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Biomedical subjects

I D Clark

Publications and source records attributed to I D Clark.

17 recordsLinked to original sources
PubMed1996

Site-specific replacement of amino acid residues in the CD site of rat parvalbumin changes the metal specificity of this Ca2+/Mg(2+)-mixed site toward a Ca(2+)-specific site.

Rat parvalbumin (PV) and oncomodulin (OM) display considerable sequence similarity and structural similarity, but differ in the affinity and selectivity of metal binding to their CD site, a Ca2+/Mg(2+)-mixed site in PV and a Ca(2+)-specific site in OM. In an attempt to identify the structural basis for these differences, mutations were introduced in the previously generated [W102]PV mutant, which contains a unique tryptophan as a conformational-sensitive fluorescent probe inside the hydrophobic core. In the present report, we substituted selected amino acid residues in the CD site of PV by those present at identical positions in OM. One mutant protein, named [F66, W102]PV, has one new substitution in which isoleucine at position 66 was exchanged by phenylalanine. The second mutant protein, [I46, I50, L58, F66, W102]PV, has four new substitutions, namely V46-->I, L50-->I, I58-->L and I66-->F. Tryptophan fluorescence and difference spectrophotometry indicated that the mutations do not alter significantly the hydrophobic core. Both mutant proteins display two metal-binding sites of identical affinities with intrinsic affinity constants K'Ca2+ of 2.9 x 10(7) M-1 for [F66, W102]PV and 1.7 x 10(7) M-1 for [I46, I50, L58, F66, W102]PV and K'Mg2+ of 3.1 x 10(4) M-1 for [F66, W102]PV and 1.9 x 10(4) M-1 for [I46, I50, L58, F66, W102]PV. Thus, the five-residue substitution, but not the two-residue one, leads to a small decrease of affinity compared to [W102]PV (K'Ca2+ = 2.7 x 10(7) M-1, K'Mg2+ = 4.4 x 10(4) M-1). Despite these similarities, the Mg2+ effect on Ca2+ binding is different for the two mutant parvalbumins: the Ca(2+)-binding isotherms of [F66, W102]PV undergo a parallel shift upon increasing Mg2+ concentrations, which indicates that the Mg2+ effect on the two Ca(2+)-binding sites is the same and quantitatively very similar to that described for [W102]PV. In [I46, I50, L58, F66, W102]PV, Mg2+ antagonizes the binding of the second Ca2+ (likely at the EF site) much more than that of the first Ca2+ (likely the CD site). According to the competition equation, the two sites display KMg2+.compet values of 390 M-1 and 3.9 x 10(3) M-1, respectively. These data indicate that (a) the single I66-->F mutation does not modify the cation binding parameters. (b) Multiple modifications in the hydrophobic core still do not change the affinity for Ca2+ and Mg2+, but strongly affect the Mg2+ antagonism and probably the selectivity of the CD site.

Amino Acid Sequence↗
PubMed1995

A protease assay using time-resolved lanthanide luminescence from an engineered calcium binding protein substrate.

OBJECTIVES: The objective of this work was to demonstrate the utility of luminescence from lanthanides bound to a mutant of the Ca2+ binding protein, oncomodulin, to monitor protease activity. DESIGN AND METHODS: A mutant of oncomodulin with a cysteine residue at position 57 located in the CD binding loop was conjugated to a salicylic acid group. The luminescence of Tb3+ resulting from electronic energy transfer from the salicylic acid group was monitored using time resolved lanthanide luminescence in the presence of proteolytic enzymes. RESULTS: Low detection limits for subtilisin (150 pg), chymotrypsin (2.5 ng), cathepsin B (3.5 ng), and HIV-1 protease (25 ng) were found. CONCLUSION: The simplicity of the assay coupled with its high level of sensitivity make it useful for the detection of protease at very low concentrations.

Animals↗
PubMed1995

Bifunctional fusion proteins consisting of a single-chain antibody and an engineered lanthanide-binding protein.

