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PubMed2003

General methods for analysis of sequential "n-step" kinetic mechanisms: application to single turnover kinetics of helicase-catalyzed DNA unwinding.

Helicase-catalyzed DNA unwinding is often studied using "all or none" assays that detect only the final product of fully unwound DNA. Even using these assays, quantitative analysis of DNA unwinding time courses for DNA duplexes of different lengths, L, using "n-step" sequential mechanisms, can reveal information about the number of intermediates in the unwinding reaction and the "kinetic step size", m, defined as the average number of basepairs unwound between two successive rate limiting steps in the unwinding cycle. Simultaneous nonlinear least-squares analysis using "n-step" sequential mechanisms has previously been limited by an inability to float the number of "unwinding steps", n, and m, in the fitting algorithm. Here we discuss the behavior of single turnover DNA unwinding time courses and describe novel methods for nonlinear least-squares analysis that overcome these problems. Analytic expressions for the time courses, f(ss)(t), when obtainable, can be written using gamma and incomplete gamma functions. When analytic expressions are not obtainable, the numerical solution of the inverse Laplace transform can be used to obtain f(ss)(t). Both methods allow n and m to be continuous fitting parameters. These approaches are generally applicable to enzymes that translocate along a lattice or require repetition of a series of steps before product formation.

Algorithms↗
PubMed1980

Study of kinetics of epithelial cell populations in normal tissues of the rat's intestines and in carcinogenesis. I. A comparison of enterocyte population kinetics in different segments of the small intestine and colon.

The peculiarities of enterocyte proliferation in the duodenum, jejunum, ileum, caecum, ascending, transverse and descending colon in the rat were studied by different methods of analysis of cell population kinetics (percentage-labelled mitosis curves, cumulative labelling curves, distribution of labelling index curves, etc.). The majority of proliferating cells in the small intestine are homogenous, as far as mitotic cycle mean duration (11-12 hrs) is concerned. All proliferating cells in all the zones of the colonic crypts and the bottom of the small intestine crypts are divided into subpopulations, having different mean durations of the mitotic cycle. It is suggested that, in the crypt bottom in all intestines as well as the crypt's maximum proliferation zones in most of the colonic segments, a considerable fraction of cells has a very long mitotic cycle or enters resting phase R1. The average value of the mean durations of the mitotic cycle of all colonic enterocyte subpopulations is 18-22 hours. On the basis of the authors' findings and literature data, a model for the enterocyte life cycle is proposed, according to which the cell flux is branched during the mitotic cycle and the crypt develops from a stem enterocyte population located at its bottom.

Animals↗
PubMed1988

Kinetic analysis of covalent hybrid plasminogen activators: effect of CNBr-degraded fibrinogen on kinetic parameters of Glu1-plasminogen activation.

The kinetic parameters of three activator species of Glu1-plasminogen (Glu1-Plg) were compared in their reaction at pH 7.4 and 37 degrees C, in the presence and absence of CNBr-digested fibrinogen (CNBr-Fg). The urokinase- (u-PA-) derived covalent hybrid activator PlnA-u-PAB had an apparent Michaelis constant (Kplg) of 7.44 microM, a catalytic rate constant (kplg) of 51.1 min-1, and a second-order rate constant (kplg/Kplg) of 6.87 microM-1 min-1. The tissue plasminogen activator (t-PA) derived covalent hybrid activator PlnA-t-PAB was characterized by a Kplg of 3.33 microM, a kplg of 1.03 min-1, and a kplg/Kplg of 0.309 microM-1 min-1. The kplg/Kplg values for the parent u-PA and t-PA activators were 6- and 16-fold higher than the respective hybrids, mainly due to an approximately 10-fold increase in the apparent Kplg for the hybrids. In the presence of CNBr-Fg, the increase of the kplg/Kplg values for u-PA and its hybrid was 1.1-fold, but for t-PA and its hybrid, the increases were 7- and 12-fold, respectively. In both the absence and presence of CNBr-Fg, activator t-PAB had an apparent Kplg of 19.1 and 27.6 microM and a kplg of 2.9 and 5.0 min-1, respectively. The increase in the kplg/Kplg value with CNBr-Fg was 1.2-fold. The streptokinase- (SK-) derived activators Glu1-plasmin.SK (Glu1-Pln.SK), Val442-Pln.SK, and Val561-Pln.SK had apparent Kplg values of 0.458, 0.268, and 0.121 microM and kplg values of 20.0, 126.0, and 63.3 min-1, respectively. In the presence of CNBr-Fg, the first two activators showed an approximately 1.4-fold increase and the last showed a 1.4-fold decrease in their kplg/Kplg values. The catalytic efficiency (kplg/Kplg) of the various activator species fell in the decreasing order SK greater than u-PA greater than t-PA, in either the presence or absence of CNBr-Fg. CNBr-Fg enhanced significantly the activities of only two activators, t-PA and PlnA-t-PAB.

