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Biomedical subjects

I M Henderson

Publications and source records attributed to I M Henderson.

11 recordsLinked to original sources
PubMed2001

Baculoviral expression and characterization of rodent cathepsin S.

The cysteinyl proteinase cathepsin S is implicated as a key enzyme in the processing of major histocompatability complex (MHC) class II molecules expressed on antigen presenting cells and thus is a potential therapeutic target for modulation in immune system-based disease. We have identified a form of rat cathepsin S, similar to a published mouse form with an eight-amino acid extended presequence relative to the human enzyme and the previously published rat enzyme. In addition, we have expressed these mouse and rat proteins in baculovirally infected Sf9 insect cells along with "humanized" forms truncated by eight residues at the amino-terminus. All forms of the rodent proteinases were overexpressed and milligram per litre amounts of functional enzyme could be isolated from the cells and/or the cell culture supernatant. Furthermore, addition of a carboxy-terminal hexahistidine purification tag had no effect on the kinetic characteristics of any of the enzyme forms against the Boc-Val-Leu-Lys-AMC peptide substrate (rat k(cat) s(-1) approximately 30; mouse k(cat) s(-1) approximately 65). Differences were seen in the potency of the generic cysteine proteinase inhibitor, E64, against the human, mouse, or rat form of the enzyme (13.3 x 10(4), 43.2 x 10(4), and 25 x 10(4) K(obe)/[I] M(-1) s(-1), respectively). Such data highlights the need for greater awareness of species variation in inhibitor potency. These reagents are vital for confirming inhibitor potency against the endogenous form of the enzyme prior to evaluation of drug candidates in rodent model systems.

Animals↗
PubMed2000

The kinetic and structural characterization of the reaction of nafamostat with bovine pancreatic trypsin.

Nafamostat mesilate (FUT-175), a synthetic serine protease inhibitor, is active against a number of the serine proteases involved in coagulation. This has been proposed as the basis of its anticoagulant activity. We investigated the reaction of Nafamostat with bovine pancreatic trypsin as a model system. It was shown to act as a time-dependent competitive inhibitor, and the inhibition constants for the binding of Nafamostat to trypsin (i.e., Ki) and the overall inhibition constants (i.e., Ki*) were calculated to be 11.5 microM and 0.4+/-0.14 nM, respectively. The second-order rate constant for the reaction was 4.5+/-0.19x10(5) M(-1)s(-1), and the product released following the acylation step, 6-amidino2-naphthol, showed mixed-type inhibition. The competitive (Kic) and uncompetitive (Kiu) inhibition constants were 14.7 microM and 19.5 microM, respectively. Formation of the acyl-enzyme intermediate was dissected into at least two steps, with rates of 0.9 s(-1) and 195 s(-1). The deacylation step was relatively much slower (3.2+/-0.19x10(-5) s(-1), enabling the mass spectroscopic analysis of the acyl-enzyme intermediate, which confirmed the covalent attachment of 4-guanidinobenzoic acid to trypsin. The product of the deacylation step, 4-guanidinobenzoic acid, showed no inhibition up to a concentration of 200 microM. These data strongly suggest that while Nafamostat is a potent inhibitor of trypsin, it is actually an extremely poor substrate, and that apparent inhibition is due to the competitive formation of a very stable acyl-enzyme intermediate, analogous to some other active site titrants.

Animals↗
PubMed1998

Omasal anatomy in New Zealand red and fallow deer: an exploratory multivariate analysis.

We measured the weight and volume of omasae, and the number, weight and area of omasal laminae from wild fallow (Cervus dama), and wild and farmed red deer (Cervus elaphus) whose diet, as indicated by rumen content, was known. A multivariate analysis yielded two principal components, interpreted as indices of overall omasal size and laminar leafiness. The second component showed significantly greater variance in wild red deer than in farmed red or wild fallow deer. Deer feeding on a mixed diet of browse and grasses may have greater variability of omasal from than those feeding on grass alone. A relationship is derived by which the laminar area can be estimated from counts of laminae in different size classes.

