PubMed Health⌕ Search

Biomedical subjects

R M Krupka

Publications and source records attributed to R M Krupka.

At least 55 records · Page 3Linked to original sources
PubMed1980

Asymmetric binding of steroids to internal and external sites in the glucose carrier of erythrocytes.

Steroids inhibit glucose transport in erythrocytes by binding to sites in the carrier which are exposed on both the outer and inner surfaces of the cell membrane. Some steroids are bound almost exclusively at inner sites (androstendione and androstandione), while others are bound about as firmly on one side as the other (corticosterone). Still others exhibit a moderate preference for the internal site (deoxycorticosterone). The inhibition is in all cases competitive with respect to a substrate which is bound at the same surface of the membrane as the inhibitor. However, in experiments on substrate entry, internally bound inhibitors act in an apparently non-competitive fashion, as expected if the carrier model is valid. This behaviour explains the appearance of competitive, non-competitive and mixed inhibitions with different steroids (Lacko, L., Wittke, B. and Geck, P. (1975) J. Cell Physiol. 86, 673--680).

Binding Sites↗
PubMed1979

The binding and translocation steps in transport as related to substrate structure. A study of the choline carrier of erythrocytes.

The relationships between structure, affinity and transport activity in the choline transport system of erythrocytes have been investigated in order to (i) explore the nature of the carrier site and its surroundings, and (ii) determine the dependence of the carrier reorientation process on binding energies and steric restraints due to the substrate molecule. Affinity constants and maximum transport rates for a series of trialkyl derivatives of ethanolamine were obtained by a method that involves measuring the trans effect of unlabeled analogs upon the movement of radioactive choline. The main conclusions are as follows: (1) An analysis of transport kinetics shows that the affinity constants determined experimentally differ from the actual dissociation constants in a predictable way. The better the substrate, the higher the apparent affinity relative to the true value, whereas the affinity of non-transported inhibitiors is underestimated by a constant factor. (2) The carrier-choline complex undergoes far more rapid reorientation (translocation) than the free carrier. (3) The carrier imposes a strict upper limit upon the size of a substrate molecule that can participate in the carrier reorientation process; this limit corresponds to the choline structure. A smaller substrate such as tetramethylammonium, despite relatively weak binding forces , is unhindered in its translocation, suggesting that a carrier conformational change, dependent upon substrate binding energy, is not required for transport. (4) Small increases in the size of the quaternary ammonium head, as in triethylcholine, sharply lower affinity, consistent with a high degree of specificity for the trimethylammonium group. (5) Lengthening the alkyl substituent in derivatives of dimethyl- and diethylaminoethanol causes a regular increase in affinity, suggestive of unspecific hydrophobic bonding in a region very near the substrate site.

Binding, Competitive↗
PubMed1979

A simple experimental approach to the determination of carrier transport parameters for unlabeled substrate analogs.

A method is described by which affinities and transport rates for unlabeled substrate analogs are readily determined, and which is based on the effect of an unlabeled analog upon the rate of transport of a labeled substrate present at a low concentration on the trans side of the membrane. The procedure is widely applicable since it does not depend on assumptions about rrate-limiting steps and holds for both active and non-active systems. Here it is applied in an experimental study of the facilitated diffusion system for choline in erythrocytes, and it is shown that the transport parameters for a test substrate obtained by this method are the same as those found when the transport of the substrate is followed directly.

Biological Transport↗
PubMed1979

A general kinetic analysis of transport. Tests of the carrier model based on predicted relations among experimental parameters.

The analysis of transport kinetics has lacked both a unified treatment in which general rate equations are written entirely in terms of experimental parameters, and a convention by which these parameters may be designated in a concise yet immediately recognizable manner. Such a treatment is presented here in an easily accessible form, and a simple system of nomenclature is proposed resembling that in use in enzyme kinetics. The treatment is independent of assumptions about rate-limiting steps in transport, and applies to both active and facilitated systems, including obligatory exchange. A single substrate is characterized by twelve different parameters, only five of which are required in theory to calculate the others. If a second substrate is present on the trans side of the membrane there are six more parameters. All eighteen parameters are linked by multiple relationships which provide a complete set of rejection criteria for the generalized form of the mobile carrier. Relationships among parameters are also defined that give information on the rate-limiting steps in transport. Equations governing any individual experiment, involving only experimental parameters, are easily written out from the general expressions, for example under conditions of zero trans and infinite trans flux, equilibrium exchange, or competitive inhibition.

Biological Transport↗
PubMed1979

The membrane valve: a consequence of asymmetrical inhibition of membrane carriers. I. Equilibrating transport systems.

Facilitated membrane transport systems act as valves, or rectifiers, when the substrate affinities on the two sides of the membrane differ substantially, i.e. when the system is strongly asymmetric. The asymmetry may be intrinsic or imposed by a reversible competitive inhibitor acting on only one side of the membrane. Under non-equilibrium conditions such systems allow net movements of substrate to proceed faster, sometimes much faster, in one direction than the other, though the final equilibrium is unaffected. Obligatory exchange systems may also function as valves when inhibited unsymmetrically, permitting exchange to occur more rapidly with one distribution of substrates than with the reversed distribution. Here, unequal flux rates do not depend on unequal concentrations of the substrate on either side of the membrane, but may also occur with equal concentrations, provided the affinities of the two substrates differ. The kinetic theory leading to these conclusions is given here, and it is shown how individual parameters of a carrier system affect the efficiency, or tightness, of the valve. In addition, simple kinetic tests for the operation of a valve are outlined. Examples are cited of transport systems having inhibitor-binding sites on only one surface of the cell membrane, which could function normally as valves. Systems implicated are glucose transport in various cells, the ADP-ATP exchanger of mitochondria, the anion transporter of erythrocytes, and the Na+-K+ pump.