Amidohydrolases↗
PubMed1982

Incorporation kinetics of lysolecithin into lecithin vesicles. Kinetics of lysolecithin-induced vesicle fusion.

The incorporation kinetics of L-palmitoylphosphatidylcholine (lysolecithin) into dimyristoyl- and dipalmitoylphosphatidylcholine vesicles and the subsequent aggregation and fusion of the vesicles into larger aggregates were studied by using stopped-flow rapid-mixing techniques. The half-times for the lysolecithin incorporation vary between 50 and 500 ms. The incorporation rate has a maximum in the temperature range of the vesicle phase transition. This process is not diffusion controlled. The rate-limiting step is the incorporation of the lysolecithin monomer into the liquid bilayer. After this fast process, a slow reaction in the 10-50-min time range is observed. The large irreversible increase in turbidity indicates aggregation and fusion of the vesicles. The initial step is a second-order reaction with respect to the vesicle concentration, indicating aggregation or fusion of two vesicles. The aggregation rate passes through a maximum at the phase transition temperature.

Dimyristoylphosphatidylcholine↗
PubMed2004

Kinetics of turnover of cefotaxime by the Enterobacter cloacae P99 and GCl beta-lactamases: two free enzyme forms of the P99 beta-lactamase detected by a combination of pre- and post-steady state kinetics.

Third-generation cephalosporins bearing oximino side chains are resistant to hydrolysis by class C beta-lactamases such as that from Enterobacter cloacae P99. For example, steady state parameters for hydrolysis of cefotaxime by this enzyme are as follows: k(cat) = 0.41 s(-1), K(m) = 17.2 microM, and k(cat)/K(m) = 2.3 x 10(4) s(-1) M(-1). On the other hand, however, the K(i) value for cefotaxime as an inhibitor of cephalothin hydrolysis is 27 nM. The discrepancy between K(m) and K(i) indicated that a real steady state had not been achieved in at least one of these experiments. Analysis indicated that only two to three cefotaxime turnovers occurred during the K(i) determination. This suggested that the first few turnovers of cefotaxime by the P99 beta-lactamase may be different from those in the subsequent steady state. A direct pre-steady state experiment confirmed this hypothesis. The simplest reaction scheme that fitted the data involved replacement of the initial enzyme form, E, which bound cefotaxime tightly, with a second more weakly binding form, E', by partitioning of the acyl-enzyme intermediate during the first few turnovers. Steady state turnover of cefotaxime then largely involved E' as the free enzyme form. E' slowly reverted to E in the post-steady state regime. Further evidence for this scheme included quantitative analysis of the post-steady state and observation of a difference in the catalytic activity of E and E' in 2 M ammonium sulfate. The kinetics of P99 beta-lactamase-catalyzed hydrolysis of an acyclic depsipeptide substrate bearing a third-generation cephalosporin side chain showed that the side chain is necessary but not sufficient for production of resistance to beta-lactamase; a combination of the side chain and the dihydrothiazine ring of a cephalosporin is required. The beta-lactamase of E. cloacae GC1, an extended spectrum mutant of the P99 enzyme, rapidly hydrolyzes third-generation cephalosporins, without the structural transition described above. The flexibility of the extended Omega loop of the GC1 enzyme probably leads to this situation. Conformational restriction of the loop in the P99 enzyme is probably responsible for the long-lived acyl-enzyme intermediate and the transition to E' induced by cefotaxime.