Animals↗
PubMed1997

Pre-steady-state kinetic analysis of 2-hydroxy-6-keto-nona-2,4-diene-1,9-dioic acid 5,6-hydrolase: kinetic evidence for enol/keto tautomerization.

The reaction catalyzed by 2-hydroxy-6-keto-nona-2,4-diene-1,9-dioic acid 5,6-hydrolase (MhpC) was analyzed by stopped-flow UV-visible kinetics at 317 nm (substrate depletion) and 270 nm (product formation) at pH 5.0 and 4.0. Comparison of the rates and amplitudes of product formation versus substrate depletion provided evidence for the formation of a discrete keto-intermediate, as predicted from previous isotope exchange experiments [Lam, W. W. Y., & Bugg, T. D. H. (1997) Biochemistry, 36, 12242-12251]. Accurate modeling of the concentration data could only be achieved using a branched kinetic mechanism in which the intermediate is released at a rate comparable to its catalytic turnover, consistent with the earlier isotope exchange data. The apparent "leakiness" of the active site and relatively weak substrate binding (Kd = 30 microM) are consistent with a mechanism in which the enzyme binds the dienol substrate in a strained, nonplanar conformation which promotes ketonization in the C-5 position to give a keto-intermediate.

Catalysis↗
PubMed1997

The effect of formalin on rumen surface area in red deer.

Previously published studies have related the surface area of rumen wall to diet. The validity of studies that utilize preserved material depends upon the predictability of any change in rumen dimension brought about by preservation. Changes in the surface are of different sections of the rumen wall of red deer (Cervus elaphus) were monitored after immersion in 10% formal saline solution at room temperature for 2, 7 and 14 days. There was a high degree of variability (0-39.5%) in wall-area reduction. Ignoring such changes when calculating the factor of increase in surface area (FISA), a composite of papillary surface area and density, can result in errors of up to 39.5%. This study questions the validity of using FISA calculations when formalin-preserved specimens are used in studies of rumen response to diet. Rumen papillary dimensions were not significantly changed by preservation in formalin.

Animals↗
PubMed1994

Binding of Ca2+ to the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum: equilibrium studies.

Equilibrium fluorescence methods have been used to establish a model for Ca2+ binding to the (Ca(2+)-Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum and to define the effects of H+ and Mg2+ on Ca2+ binding. The basic scheme proposed is: E2 <--> E1 <--> E1Ca <--> El'Ca <--> E1'Ca2. The E1 conformation of the ATPase initially has one high-affinity binding site for Ca2+ exposed to the cytoplasmic side of the sarcoplasmic reticulum, but in the E2 conformation this site is unable to bind Ca2+; Ca2+ does not bind to luminal sites on E2. The second, outer, Ca(2+)-binding site on the ATPase is formed after binding of Ca2+ to the first, inner, site on E1 and the E1Ca <--> E1'Ca conformation change. The pH- and Mg(2+)-dependence of the E2 <--> E1 equilibrium has been established after changes in the fluorescence of the ATPase labelled with 4-nitrobenzo-2-oxa-1,3-diazole. It is proposed that Mg2+ from the cytoplasmic side of the sarcoplasmic reticulum can bind to the first Ca(2+)-binding site on both E1 and E2. It is proposed that the change in tryptophan fluorescence intensity after binding of Ca2+ follows from the E1Ca <--> E1'Ca change. The pH- and Mg(2+)-dependence of this change defines H(+)- and Mg(2+)-binding constants at the two Ca(2+)-binding sites. It is proposed that the change in tryptophan fluorescence observed on binding Mg2+ follows from binding at the second Ca(2+)-binding site. Effects of pH and Mg2+ on the fluorescence of the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin are proposed to follow from binding to a site on the ATPase, the 'gating' site, which affects the affinity of the first Ca(2+)-binding site for Ca2+ and affects the rate of dissociation of Ca2+ from the ATPase.

Animals↗
PubMed1994

Binding of Ca2+ to the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum: kinetic studies.

