAM.PEC10/C3 and this surface contains a monolayer of immobilized phosphatidyl-ethanolamine (PE). Since PE is an analogue of the natural substrate for NAPES, etherIAM.PEC10/C3 columns function as an affinity column for this enzyme. Detergent-solubilized microsomal proteins from cotton were loaded on to the etherIAM.PEC10/C3 column and eluted with buffered mobile phases containing 0.2 mM dimyristoylphosphatidylethanolamine (DMPE) and 2 mM dodecylmaltoside. Little NAPES functional activity eluted if DMPE was removed from the mobile phase. Mobile phase DMPE is also a substrate for NAPES, both the mobile phase and IAM surface contains NAPES substrates. Mobile phase DMPE may function as both a surfactant-type affinity displacing ligand effecting protein elution and also a stabilizing factor of NAPES functional activity. The loading capacity on semi-preparative etherIAM.PEC10/C3 (6.5 x 1.0 cm) columns was ca. 5 mg of total detergent solubilized microsomal proteins, and protein recovery was quantitative. This one-step IAM purification of NAPES resulted in a single band on silver-stained polyacrylamide gels, and 3940 fold increase in NAPES specific activity. The molecular mass of the purified NAPES protein is 64,000. 125I labeled [12-(4-azidosalicyl)amino]dodecanoic acid is a photoreactive fatty acid substrate of NAPES that was used to confirm protein purity.

Fourier transform infrared assay of liposomal lipids.

Quantitating the lipid content in organic lipid solutions and extracted membrane preparations is described. Fourier transform infrared analysis of thin films using perdeuterated nonadecane as an internal standard permitted quantitation with greater than 95% accuracy.

Immobilized Artificial Membrane Chromatography: Surface Chemistry and Applications

Immobilized Artificial Membranes are solid surfaces containing phospholipids immobilized on silica particles at surface densities similar to the ligand density of reversed phase Chromatographic surfaces. Chromatographic and non-chromatographic applications of Immobilized Artificial Membrane surfaces are reviewed and compared to the Chromatographie and non-chromatographic applications of reversed phase columns. The methodology for synthesizing Immobilized Artificial Membranes and the stability of Immobilized Artificial Membranes are also described. Several examples are presented regarding the ability of Immobilized Artificial Membrane surfaces to model biological processes. Examples include predicting the transport of solutes across human skin, predicting the transport of amino acids across the blood brain barrier, and predicting the binding of solutes to liposome membranes. In addition, the purification of several membrane proteins, including cytochrome P450 from rat adrenals and rat livers, NADH oxidase, and rabbit intestinal phospholipid binding protein, are discussed.

Introduction to Fourier Transform Infrared Spectroscopy and Applications in the Pharmaceutical Sciences

The applications of infrared spectroscopy to pharmaceutical sciences is small compared to the applications of infrared spectroscopy to the fields of chemistry, biology, and biochemistry. This is unfortunate because modern routine infrared spectrometers are excellent research tools that provide very high signal-to-noise, high resolution, and extensive data-manipulation computer software packages. This review summarizes basic principles of infrared spectrometers and the use of Fourier self-deconvolution.

Mobile phase effects on membrane protein elution during immobilized artificial membrane chromatography

The eluotropic strength of different mobile phases for eluting membrane proteins from immobilized artificial membrane (IAM) chromatography surfaces was studied. Two protein mixtures containing bovine pancreatic PLA2 were used in this study. Protein mixture I was PLA2 obtained from Sigma which contained approximately 5-10 major protein bands in electrophoretic gels. Protein mixture II was obtained from flesh bovine pancreatic tissue and contained > 100 proteins including the target protein, PLA2. After adsorbing Sigma PLA2 to IAM columns, the elution conditions common to conventional chromatographic methods were evaluated for their ability to selectively purify PLA2. Elution conditions tested were (i) detergent gradients, (ii) salt gradients used during ion-exchange chromatography, (iii) salt conditions used during hydrophobic interaction chromatography, (iv) acetonitrile gradients used during reversed-phase chromatography, and (v) a two-step gradient consisting of first a detergent gradient followed by an acetonitrile gradient. Based on silver-stained electrophoretic protein gels. PLA2 from protein mixture I was purified to electrophoretic homogeneity with 417-fold increase in specific activity in one step using elution condition (v), and PLA2 from protein mixture II was purified in one step (660-fold increase in specific activity) using elution condition (iv). Total protein recovery from IAM columns is 70-100%.