Acylation↗
PubMed2001

Kinetic analysis of the stepwise formation of a long-range DNA interstrand cross-link by a dinuclear platinum antitumor complex: evidence for aquated intermediates and formation of both kinetically and thermodynamically controlled conformers.

Reported here is a detailed study of the kinetics and mechanism of formation of a 1,4 GG interstrand cross-link by [(trans-PtCl(NH(3))(2))(2)(mu-NH(2)(CH(2))(n)NH(2))](2+) (1,1/t,t (n = 6), 1), the prototype of a novel class of platinum antitumor complexes. The reaction of the self-complementary 12-mer duplex 5'-[d(ATATGTACATAT)(2)] with (15)N-1 has been studied at 298 K, pH 5.4, by [(1)H,(15)N] HSQC 2D NMR spectroscopy. Initial electrostatic interactions with the duplex are observed for 1 and the monoaqua monochloro species (2). Aquation of 1 to yield 2 occurs with a pseudo-first-order rate constant of (4.15 +/- 0.04) x 10(-5) s(-1). 2 then undergoes monofunctional binding to the guanine N7 of the duplex to form 3 (G/Cl) with a rate constant of 0.47 +/- 0.06 M(-(1) s(-1). There is an electrostatic interaction between the unbound [PtN(3)Cl] group of 3 and the duplex, which is consistent with H-bonding interactions observed in the molecular model of the monofunctional (G/Cl) adduct. Closure of 3 to form the 1,4 GG interstrand cross-link (5) most likely proceeds via the aquated (G/H(2)O) intermediate (4) (pseudo-first-order rate constant = (3.62 +/- 0.04) x 10(-5) s(-1)) followed by closure of 4 to form 5 (rate constant = (2.7 +/- 1.5) x 10(-3) s(-1)). When closure is treated as direct from 3 (G/Cl) the rate constant is (3.39 +/- 0.04) x 10(-5) s(-1). Closure is ca. 10-55-fold faster than that found for 1,2 GG intrastrand cross-link formation by the diaqua form of cisplatin. Changes in the (1)H and (15)N shifts of the interstrand cross-link 5 indicate that the initially formed conformer (5(i)) converts irreversibly into other product conformer(s) 5(f). The NMR data for 5(i) are consistent with a molecular model of the 1,4 GG interstrand cross-link on B-form DNA, which shows that the NH(2) protons have no contacts except with solvent. The NMR data for 5(f) show several distinct NH(2) environments indicative of interactions between the NH(2) protons and the DNA. HPLC characterization of the final product showed only one major product peak that was confirmed by ESI-FTICR mass spectroscopy to be a cross-linked adduct of (15)N-1 and the duplex. The potential significance of these findings to the antitumor activity of dinuclear platinum complexes is discussed.

Antineoplastic Agents↗
PubMed2003

Similarities between the thermal inactivation kinetics of Bacillus amyloliquefaciens alpha-amylase in an aqueous solution of sodium dodecyl sulphate and the kinetics in the solution of anionic-phospholipid vesicles.

An anionic surfactant, SDS, is commonly used to model compounds of negatively charged phospholipids. The effect of SDS on the thermal inactivation of BAA (alpha-amylase from Bacillus amyloliquefaciens ) was compared with the effect of negatively charged phospholipid vesicles of DOPA (dioleoylphosphatidic acid) at 40-55 degrees C. The inactivation kinetics revealed that both SDS below its c.m.c. (critical micellar concentration) and DOPA vesicles accelerated the inactivation and the unfolding of the enzyme structure. Both SDS and DOPA vesicles lowered the activation enthalpy and entropy for the inactivation. The lowered activation parameters might be due to the relatively small energy requirement needed to reach the transition state from the ground state of BAA in the presence of the negatively charged hydrophobic environments. This study suggests an accelerated unfolding of BAA in the presence of SDS to be energetically similar to the accelerated unfolding in the presence of DOPA vesicles that involve a molten-globule-like state.