The combination of an antibody fragment with a lanthanide chelating protein has desirable characteristics for fluorescence-based immunoassays and tumor radioimmunotherapy. As a model for this design, a fusion protein consisting of a single-chain antibody linked to an engineered version of oncomodulin, a protein with two Ca(2+)-binding motifs (the CD and EF loops), was produced by secretion from Escherichia coli in good yield. The single-chain antibody was specific for a Salmonella O-polysaccharide. The CD loop of oncomodulin had been redesigned to bind lanthanide ions with high affinity. The fusion protein was shown to have antigen-binding activity that was comparable to that of the unfused single-chain antibody, to bind Tb3+ with very high affinity and to give strong, sensitized Tb3+ luminescence via excitation of the tryptophan residue in the CD loop. A second fusion protein containing a 30-residue helix-loop-helix motif as the lanthanide-binding component was also prepared, but showed considerably lower solubility. Competition for Tb3+ binding by a series of metal chelators indicated that the affinities of the oncomodulin and 30 residue fusions for Tb3+ were approximately 10(11) M-1 and 10(7) M-1, respectively. Time-resolved lanthanide luminescence photography of electrophoresis gels demonstrated that the helix-loop-helix Ca(2+)-binding could be used to specifically visualize the scFv fragment.

Amino Acid Sequence↗
PubMed1994

Detection of calcium binding proteins on polyacrylamide gels using time-resolved lanthanide luminescence photography.

Methods were developed for using the luminescent lanthanides Tb3+ and Eu3+ for the specific staining of calcium-binding proteins, as well as the nonspecific staining of proteins, on polyacrylamide gels. These methods involve equilibration of the gel after electrophoresis in solutions containing the appropriate lanthanide and a weak competitive chelating agent, such as N-(2-hydroxyethyl)iminodiacetic acid or nitrilotriacetic acid. This staining has the potential for complete reversibility using stronger chelating agents such as EDTA or diethylenetriaminepentaacetic acid, to allow for recovery of the protein. Specific staining produces an intense luminescent signal from those metal-binding proteins which have been modified either chemically or via site-directed mutagenesis. Gels were photographed using a time-resolved fluorescence camera system.

Acrylic Resins↗
PubMed1994

The prevalence of stroke in those at home and the need for care.

This paper highlights the severity of disability, social context and care needs of stroke survivors living outside of institutional care. It provides an estimate of the prevalence of stroke in the community, and offers an operational rationale to purchasers of community care services.

Activities of Daily Living↗
PubMed1993

A novel peptide designed for sensitization of terbium (III) luminescence.

Several synthetic peptides, modelled from a Ca(2+)-binding loop of the EF-hand family of proteins, were prepared containing cysteine residues. The peptide, GDKNADGFICFEEL, was labelled covalently at the cysteine residue (loop position 9) with iodoacetamidosalicylic acid. This novel conjugate is a metal-binding loop containing a salicylic acid side chain that could not only chelate Tb3+ in conjunction with the other chelating groups in the sequence, but could also sensitize Tb3+ luminescence. The loop had a high Tb3+ affinity, with stoichiometric binding observed under experimental conditions. The luminescence from the Tb(3+)-peptide complex was more than 10-fold greater than the luminescence reported from a related peptide which contained Trp as the Tb3+ donor at loop position 7. This peptide has significant potential for use in lanthanide-based time-resolved luminescence immunoassays.

Amino Acid Sequence↗
PubMed1993

Metal binding properties of recombinant rat parvalbumin wild-type and F102W mutant.