Adenosine Diphosphate↗
PubMed1978

Testing transport models with substrates and reversible inhibitors.

The kinetic behavior of five models for biological transport, only one of which is based on the classical carrier mechanism, is investigated. All give hyperbolic substrate saturation curves in accord with experimental observations on many systems. Several simple kinetic tests with substrates and competitive inhibitors serve to exclude or confirm proposed models. The tests involve measuring rates of efflux of radioactive substrate in the presence of (i) a competitive inhibitor outside the cell; (ii) inhibitor inside and outside; and (iii) unlabeled substrate outside. Rules for testing hypothetical mechanisms are presented in tables which may be consulted directly, disregarding the mathematical derivation.

Biological Transport↗
PubMed1978

Cytochalasin B and the kinetics of inhibition of biological transport: a case of asymmetric binding to the glucose carrier.

Cytochalasin B inhibits glucose transport in human erythrocytes by competing with glucose for the carrier on the inner surface of the cell membrane, but there is no cytochalasin site associated with the outware-facing form of the carrier. Such asymmetry may be demonstrated by zero trans exit and entry experiments, whereas Sen-Widdas exit experiments are not easily interpretable. The orientation of the transport system appears to be reversed in certain other cell types: chich embryo fibroblasts, Novikoff hepatoma cells and HeLa cells. Here the cytochalasin site is present in the external but not internal carrier form.

Binding Sites↗
PubMed1978

A new approach in the kinetics of biological transport. The potential of reversible inhibition studies.

Kinetic equations are derived for reversible inhibition of both active and facilitated transport systems for seven common experimental arrangements. It is shown that the unique features of transport kinetics may be exploited to give new kinds of information. It is also shown that that the familiar rules of enzyme kinetics, though often applied to transport, can be seriously misleading. The analysis leads to the following general conclusions: (1) A competitive mechanism frequently gives rise to non-competitive kinetics, depending on the experimental design, but a non-competitive mechanism never produces competitive kinetics. (2) Inhibition studies on exchange diffusion at equilibrium in non-active systems or in the final steady state in active systems are the only unambiguous kinetic tests to distinguish competitive from non-competitive mechanisms. (3) Substrate analogs that are bound to the carrier and transported are readily distinguished by inhibition kinetics from those not transported, even though both may rapidly enter the cell by another route. (4) Even in non-active systems competitive inhibitors commonly have far different affinities for the substrate sites on the two membranes faces: where sufficient non-polarity allows their penetration into the cell, inhibition kinetics readily establish such sideness in their action. (5) Inhibition kinetics of the mixed competitive and non-competitive type result from moderately asymmetrical binding of inhibitor at the substrate site. (6) Asymmetry is a necessary feature of active transport: hence studies of inhibition kinetics should provide important insights into its mechanism.

Binding, Competitive↗
PubMed1975

Differential effects of denaturing agents on acetylcholinesterase: insensitivity of the reaction of methanesulfonyl fluoride compared to diisopropylphosphorofluoridate and p-nitrophenyl acetate.

The effects of various agents that could be expected to perturb enzyme structure in a non-specific and reversible manner (alcohols, dimethylsulfoxide, dimethylformamide, dinitrobenzene, urea and guanidine - HCl) have been determined on reaction of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) with a substrate, p-nitrophenyl acetate, and two irreversible inhibitors, diisopropylphosphorofluoridate and methanesulfonyl fluoride. In all three of these reactions an acyl group (acetyl, phosphoryl or sulfonyl respectively) bonds covalently with the active center of the enzyme. As expected, the reactions of p-nitrophenyl acetate and diisopropylphosphorofluoridate were severely retarded by most of these agents. By contrast, reaction of methanesulfonyl fluoride was usually depressed to a far smaller degree, and in two cases was faster. These findings are of interest in connection with: (1) differing requirements for the integrity of tha active center in catalysis with various substrate analogs, and (2) the mechanism by which cationic substrate analogs accelerate reaction of the enzyme with methanesulfonyl fluoride.

Acetylcholinesterase↗
PubMed1975

Indophenyl acetate and acetylcholinesterase: binding of a non-specific substrate on the margin of the active center.

1. Indophenyl acetate is a very poor substrate of eel or bovine acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7), with a V less than 5% of that of phenyl acetate, but it is a labile ester and in imidazole buffer is hydrolyzed, non-enzymically, even faster than phenyl acetate. 2. Indophenyl acetate completely protects the enzymes against inhibition by diisopropylphosphorofluoridate but promotes inhibition by methanesulfonyl fluoride. 3. With either of these inhibitors the measured rate of inactivation of eel acetylcholinesterase is the same whether activity is determined with this poor substrate or with a good substrate, acetylthiocholine. With bovine enzyme the inactivation rate is 25% lower when assayed with the former substrate. However this preparation contains a minor enzyme component which is involved in hydrolysis of indophenyl acetate but not good substrates, and which is not readily inhibited. When this is taken into account the inactivation rates for bovine acetylcholinesterase, too, are found to be the same in either assay. These and other observations in the literature can be explained if indophenyl acetate, because of its size, cannot fully penetrate into the active center and is bound in adjoining non-polar regions of the protein. From this external position it makes only intermittent contact with the esteratic site. Hence it is slowly hydrolyzed and fails to protect the enzyme against methanesulfonyl fluoride, though it does protect, possibly sterically, against the larger inhibitor diisopropylphosphorofluoridate.

Acetates↗