Animals↗
PubMed1974

Kinetics and mechanism of catalysis by proteolytic enzymes. A comparison of the kinetics of hydrolysis of synthetic substrates by bovine alpha- and beta-trypsin.

Several esters of the alpha-N-toluene-p-sulphonyl and alpha-N-benzoyl derivatives of S-(3-aminopropyl)-l-cysteine and the methyl ester of S-(4-aminobutyl)-N-toluene-p-sulphonyl-l-cysteine were synthesized. The kinetics of hydrolysis of these and esters of the alpha-N-toluene-p-sulphonyl and alpha-N-benzoyl derivatives of l-arginine, l-lysine, S-(2-aminoethyl)-l-cysteine and esters of gamma-guanidino-l-alpha-toluene-p-sulphonamidobutyric acid and alpha-N-toluene-p-sulphonyl-l-homoarginine by alpha- and beta-trypsin were compared. On the basis of values of the specificity constants (k(cat.)/K(m)), the two enzymes display similar catalytic efficiency towards some substrates. In other cases alpha-trypsin is less efficient than beta-trypsin. It is possible that alpha-trypsin possesses greater molecular flexibility than beta-trypsin.

Animals↗
PubMed1976

Kinetic mechanism from steady-state kinetics of the reaction catalysed by baker's-yeast glucose 6-phosphate dehydrogenase in solution and covalently attached to sepharose.

1. The reaction catalysed by glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate-NADP+ oxidoreductase, EC 1.1.1.49) from baker's yeast was studied in 42mM-glycylglycine buffer, pH7.4 at 25 degrees C, by initial-velocity studies and by the use of NADPH as a product inhibitor. 2. The reactions catalysed by both the soluble enzyme and a stable enzyme covalently attached to CNBr-activated Sepharose 4B probably follow an ordered reaction mechanism with NADP+ and NADPH as the leading reactants. 3. The kinetic constants obtained for the soluble enzyme lere: KNADP+m, 19 muM; KNADP+s, 23 muM; KNADPHs, 15 muM. Similar values were obtained for the immobilized enzyme. 4. The assay of the immobilized enzyme was done by using a micro packed-bed recirculation reactor, and the advantages of this technique are discussed.

Cyanogen Bromide↗
PubMed1982

Kinetic mechanism of mitochondrial adenosine triphosphatase. ADP-specific inhibition as revealed by the steady-state kinetics.

1. A substantial increase of the initial rate of ATP hydrolysis was observed after preincubation of bovine heart submitochondrial particles with phosphoenolpyruvate and pyruvate kinase. 2. The activation was accompanied by an increase of Vmax, without change of Km for ATP. 3. The activated particles catalysed the biphasic hydrolysis of ATP in the presence of an ATP-regenerating system; the initial rapid phase was followed by a second, slower, phase in a time-dependent fashion. 4. The higher the ATP concentration used as a substrate, the higher is the rate of transition between these two phases. 5. The particles catalysed the hydrolysis of ITP with a lag phase; after preincubation with phosphoenolpyruvate and pyruvate kinase, ITP was hydrolysed at a constant rate. 6. Qualitatively the same phenomena were observed when soluble mitochondrial ATPase (F1-ATPase) prepared by the conventional method in the presence of ATP was used as nucleotide triphosphatase. 7. A kinetic scheme is proposed, in which the intermediate active enzyme-product complex (E.ADP) formed during ATP hydrolysis is in slow equilibrium with the inactive E*.ADP complex forming as a result of dislocation of ADP from the active site of ATPase to the other site, which is not in rapid equilibrium with the surrounding medium.