Rat parvalbumin (PV), an EF-hand type Ca(2+)-binding protein, was expressed in Escherichia coli and mutated by replacing a Phe at position 102 with a unique Trp in order to introduce a distinct fluorescent label into the protein. Mass spectroscopy and NMR data indicate that the recombinant wild-type (PVWT) and F102W mutant (PVF102W) proteins have the expected molecular weight and retain the native structure. Both proteins contain two non-cooperative Ca2+/Mg(2+)-binding sites with intrinsic affinity constants, KCa and KMg, of 2.4 +/- 0.9 x 10(7) M-1 and of 2.9 +/- 0.2 x 10(4) M-1, respectively, for PVWT, and KCa and KMg, of 2.7 +/- 1.1 x 10(7) M-1 and of 4.4 +/- 0.3 x 10(4) M-1, respectively, for PVF102W. Based on the highly similar metal binding properties of PVWT and PVF102W the latter protein was used to study cation-dependent conformational changes. Trp fluorescence emission and UV difference spectra of PVF102W indicated that the Trp residue at position 102 is confined to a hydrophobic core and conformationally strongly restricted. Upon Ca2+ or Mg2+ binding the structural organization of the region around the Trp is hardly affected, but there are significant changes in its electrostatic environment. The conformational change upon binding of Ca2+ and Mg2+, as monitored by UV difference spectrophotometry, increases linearly from 0 to 2 cations bound, indicating that the binding of both ions contributes equally to the structural organization in this protein.

Animals↗
PubMed1993

A study of sensitized lanthanide luminescence in an engineered calcium-binding protein.

In this study, the CD loop of the Ca(2+)-binding protein oncomodulin was replaced by a high-affinity, metal-binding sequence that was found to reverse the order of fill of the two sites in the protein. A cysteine was included at position 7 of this sequence, i.e., DKNADGCIEFEE. The cysteine allowed covalent attachment of chromophores to the loop that could subsequently be tested for their ability to sensitize the luminescence of Tb3+ or Eu3+ bound in the loop. 7-Diethylamino-3-((4'-iodoacetylamino)phenyl)-4-methylcoumarin was the most efficient Eu3+ sensitizer studied, consistent with a mechanism of energy transfer that involves the triplet state of the donor. 4-Iodoacetamidosalicylic acid was the most efficient Tb3+ donor tested. Levels of lanthanide ion and labeled C3 as low as 5 x 10(-10) mol/liter could be detected. This protein chelator system has potential to be a useful, flexible complement to the organic chelators currently used in lanthanide-based, time-resolved luminescence immunoassays.

Amino Acid Sequence↗
PubMed1993

Self-association of Ca(2+)-binding peptides induced by lanthanide ions: a fluorescence study.

In this study a Ca(2+)-binding 14mer peptide was synthesized with the sequence GDKNADGCIEFEEL, allowing covalent attachment of sulfhydryl-reactive fluorescent molecules at position 7 of the 12-residue, metal-binding loop (underlined). This provided the opportunity to select donor molecules with suitable spectral characteristics for sensitized excitation of chelated terbium (Tb3+) or europium (Eu3+) ions. N-(1-Pyrene)-iodoacetamide and 7-diethylamino-3-((4'-iodoacetylamino)phenyl)-4-methylcoumarin were attached to the peptide and titrations carried out with terbium or europium stock solutions. It was possible to observe lanthanide ion binding to the loop in stoichiometric quantities, but maximal lanthanide luminescence was achieved with a large excess of lanthanide present, due to metal-induced peptide association. Obtaining maximal lanthanide luminescence is important in the development of systems for use in sensitive clinical diagnostic and time-resolved luminescence-based immunoassay applications.

Amino Acid Sequence↗
PubMed1991

Hybridization of rabbit cardiac tropomyosin with nonpolymerizable tropomyosin species.

Rabbit cardiac tropomyosin was hybridized with its nonpolymerizable form, produced by treatment with carboxypeptidase A, and with a naturally occurring nonpolymerizable tropomyosin from horse platelets. Hybridization was achieved by heating equimolar mixtures to 60 degrees C in the presence of 10 mM dithiothreitol, followed by recooling. Samples of intact and carboxypeptidase-truncated tropomyosins treated in this way show lower viscosities at low ionic strength than predicted assuming random reformation of the coiled coils, suggesting that hybrids formed with one intact COOH-terminus are unable to polymerize normally. Hybridization of cardiac and platelet tropomyosins was detected by observation of the fluorescence of pyrene groups attached to cysteine residues on platelet tropomyosin.

