George P. Hess

Protecting group for carboxylic acids that can be photolyzed by visible light

We report on a photolabile protecting (caging) group that is new for carboxylic acids. Unlike previously used caging groups for carboxylic acids, it can be photolyzed rapidly and efficiently in the visible wavelength region. The caging group 7-N,N-diethyl aminocoumarin (DECM) was used to cage the gamma-carboxyl group of glutamic acid, which is also a neurotransmitter. The caged compound has a major absorption band with a maximum at 390 nm (epsilon(390) = 13651 M(-)(1) cm(-)(1)). Experiments are performed at 400 nm (epsilon(400) = 12232 M(-)(1) cm(-)(1)) and longer wavelengths. DECM-caged glutamate is water soluble and stable at pH 7.4 and 22 degrees C. It photolyzes rapidly in aqueous solution to release glutamic acid within 3 micros with a quantum yield of 0.11 +/- 0.008 in the visible region. In whole-cell current-recording experiments, using HEK-293 cells expressing glutamate receptors and visible light for photolysis, DECM-caged glutamate and its photolytic byproducts were found to be biologically inert. Neurotransmitter receptors that are activated by various carboxyl-group-containing compounds play a central role in signal transmission between approximately 10(12) neurons of the nervous system. Caged neurotransmitters have become an essential tool in transient kinetic investigations of the mechanism of action of neurotransmitter receptors. Previously uncaging the compounds suitable for transient kinetic investigations required ultraviolet light and expensive lasers, and, therefore, special precautions. The availability of caged neurotransmitters suitable for transient kinetic investigations that can be photolyzed by visible light allows the use of simple-to-use, readily available inexpensive light sources, thereby opening up this important field to an increasing number of investigators.

Interaction between calcium and ligand-binding sites of the purified acetylcholine receptor studied by use of a fluorescent lanthanide.

Abstract The acetylcholine receptor isolated from Torpedo ocellata binds about 10 moles of a fluorescent lanthanide, terbium, per mole α-bungarotoxin-binding site, a process which is accompanied by a fluorescence enhancement (λexcitation 295 nm, λemission 546 nm) which allows detection of receptor-Tb 3+ complexes at μM concentrations. In presence of calcium two types of terbium-binding site are revealed, both with terbium dissociation constants of 18 ± 0.5 μM. About 60% of the sites bind calcium with an apparent dissociation constant of 1.1 ± 0.1 mM. Sites which interact with calcium also interact with activators of neural transmission, carbamylcholine and decamethonium, but not with the inhibitors, d-tubocurarine and α-bungarotoxin. Whether the displacement of calcium by chemical mediators is directly responsible for activator-induced changes in ion permeability of neural membranes is an important question raised by our experiments. The results show that fluorescent lanthanides can be an important tool in such studies.

Conformation of the high pH form of chymotrypsin

Abstract Optical rotatory dispersion and circular dichroism spectra of chymotrypsin, its precursor, and an inhibition product have been measured. The results, together with previous information on chemical and physical properties of these proteins in solution, and on the conformation of crystalline α-chymotrypsin at neutral pH, permit some deductions about conformational features of catalytically inactive species—chymotrypsinogen and the form of chymotrypsin that predominates at high pH. The data are in agreement with the suggestion (Sigler, Blow, Matthews & Henderson, 1968) that in these forms of the protein, the carboxyl group of aspartate residue 194—which according to Blow & co-workers (Matthews, Sigler, Henderson & Blow, 1967) forms an internal ion pair with the α-amino group of isoleucine residue 16 in chymotrypsin at neutral pH—protrudes into the active site of the enzyme and thereby interferes with substrate binding. It has also been concluded that this position of the carboxyl group disrupts the bondbreaking site; this is in accord with previous studies which have indicated that in chymotrypsinogen and chymotrypsin at high pH, the bond-breaking site is unreactive.

Quenched flow technique with plasma membrane vesicles: Acetylcholine receptor-mediated transmembrane ion flux

Abstract The construction and use of a versatile quenched flow machine suitable for use with small volumes of membrane vesicle suspension with a wide range of incubation times is described. Several sequential incubations can be made and the mixing can be performed with different dilutions. The design allows great versatility in plumbing which can be changed readily during an experiment. The time course of ion influx and efflux mediated by acetylcholine receptor was followed with varying ligand concentration using membrane vesicles with reaction times ranging from 5 ms to minutes. These measurements expose the varying response to different ligands, and the different ligand dependencies of ion flux and inactivation of receptor. Inactivation of the receptor by ligand and its recovery after dilution of ligand were studied by double incubation and concentration jump techniques. Problems particularly associated with the use of membrane vesicles in a rapid mixing system are considered. Breakage of the vesicles, studied with labeled membrane as an internal standard, was usually small but could be significant depending on the tubing arrangement and membrane preparation. Breakage, when observed, occurred very reproducibly. The mixing-quenching dead time with the vesicle system was apparently greater than that measured with a model hydrolysis reaction. This discrepancy may be attributable to the low diffusion rate of the vesicles.

A rapid method for the quantitative assay of proteolytic enzymes

Abstract 1. 1. Methods for the preparation of stable dye-protein complexes for use in proteolytic enzymes are described. 2. 2. Indigo carmine-fibrin provides a suitable substrate for the quantitative assay of proteolytic enzymes of the pepsin type, whereas, Congo redhide powder provides a suitable substrate for the quantitative assay of proteolytic enzymes of the trypsin type. 3. 3. The dye concentration, as measured spectrophotometrically at 620 mμ, parallels the concentration of the enzymically obtained hydrolysis products as measured at 273 mμ. This suggests that the dye concentration is directly proportional to the concentration of hydrolysis products.