Adenosine Diphosphate↗
PubMed1983

Effects of conformational selectivity and of overlapping kinetically influential ionizations on the characteristics of pH-dependent enzyme kinetics. Implications of free-enzyme pKa variability in reactions of papain for its catalytic mechanism.

The effects of selection by a small molecule, when binding to a protein, of a particular conformation from an equilibrium stereopopulation on the characteristics of the pH-dependence of reaction with a reactivity probe or substrate were determined by analysis of an appropriate kinetic model. For reaction in one protonic state containing an equilibrium mixture of two conformational isomers, the pH-second-order rate constant (k) profile is of conventional sigmoidal form. The apparent pKa value is a composite of the pKa values of the two conformational states. The value of pKapp. for a given enzyme under given experimental conditions will always be the same (provided that the site-specificity assumed in the model is maintained) irrespective of whether only one conformation reacts or both react, with the same or with different rate constants. The experimentally determined pH-independent rate constant (kapp.) is an average of the reactivities of the two conformational states weighted in favour of the predominant form. The presence of an additional but unreactive conformational state also affects the value of kapp. The possibility that overlapping acid dissociations that affect the reactivity of the enzyme might provide pH-k profiles often indistinguishable in practice from simple sigmoidal dissociation curves and subject to variability in apparent pKa values was evaluated by a simulation study. If two reactive protonic states of the enzyme respond differently to changes in the structure of the substrate or site-specific reactivity probe, differences in apparent pKa values of up to approx. 1 unit can be exhibited without deviation from sigmoidal behaviour being reliably observed. Differences in apparent pKa values observed in some site-specific reactions of papain and their possible consequences for its catalytic mechanism are discussed.

Hydrogen-Ion Concentration↗
PubMed1989

The kinetics of transport of lactate and pyruvate into isolated cardiac myocytes from guinea pig. Kinetic evidence for the presence of a carrier distinct from that in erythrocytes and hepatocytes.

1. Time courses for the uptake of L-lactate, D-lactate and pyruvate into isolated cardiac ventricular myocytes from guinea pig were determined at 11 degrees C or 0 degrees C (for pyruvate) in a citrate-based buffer by using a silicone-oil-filtration technique. These conditions enabled initial rates of transport to be measured without interference from metabolism of the substrates. 2. At a concentration of 0.5 mM, transport of all these substrates was inhibited by approx. 90% by 5 mM-alpha-cyano-4-hydroxycinnamate; at 10 mM-L-lactate a considerable portion of transport could not be inhibited. 3. Initial rates of L-lactate and pyruvate uptake in the presence of 5 mM-alpha-cyano-4-hydroxycinnamate were linearly related to the concentration of the monocarboxylate and probably represented diffusion of the free acid. The inhibitor-sensitive component of uptake obeyed Michaelis-Menten kinetics, with Km values for L-lactate and pyruvate of 2.3 and 0.066 mM respectively. 4. Pyruvate and D-lactate inhibited the transport of L-lactate, with Ki values (competitive) of 0.077 and 6.6 mM respectively; the Ki for pyruvate was very similar to its Km for transport. The Ki for alpha-cyano-4-hydroxycinnamate as a non-competitive inhibitor was 0.042 mM. 5. These results indicate that L-lactate, D-lactate and pyruvate share a common carrier in guinea-pig cardiac myocytes; the low stereoselectivity for L-lactate over D-lactate and the high affinity for pyruvate distinguish it from the carrier in erythrocytes and hepatocytes. The metabolic roles for this novel carrier in heart are discussed.

Animals↗
PubMed2005

Structure and kinetics of a transient antibody binding intermediate reveal a kinetic discrimination mechanism in antigen recognition.

