Rudolf A. Lutz

Computer analysis of enzyme-substrate-inhibitor kinetic data with automatic model selection using IBM-PC compatible microcomputers.

A weighted nonlinear least-squares curve-fitting program, implemented in compiled BASIC for the IBM-PC is described to estimate the parameters of enzyme kinetics obeying Michaelis-Menten kinetics and seven inhibition models. The effects of the inhibitor on the maximal velocity (Vm) and the Michaelis-Menten constant (Km) are used to select automatically the most plausible model of inhibition and to calculate initial estimates of parameters. The program is used to demonstrate that the inhibition of carbamyl-phenylalanine hydrolase by the product phenylalanine is consistent with the pure mixed noncompetitive model.

MU1: A very high affinity subtype of enkephalin binding sites in rat brain

Displacement studies of [3H]-[D-Ala2-MePhe4-Gly-ol5]-enkephalin ([3H]-DAGO) and [3H]-[D-Ala2-D-Leu5]-enkephalin ([3H]-DADL) by the corresponding unlabeled ligands show that there are at least three classes of sites which bind these enkephalin analogs with high affinity. Using computer modeling, the introduction of the third site significantly improved the goodness of fit in ten consecutive experiments. These sites appear to correspond to the mu, delta and mu 1 sites, with mean dissociation constants of 11, 1.3 and 0.9 nM for DADL and 2.5, 300 and 0.3 nM for DAGO, respectively.

Increased affinity and selectivity of enkephalin tripeptide (Tyr-D-Ala-Gly) dimers

The binding of alkylendiamide dimers of the three N-terminal residues of [D-Ala2,D-Leu5]enkephalin (DADL) to rat brain and Ng108-15 neuroblastoma-glioma cell membranes was compared with that of DADL, Tyr-D-Ala-Gly-NMe-Phe-Gly-ol (DAGO) and morphiceptin. Tritiated DADL and DAGO were used as labeled ligands for delta- and mu-receptors, respectively. Dimerization of the tripeptides resulted in dramatic increases in both mu and delta binding. The binding to mu-receptors showed two peaks at an alkyl chain length of n = 2 and approximately n = 16. In contrast, delta binding (NG108-15 cells) increased steadily with increasing chain length. The dimers with n less than 18 were mu-preferential, and the one with n = 2 showed the most dramatic increase in mu selectivity with a 400 fold higher affinity to mu- than to delta-receptors. For long-chain alkyl spacers the compounds became delta selective.

New methods for characterization of complex receptor systems involving 3 or more binding sites: application to brain opiate receptors.

The problems of evaluating results based on the analysis of complex binding models are considered and new methods are proposed to provide independent confirmation of the existence of multiple sites. A new plotting format for the results from experiments involving two ligands is introduced, and its utility is demonstrated for a) finding initial estimates for nonlinear least squares curve fitting; b) presenting the results of multiple experiments; and c) giving a new means for evaluating the significance of a third site. The general problem of finding initial estimates for models involving three classes of sites, and strategies for using nonlinear least squares curve fitting algorithms to optimize the fit are considered.

BINDING OF INTERLEUKIN-13 AND INTERLEUKIN-4 TO THE INTERLEUKIN (IL)-4/IL-13 RECEPTOR OF HUMAN SYNOVIAL FIBROBLASTS

Synovial fibroblasts expressed transcripts for IL-4R alpha, and IL-13R alpha 1 and IL-13R alpha 2. Using weighted nonlinear computer modeling of the data from equilibrium binding studies, a 2 bindings sites model fitted the data best. After occupation of the shared high affinity receptors by the non-signaling, double mutant IL-4(121)R-->D, 124Y-->D (RY-IL-4) the high affinity binding of IL-13 could be abolished. A 2 binding site model still could be fitted, however the improvement in fit over a onesite model was not statistically significant. Using affinity spectra, at least 2 binding sites are apparent. After treatment with RY-IL-4, some of the high affinity binding was abolished, however not completely. A correlation between the number of binding sites and the affinity is apparent, which seriously casts doubt on the classical evaluation of binding isotherms, where the parameters are assumed to be independent. In a previous study we suggested that the large number of IL-13R alpha 2 monomers are silent receptors, likely representing a decoy target for IL-13. The high affinity binding therefore most likely represents the binding to the heterodimer consisting of IL-4R alpha and IL-13R alpha 1 or IL-13R alpha 2. The low affinity binding may represent the IL-13R alpha 2.

