PubMed Health⌕ Search

Biomedical subjects

M P Jackman

Publications and source records attributed to M P Jackman.

6 recordsLinked to original sources
PubMed1993

Evidence for common structural changes in thrombin induced by active-site or exosite binding.

The gamma-loop of thrombin is a flexible, surface-accessible loop in free thrombin that appears to be one of several sites participating in the interaction of the enzyme with macromolecular substrates and inhibitors. Using limited proteolysis and intrinsic fluorescence measurements, we have studied changes in thrombin structure induced by small, site-specific ligands. Binding of a C-terminal peptide of hirudin to the anion-binding exosite of thrombin induced a structural change in the gamma-loop, which caused a 6-fold reduction in the susceptibility of the enzyme to limited proteolysis by elastase and chymotrypsin. Binding of several active site-specific thrombin inhibitors conferred an even greater protection from proteolysis at the gamma-loop. For example, the covalent complex of thrombin with D-Phe-Pro-Arg-CH2Cl was 95-fold less susceptible to cleavage by chymotrypsin than the free enzyme. Furthermore, binding of either exosite or active-site probes induced a common intrinsic fluorescence change in thrombin (a fractional increase of 0.13). These results are surprising because crystallographic studies indicate that direct contact between the bound probes and relevant residues of the gamma-loop is very unlikely. Thus we have identified an allosteric interaction that couples the active site of thrombin to the gamma-loop. An interaction of this nature may be one way in which thrombomodulin modulates the reactivity of thrombin.

Amino Acid Sequence↗
PubMed1992

Intrinsic fluorescence changes and rapid kinetics of the reaction of thrombin with hirudin.

Stopped-flow fluorescence spectroscopy has been used to study the reaction of human alpha-thrombin with recombinant hirudin variant 1 (rhir) at 37 degrees C and an ionic strength of 0.125 M. A 35% enhancement in intrinsic fluorescence accompanied formation of the thrombin-rhir complex. Over one third of this enhancement corresponded to a structural change that could be induced by binding of either the NH2-terminal fragment (residues 1-51) or the COOH-terminal fragment (residues 52-65) of rhir. Three kinetic steps were detected for reaction of thrombin with rhir. At high rhir concentrations (greater than or equal to 3 microM), two intramolecular steps with observed rate constants of 296 +/- 5 s-1 and 50 +/- 1 s-1 were observed. By using the COOH-terminal fragment of rhir as a competitive inhibitor, it was possible to obtain an estimate of 2.9 x 10(8) M-1 s-1 for the effective association rate constant at low rhir concentrations. At higher ionic strengths, this rate constant was lower, which is consistent with the formation of the initial complex involving an ionic interaction. The mechanism for the reaction of both the COOH- and NH2-terminal fragments of rhir appeared to involve two steps. When thrombin was reacted with the COOH-terminal fragment at high concentrations (greater than or equal to 6 microM), the bimolecular step occurred within the dead time of the spectrometer and only one intramolecular step, with a rate constant of 308 +/- 5 s-1 was observed. At concentrations of NH2-terminal fragment below 50 microM, its binding to thrombin appeared to be a bimolecular reaction with an association rate constant of 8.3 x 10(5) M-1 s-1. In the presence of saturating concentrations of the COOH-terminal fragment, a 1.7-fold increase in this rate constant was observed. At concentrations of NH2-terminal fragment greater than 50 microM, biphasic reaction traces were observed which suggests a two-step mechanism. By comparing the reaction amplitudes and dissociation constants observed with rhir and its COOH-terminal fragment, it was possible to obtain approximate estimates for the values of the rate constants of different steps in the formation of the rhir-thrombin complex.

Genetic Variation↗
PubMed1992

Stabilization of the oxy form of tyrosinase by a single conservative amino acid substitution.

Asp-208 of Streptomyces glaucescens tyrosinase (an invariant residue in the CuB-binding region of tyrosinases and haemocyanins) was conservatively substituted by glutamic acid. Although having little effect on spectroscopic or kinetic properties of the enzyme, the mutation greatly decreased the lability of Cu-bound O2. A rationalization for these results is given, based on the crystal structure of Panuliris interruptus haemocyanin in the conserved CuB-binding region.

Amino Acid Sequence↗
PubMed1991

Albino mutants of Streptomyces glaucescens tyrosinase.

Site-directed mutagenesis was used to determine the functional role of several residues of Streptomyces glaucescens tyrosinase. Replacement of His-37, -53, -193 or -215 by glutamine yields albino phenotypes, as determined by expression on melanin-indicator plates. The purified mutant proteins display no detectable oxy-enzyme and increased Cu lability at the binuclear active site. The carbonyl derivatives of H189Q and H193Q luminesce, with lambda max. displaced more than 25 nm to a longer wavelength compared with native tyrosinase. The remaining histidine mutants display no detectable luminescence. The results are consistent with these histidine residues (together with His-62 and His-189 reported earlier) acting as Cu ligands in the Streptomyces glaucescens enzyme. Conservative substitution of the invariant Asn-190 by glutamine also gives an albino phenotype, no detectable oxy-enzyme and labilization of active-site Cu. The luminescence spectrum of carbonyl-N190Q, however, closely resembles that of the native enzyme under conditions promoting double Cu occupancy of the catalytic site. A critical role for Asn-190 in active-site hydrogen-bonding interactions is proposed.

Albinism↗
PubMed1978

Crown construction to the lingual margin of a partial denture.

A technique is described which ensures that the interface between a newly constructed crown and an existing partial denture is accurate and neither crown nor denture need adjustment. At no time does the patient have to be without the denture. A silver plated die with a double dowelling technique is used.

Calcium Sulfate↗