Stop-flow fluorescence and rapid-filtration methods have been used to establish the kinetics of Ca2+ binding to, and dissociation from, the (Ca(2+)-Mg2+)-ATPase of skeletal-muscle sarcoplasmic reticulum and to define the effects of H+ and Mg2+ on Ca2+ binding and dissociation rates. The kinetics have been interpreted in terms of the scheme: E2 E2<==>E1<==>E1Ca<==>E1'Ca<==>E1'Ca2. The kinetics of the E2<==>E1 E1 transition have been determined by measuring the rate of change of the fluorescence of the ATPase labelled with 4-nitrobenzo-2-oxa-1,3-diazole after a pH jump or the addition of Ca2+ to the labelled ATPase in the presence of thapsigargin or thapsivillosin A. It has been shown that Mg2+ has a marked effect on Ca2+ dissociation at pH 7.2 and that changes in the tryptophan fluorescence of the ATPase follow the same time course as the dissociation of 45Ca2+. It is proposed that the effect of Mg2+ follows from binding to a 'gating' site, as detected by changes in the fluorescence of the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin. The rate of dissociation of Ca2+ from the ATPase increases with increasing pH. The rate of dissociation of Ca2+ decreases with increasing Ca2+ concentration in the medium, with an apparent affinity for Ca2+ greater than that seen for the change in fluorescence amplitude. It is shown that this follows if the first, inner, Ca(2+)-binding site on the ATPase has a lower affinity for Ca2+ than the second, outer, site. Effects of H+ and Mg2+ on Ca2+ dissociation can be treated by the quasiequilibrium approach. Mg2+ and H+ also affect the rate of Ca2+ binding to the ATPase, and effects of H+ and Mg2+ on the E2<==>E1 equilibrium explain the results of experiments in which the concentrations of H+ and Mg2+ are jumped.

Animals↗
PubMed1994

An unequivocal example of cysteine proteinase activity affected by multiple electrostatic interactions.

The role of electrostatic interactions between the ionizable Asp158 and the active site thiolate-imidazolium ion pair of some cysteine proteinases has been the subject of controversy for some time. This study reports the expression of wild type procaricain and Asp158Glu, Asp158Asn and Asp158Ala mutants from Escherichia coli. Purification of autocatalytically matured enzymes yielded sufficient fully active material for pH (kcat/Km) profiles to be obtained. Use of both uncharged and charged substrates allowed the effects of different reactive enzyme species to be separated from the complications of electrostatic effects between enzyme and substrate. At least three ionizations are detectable in the acid limb of wild type caricain and the Glu and Asn mutants. Only two pKa values, however, are detectable in the acid limb using the Ala mutant. Comparison of pH activity profiles shows that whilst an ionizable residue at position 158 is not essential for the formation of the thiolate-imidazolium ion pair, it does form a substantial part of the electrostatic field responsible for increased catalytic competence. Changing the position of this ionizable group in any way reduces activity. Complete removal of the charged group reduces catalytic competence even further. This work indicates that hydronations distant to the active site are contributing to the electrostatic effects leading to multiple active ionization states of the enzyme.

Amino Acid Sequence↗
PubMed1989

Influenza viruses, comets and the science of evolutionary trees.

The study of phylogeny is becoming increasing scientific in that hypotheses can be tested quantitatively. We report a method of estimating the probabilities of obtaining a tree of a given length from nucleic acid sequence data. The method is applied to the hypothesis of Hoyle & Wickramasinghe that the earth is being continually bombarded by influenza (and other) viruses which originate from comets. A quantitative analysis of sequences from the H1 strain of human influenza viruses contradicts three versions of the Hoyle-Wickramasinghe model. One non-evolutionary version of their model has less than one chance in 10(66) of being correct. A version that allowed extraterrestrial evolution has less than one change in 10(6) of being correct. The sequence data is in agreement with the biological (evolutionary) model. The results are discussed from the aspect of the falsifiability of evolutionary theory.

Base Sequence↗