Multiple Interactions of Unsaturated Fatty Acids with Opiate and Ouabain Binding Sites and β-Adrenergic Sensitive Adenylate Cyclase System

The unsaturated fatty acids oleic, linoleic and arachidonic inhibited binding of ligands to the ouabain, opiate, and beta-adrenergic plasma membrane receptors. Low concentrations of fatty acids slightly increased the binding of ouabain to its binding sites. The effect of these fatty acids on beta-adrenergic sensitive adenylate cyclase was more complex. 0.2-0.3 mM fatty acids increased adenylate cyclase activity, while higher concentrations of arachidonic and linoleic acids, but not oleic acid, inhibited basal, beta-agonist- and NaF-stimulated activities in membranes of A431 and C6 cells. To evaluate which aspects of the unsaturated fatty acid molecules might be responsible for the observed effects, myristic acid, monoolein and taurodeoxycholic acid were studied. They also inhibited binding to the opiate receptor. Myristic acid, did not inhibit ouabain binding, binding to beta-receptor, nor adenylate cyclase activity. Monoolein, had no inhibitory effect on ouabain binding but behaved similar to oleic acid in the beta-receptor/adenylate cyclase system. Taurodeoxycholic acid inhibited binding to all three receptors as well as adenylate cyclase activity. We conclude that the effects of unsaturated fatty acids on ligand binding and adenylate cyclase activity are the result of their multiple interactions with various molecular processes rather than any unique property of long chain unsaturated fatty acids, per se.

Interaction of dimeric and monomeric enkephalins with NG108-15 hybrid cells

The binding of the enkephalin dimer [d-Ala2, Leu5-NH-CH2-]2 (DPE2) is characterized by (1) its high affinity for receptors on NG108-15 hybrid cells, the affinity constantK=4.7×109 M−1 is up to 8-fold that of monomers (0.6 to 1.0×109 M−1), and (2) a maximal binding capacity equal to one half that of the monomers. Kinetic studies showed that DPE2 binds with a 2-fold higher rate, k1=6.3×107 M−1min−1, than monomers (2.4 to 3.8×107 M−1min−1), and dissociates at a slower rate than monomers. Dissociation of DPE2 was consistently bi- or multiphasic but increased about 12% only after 3 hr of dissociation in the presence of a large excess of unlabeled enkephalin. The dissociation kinetics of monomers varied with enkephalin and experimental conditions used. Consistent with the value for the maximal binding capacity, the kinetic studies are interpreted in support of the hypothesis that DPE2 binds by cross-linking two subunits of one receptor.

A Computer Controlled Device to Facilitate Studies of the Kinetics of Ligand-Binding: Binding of Diazepam to Bovine Brain Membranes

A device to facilitate kinetic receptor filtration assays is described. The receptor containing membranes and the labeled ligand are placed in two separate syringes and are rapidly mixed into a collecting syringe using a pneumatic ram. Shortly after the start of mixing, a pneumatically controlled valve switches the collecting syringe containing the receptor-ligand mixture to the filtration unit. Filtration is performed on glass/microfiber filters or equivalent by pushing the plunger of the collecting syringe by a stepper motor. A valve positioner controlling several valves allows the filtered membranes to be washed and dried by pressure in any user programmable sequence. Further filtration of the receptor-ligand mixture can be programmed at selected time points. The entire system is controlled by an IBM-PC. With this system, the association and dissociation of diazepam from crude bovine-brain membranes has been studied at 4 degrees C. The dissociation shows a biphasic pattern with half lifes of 1.3 and more than 23 minutes respectively. Association appears to be into a single compartment.

Demonstration and Characterization of two Classes of Cardiac Glycoside Binding Sites to Rat Heart Membrane Preparations Using Quantitative Computer Modeling

Cardiac glycoside binding to rat heart membrane preparations was measured by rapid filtration technique. The binding data were analyzed using quantitative computer analysis. The experimental results using [3H]-ouabain as the labeled ligand were consistent with a model in which cardiac glycoside specific binding occurs at two independent classes of sites. The high affinity sites were characterized by a dissociation constants of 40 nM, 50 nM, and 61 nM for ouabain, digoxin and digitoxin, respectively, with a binding capacity of 1.3 pmoles/mg protein. The lower affinity sites for ouabain were characterized by dissociation constants of 2.3 microM, 67 nM and 71 nM for ouabain, digoxin and digitoxin, respectively, with a binding capacity of 3 pmoles/mg protein. Potassium ions inhibit [3H]-ouabain binding in a dose dependent manner with an IC50 of 500 microM. Quantitative computer modelling indicated that potassium inhibits ouabain binding at both binding sites.