Animals↗
PubMed1990

Coupled responses of the regions near cysteine-190 and the carboxy terminus of rabbit cardiac tropomyosin: fluorescence and circular dichroism studies.

Rabbit cardiac tropomyosin was labeled at Cys-190 with either N-(1-pyrenyl)iodoacetamide (Py) or 6-acryloyl-2-(dimethylamino)naphthalene (AD, acrylodan). Half of the labeled sample then was treated with carboxypeptidase A to produce an identically labeled nonpolymerizable form of tropomyosin, NPTM. Investigation of temperature-dependent changes in pyrene excimer emission, acrylodan fluorescence polarization, and tyrosyl circular dichroism in different samples of tropomyosin and NPTM reveals that absence of the COOH-terminal portion of tropomyosin modifies the response of the Cys-190 region to heat. Removal of the COOH terminus releases certain conformational constraints from the coiled-coil back to and including the Cys-190 region without causing a severe drop in the net alpha-helical content of the protein. Observation of changes in pyrene excimer fluorescence and in fluorescence polarization of acrylodan with time after addition of carboxypeptidase A to samples of labeled tropomyosin directly demonstrates this relaxation process. Thermally induced reduction in tyrosyl circular dichroism, together with consideration of the distribution of tyrosyl residues on tropomyosin, also supports the proposal.

Animals↗
PubMed1989

Interaction of muscle and non-muscle tropomyosins with deoxyribonuclease I.

The sulfhydryl-selective fluorescent reagent acrylodan (6-acryloyl-2-dimethylaminonaphthalene) was used to label tropomyosins from rabbit cardiac muscle and from equine platelets. Addition of bovine pancreatic deoxyribonuclease I to solutions of acrylodan-modified tropomyosins significantly altered the emission properties of the samples. Muscle and non-muscle tropomyosin fluorescence were affected in qualitatively similar manners; emission maxima were red-shifted by about 8 nm to 522-525 nm and maximal intensities were reduced by approximately 15%. Addition of KI to each of the fluorescent samples caused a greater degree of fluorescence quenching in the presence of DNase I than in its absence. The slopes of Stern-Volmer plots were 15-25% steeper in the presence of DNase I. Fluorescence polarization values for acrylodan-labelled tropomyosin samples were 25-35% lower in the presence of DNase I. Each of these effects could be saturated by addition of about a two-fold molar excess of DNase I to tropomyosin. Together they suggest that interaction with DNase I causes localized unfolding of tropomyosin, thereby allowing the fluorescent label to become more exposed to the solvent and less restricted in its local motions. Circular dichroism measurements support this idea. Addition of DNase I to solutions of either labelled or unlabelled tropomyosin results in a net 14-18% loss in ellipticity near 220 nm, indicative of unfolding of alpha-helix.

Animals↗
PubMed1988

Fluorescence of equine platelet tropomyosin labeled with acrylodan.