Induced fit is a predominant phenomenon in protein-ligand interactions, yet it is invariably attributed without establishing the existence, let alone the structure, of the initial, low-affinity encounter complex. We determined the crystal structure of the encounter complex on the pathway of ligand binding by IgE antibody SPE7. We show that this complex is formed by a wide range of ligands that initially bind with identical affinity. Nonspecific ligands rapidly dissociate, whereupon the antibody isomerizes to a nonbinding isomer. Specific ligand complexes, however, slowly isomerize to give a high-affinity complex. This isomerization involves backbone and side-chain rearrangements of up to 14 A and the formation of specific hydrogen bonds. The postbinding conformational switch, combined with the prebinding isomerization to an energetically favorable nonbinding isomer, results in a "kinetic discrimination" mechanism that mediates selective binding, by a factor of >10(3), between highly related ligands that initially bind with the same affinity. This model may apply to proteins that bind multiple ligands in a specific manner or other proteins that, although capable of binding many ligands, are activated by only a few.

Anthracenes↗
PubMed1997

Assessment of total body water and lean body mass from anthropometry, Watson formula, creatinine kinetics, and body electrical impedance compared with antipyrine kinetics in peritoneal dialysis patients.

BACKGROUND: Indirect methods such as anthropometry (A), Watson formula (W), creatinine kinetics (CK), and body electrical impedance (BEI) are increasingly applied to determine total body water (TBW) and lean body mass (LBM) in dialysis patients. These methods share the disadvantage that they have been validated for healthy men only. We studied which of these four commonly applied methods can best be used routinely in CAPD patients. METHODS: TBW estimates obtained from A, W, CK, and BEI were compared with those obtained by a gold standard (antypirine distribution volume, ADV) in eight CAPD patients. In addition, several BEI equations to derive lean body mass (LBM) were compared with LBM estimated by ADV in order to determine which equation is the most valuable for the assessment of LBM by BEI in CAPD patients. RESULTS: TBW as ADV was 41.4 +/- 6.6 (mean +/- SD) L. TBW estimated by W, A and CK underestimated ADV by a mean +/- SD of 2.3 +/- 13, 5 +/- 8.4 and 12.3 +/- 10.9% respectively. TBW as measured by BEI overestimated ADV by 2.5 +/- 8.8%. The correlation coefficients between ADV-TBW and TBW estimated by the indirect methods were r = 0.88 (A), r = 0.87 (BEI), r = 0.82 (CK), and 0.68 (W). LBM estimated by ADV was 56.7 +/- 8.9 (mean +/- SD) kg; LBM by different BEI equations ranged from 49.9 +/- 7 to 58.1 +/- 10.7 kg. The correlation coefficient between LBM by ADV and LBM according to the various BEI equations ranged from 0.81 to 0.93. CONCLUSION: A and BEI can be used to estimate TBW, but the considerable SD (or inaccuracy) makes individual predictions hazardous. Considering the correlation coefficients and difference between LBM by ADV and LBM according to different BEI equations, Deurenberg's formula can be advocated for use in the estimation of LBM by BEI.

Adult↗
PubMed1986

Effect of intraperitoneal insulin on solute kinetics in CAPD: insulin kinetics in CAPD.

The authors evaluated the transport kinetics of insulin and inulin administered intraperitoneally to six diabetic patients undergoing continuous ambulatory peritoneal dialysis. The mass transfer coefficients (MTC) calculated from dialysate to blood for 1.5% and 4.25% dextrose dialysate were (ml/min): insulin 2.9 +/- 0.9, 2.0 +/- 0.5; inulin 3.3 +/- 1.4; 2.9 +/- 1.7, respectively. The MTC for inulin calculated from blood to dialysate was 2.0 +/- 0.7 ml/min. Because insulin disappears from the peritoneal cavity at a rate similar to inulin, it suggests that insulin transport can be defined by diffusion. The derived MTC values for glucose were not altered by the addition of intraperitoneal insulin. The derived MTCs for eight diabetic to thirteen nondiabetic patients were compared. The MTC derived for urea was less among the diabetics (16.6 +/- 2.2 vs. 24.6 +/- 2.6 p less than 0.05), but there were no differences for creatinine, uric acid, glucose, inulin, and protein. The derived values were found to be normally distributed and patients in the upper quartile for one solute were generally in the upper quartile for other solutes.