Acetylcholine receptor inactivation in torpedo californica electroplax membrane vesicles. Detection of two processes in the millisecond and second time regions

Summary The rates of inactivations of the acetylcholine receptor-controlled ion flux were measured with membrane vesicles prepared from the electric organ of Torpedo californica . A flow quench technique with a time resolution of 2 msec was used. The vesicles were preincubated with carbamylcholine for various periods of time and then the rate of 86 Rb + influx was measured. The influx rate decreased progressively with the length of preincubation time. This decrease (inactivation) in influx rate occurred in two different time zones. A fast inactivation process with a half-time of ∼300 msec resulted in at least a 60-fold decrease of ion flux rate. The remaining ion flux activity decreased to an undetectable level in a slow inactivation process with a half-time of 6–7 seconds. Previously, only one inactivation process has been observed by ion flux measurements with receptor-rich vesicles from Electrophorus electricus or Torpedo species.

Development and application of caged ligands for neurotransmitter receptors in transient kinetic and neuronal circuit mapping studies.

Publisher Summary This chapter describes the instrumentation and theory involved in transient kinetic investigations of neurotransmitter receptor-mediated reactions on the surface of nervous system cells and in the membranes of Xenopus laevis oocytes in which many receptors have been expressed. Photolabile, biologically inert precursors of neurotransmitters (caged neurotransmitters) that can be photolyzed to the neurotransmitters in the microsecond time region form an integral part of the transient kinetic techniques. The use of these techniques was illustrated in investigations of the formation of transmembrane channels, receptor-drug interactions, and integration of excitatory and inhibitory signals by single central nervous system neuron that have temporal and spatial information. The use of caged neurotransmitters in identifying cells that contain a specific receptor and cells that secrete the neurotransmitter in a circuit of cells controlling a measurable response has also been illustrated in the chapter.

Dihydropyrimidinone Positive Modulation of δ-Subunit-Containing γ-Aminobutyric Acid Type A Receptors, Including an Epilepsy-Linked Mutant Variant

Gamma-aminobutyric acid type A receptors (GABA(A) receptors) are ligand-gated chloride channels that play a central role in signal transmission within the mammalian central nervous system. Compounds that modulate specific GABA(A) receptor subtypes containing the delta-subunit are scarce but would be valuable research tools and starting points for potential therapeutic agents. Here we report a class of dihydropyrimidinone (DHPM) heterocycles that preferentially potentiate peak currents of recombinant GABA(A) receptor subtypes containing the delta-subunit expressed in HEK293T cells. Using the three-component Biginelli reaction, 13 DHPMs with structural features similar to those of the barbiturate phenobarbital were synthesized; one DHPM used (monastrol) is commercially available. An up to approximately 3-fold increase in the current from recombinant alpha1beta2delta receptors was observed with the DHPM compound JM-II-43A or monastrol when co-applied with saturating GABA concentrations, similar to the current potentiation observed with the nonselective potentiating compounds phenobarbital and tracazolate. No agonist activity was observed for the DHPMs at the concentrations tested. A kinetic model was used in conjunction with dose-dependent measurements to calculate apparent dissociation constant values for JM-II-43A (400 muM) and monastrol (200 microM) at saturating GABA concentrations. We examined recombinant receptors composed of combinations of subunits alpha1, alpha4, alpha5, alpha6, beta2, beta3, gamma2L, and delta with JM-II-43A to demonstrate the preference for potentiation of delta-subunit-containing receptors. Lastly, reduced currents from receptors containing the mutated delta(E177A) subunit, described by Dibbens et al. [(2004) Hum. Mol. Genet. 13, 1315-1319] as a heritable susceptibility allele for generalized epilepsy with febrile seizures plus, are also potentiated by these DHPMs.

Evidence for separate initiation and inhibitory sites in the regulation of membrane potential of electroplax I. Kinetic studies with α-bungarotoxin

Abstract The minimum reaction mechanism for the irreversible reaction of α-bungarotoxin with membrane preparations of Electrophorus electricus involves a rapid reversible phase (K diss = 0.2 μM) followed by an irreversible reaction (k = 0.038 min −1 ). Compounds which initiate changes in membrane potential of electroplax affect only the rate of reaction but not the binding of toxin to the membrane. d-Tubocurare which inhibits membrane potential changes, as does α-bungarotoxin, is a competitive inhibitor which affects toxin binding but does not affect the rate of reaction. The simplest explanation of this is that membrane potential changes are controlled by two different sites, one for initiators and the other for inhibitors.

A CONTINUOUS, AUTOMATIC METHOD FOR THE STUDY OF RATE OF HYDROLYSIS OF PEPTIDES AND AMIDES.

Abstract A continuous, automatic method for following the enzymic hydrolysis of amides and peptides is presented. The apparatus used is a modification of part of the Technicon automatic amino acid analyzer, and the method is based on the development of color with ninhydrin. Excellent sensitivity and precision have been demonstrated in experiments with N -acetyl- l -tryptophan amide as substrate and α-chymotrypsin as enzyme. The method permits the use of considerably lower enzyme concentrations than was possible previously, and is comparable in convenience and sensitivity to the pH-stat method used in following the kinetics of ester hydrolysis.