Equine platelet tropomyosin was labeled with the sulfhydryl-specific fluorescent reagent 6-acryloyl-2-dimethylaminonaphthalene (acrylodan). The extent of labeling at 4 degrees C could be regulated between 0.5 and 1.3 acrylodans per tropomyosin chain by varying the reaction time from 1 to 4.5 h. Acrylodan-labeled platelet tropomyosin, AD-P-TM, was highly fluorescent, having an emission maximum near 518 nm on excitation at 365 nm. Steady-state measurements of polarization of the fluorescence of AD-P-TM in both low and high ionic strength solutions gave Perrin plots that exhibited sharp changes in slope near 50 degrees C, indicative of a sharp increase in mobility of the label at that temperature. This correlates with the melting temperature of the platelet tropomyosin coiled coil observed by circular dichroism [G. P. Côté, W. G. Lewis, M. D. Pato, and L. B. Smillie, (1978) FEBS Lett. 91, 237-241]. Perrin plots of carboxypeptidase A-treated platelet tropomyosin that was labeled with acrylodan after digestion resembled more closely those of acrylodan-labeled cardiac tropomyosin rather than those of AD-P-TM, suggesting that the observed emission arose from label at Cys-153 on each truncated platelet tropomyosin chain. In solutions containing 150 mM KCl and 5 mM MgCl2, addition of actin at up to a sixfold molar excess over AD-P-TM caused both the fluorescence emission intensities and fluorescence polarization values of samples to increase. In the presence of actin, the wavelength of maximal emission was shifted to shorter values by about 5 to 7 nm. These changes indicate that actin does bind to AD-P-TM and that the binding affects the environment of the label, both by making it more hydrophobic and by reducing the freedom of the label to tumble in solution.

2-Naphthylamine↗
PubMed1986

Excimer fluorescence of equine platelet tropomyosin labeled with N-(1-pyrenyl)iodoacetamide.

Tropomyosin from equine platelets was reacted with N-(1-pyrenyl)iodoacetamide, a sulfhydryl-specific fluorescent reagent, to give an average extent of incorporation of 1.12 pyrene (Py) groups per platelet tropomyosin (P-TM) chain. The predominant site of reaction on P-TM was the penultimate COOH-terminal residue, Cys-246. The high proportion of the total emission that is due to pyrene ecximers and the pretransition observed in thermal denaturation of Py-P-TM point to a rather loose structure for the COOH-terminal amino acid residues of P-TM. The label on Cys-246 also reports on end-to-end overlap interactions that occur between two different tropomyosin molecules. Additions to a Py-P-TM solution at low ionic strength of unlabeled P-TM, rabbit cardiac tropomyosin (C-TM), or a carboxypeptidase A treated, nonpolymerizable derivative of C-TM all reduce the extent of excimer fluorescence from the sample. Addition of salt greatly reduces the effects of the unlabeled TM species on the Py-P-TM emission spectrum. Circular dichroism measurements indicate Py-P-TM still to be greater than 95% helical. However, analysis of excimer fluorescence levels in samples that contained a constant protein concentration but different mole ratios of labeled to unlabeled P-TM suggests that the bulky pyrene group may diminish the tendency of Py-P-TM to polymerize in an end-to-end manner.

Animals↗
PubMedDate not supplied

Lithium isotope geochemistry and origin of Canadian shield brines.

Hypersaline calcium/chloride shield brines are ubiquitous in Canada and areas of northern Europe. The major questions relating to these fluids are the origin of the solutes and the concentration mechanism that led to their extreme salinity. Many chemical and isotopic tracers are used to solve these questions. For example, lithium isotope systematics have been used recently to support a marine origin for the Yellowknife shield brine (Northwest Territories). While having important chemical similarities to the Yellowknife brine, shield brines from the Sudbury/Elliot Lake (Ontario) and Thompson/Snow Lake (Manitoba) regions, which are the focus of this study, exhibit contrasting lithium behavior. Brine from the Sudbury Victor mine has lithium concentrations that closely follow the sea water lithium-bromine concentration trajectory, as well as delta6Li values of approximately -28/1000. This indicates that the lithium in this brine is predominantly marine in origin with a relatively minor component of crustal lithium leached from the host rocks. In contrast, the Thompson/Snow Lake brine has anomalously low lithium concentrations, indicating that it has largely been removed from solution by alteration minerals. Furthermore, brine and nonbrine mine waters at the Thompson mine have large delta6Li variations of approximately 30/1000, which primarily reflects mixing between deep brine with delta6Li of -35 +/- 2/1000 and near surface mine water that has derived higher delta6Li values through interactions with their host rocks. The contrary behavior of lithium in these two brines shows that, in systems where it has behaved conservatively, lithium isotopes can distinguish brines derived from marine sources.

Calcium↗