Ascitic Fluid↗
PubMed1981

Evidence for the presence of two kinetically distinct active forms of ribonuclease T2. The pH dependence of the steady-state kinetic parameter, kcat, for transphosphorylation of both a natural and a synthetic substrate.

A kinetic study has been made of the RNase-T2-catalyzed transphosphorylation of two adenine nucleotides, adenylyl(3'-5')uridine and adenosine 3'-(1-naphthyl)phosphate. Rates were measured at pH values ranging from 2.6 to 8.2. The observed shape of the plot of log kcat against pH for both the natural and the synthetic substrate suggests that there exist two parallel rate-determining pathways. Two pH-independent rate constants and three ionization constants of the enzyme-substrate complexes were obtained by nonlinear iterative least-squares analysis. Detailed interpretation of the pH profiles was carried out and it is proposed that carboxylate anion is likely to deprotonate O-2' at 4 less than pH less than 6, but at pH greater than 6 an alternative general base would play this role more effectively than the carboxylate group. Another base in its protonated cationic form is responsible for the general acid catalysis.

Binding Sites↗
PubMed1988

Temperature-dependence of the kinetics of the binding of [3H]-(+)-N-methyl-4-methyldiphenhydramine to the histamine H1-receptor: comparison with the kinetics of [3H]-mepyramine.

1. The dissociation of [3H]-(+)-N-methyl-4-methyldiphenhydramine ([3H]-QMDP) from the histamine H1-receptor was markedly temperature-dependent. The t1/2 was 4 min at 37 degrees C and 16 h at 6 degrees C. The association rate constant, k1, was also temperature-dependent, but not to the same extent as k-1. 2. Plots of the observed rate constant for [3H]-QMDP-receptor complex formation, kon, versus [3H-QMDP] were linear at both 30 degrees C and 10 degrees C, consistent with the interaction of [3H]-QMDP with the H1-receptor being a simple, one-step equilibrium. 3. The ratio of the kinetic constants, k1/k-1, indicated that the affinity constant of [3H]-QMDP for the H1-receptor should increase with decreasing temperature. Measurement of (+)-QMDP antagonism of the contraction of longitudinal muscle strips from guinea-pig small intestine induced by histamine at 37 degrees C, 30 degrees C and 25 degrees C provided some evidence that the affinity of (+)-QMDP is greater at 25 degrees C than 37 degrees C. However, the flattening of the concentration-response curves for histamine at low concentrations of (+)-QMDP at 30 degrees C and 25 degrees C is consistent with a slow dissociation of the (+)-QMDP-receptor complex and hence an incomplete equilibration with the agonist. 4. Arrhenius plots for k1 and k-1 for [3H]-QMDP were linear between 37 degrees C and 6 degrees C. The activation energies, Ea, for complex formation and dissociation were 77 +/- 4 and 129 +/- 3 kJ mol-1, respectively. 5. An Arrhenius plot for k-1 for the dissociation of [3H]-mepyramine from the H1-receptor was also linear between 37 degrees C and 6 degrees C. The activation energy was 140 +/- 2 kJ mol-1. 6. Activation energies for complex formation with the H1-receptor, Eaf, and complex dissociation, Ead, were similar for [3H]-QMDP and [3H]-mepyramine. The energy difference, Eaf--Ead, equivalent to the enthalpy change, did not differ significantly for the two ligands (-52 and -48 kJ mol-1, respectively). The larger values of k1 and k-1 for [3H]-mepyramine compared to [3H]-QMDP imply the presence of an entropic component in the interaction. 7. The simplest explanation for these observations is that transfer from the aqueous phase into a hydrophobic region is a significant factor in antagonist-H1-receptor interaction. This would be entropically more favourable for [3H]-mepyramine, a tertiary amine, than for [3H]-QMDP, a quaternary amine.

Aminopyridines↗