Biomedical subjects

C J Wallace

Publications and source records attributed to C J Wallace.

At least 91 records · Page 5Linked to original sources

PubMed1991

Enhancing protein engineering capabilities by combining mutagenesis and semisynthesis.

If site-directed mutagenesis could be used to facilitate protein semisynthesis, then structural engineering goals should be achieved that are unattainable by either technique alone. We tested this possibility by mutating Ser65 of yeast cytochrome c to methionine, creating a new site for CNBr cleavage. Fragments obtained by cleaving there were found to refold cooperatively, bringing together the breakpoint termini and leading to efficient autocatalytic peptide bond synthesis. Structurally modified fragments may be substituted for natural ones. Generally, naturally occurring sites are unsuitable for autocatalytic religation, for reasons briefly discussed, and thus the power of this new approach lies in the freedom to choose sites, including enzymatic ones, that are appropriate to the semisynthetic goals.

Amino Acid Sequence↗

PubMed1991

The specificity and Kd at physiological ionic strength of an ATP-binding site on cytochrome c suit it to a regulatory role.

Cytochrome c binds ATP with marked specificity at a site that contains the evolutionarily invariant residue Arg-91. The binding of ATP to this site was studied using equilibrium gel filtration, equilibrium dialysis and affinity chromatography. At physiological ionic strength the affinity is such that the major change in occupancy coincides with the normal cellular ATP concentration range, and the degree of saturation is proportional to the ratio of [ATP]/[ADP]. The specificity of binding at this site is more a function of the degree of phosphorylation of the nucleotide, than of the nature of the nucleoside moiety. Thus under physiological conditions the degree of occupancy of this site is proportional to the energy state of the cell, providing a means for the regulation of the respiratory chain which is sensitive to cytoplasmic ATP levels.

Adenosine Triphosphate↗

PubMed1991

Quantitative imaging of osteoarthritis.

Plain-film radiography currently remains the mainstay of imaging for diagnosis and follow-up in osteoarthritis (OA). However, recent studies have questioned some aspects of its use, particularly the correlation between radiographically evident joint-space narrowing and articular cartilage loss. The results with imaging modalities such as magnetic resonance imaging and ultrasound suggest that these methods will allow accurate noninvasive definition of the structure of articular cartilage and other soft tissues of joints. Other modalities, including microfocal radiography and high-resolution computed tomography, can produce detailed images of trabecular structure and bony alterations in osteoarthritis. Improvements in image analysis and data manipulation, including three-dimensional reconstruction and digitized storage and measurement of images, will likely enable improved quantitative assessment of the abnormalities demonstrated by these techniques. One can hope that such developments will facilitate not only improved detection and definition of OA, but also better evaluation of the effectiveness of pharmacological and physical therapy in practice and in clinical trials.

Cartilage, Articular↗

PubMed1991

Magnetic resonance imaging of the brain.

Magnetic resonance imaging (MRI) of the brain has become essential to a neurologic work-up. In Canadian centres where access to MRI is limited, use of this technique is usually restricted to assessment of disorders for which it is likely to provide unique information after computed tomography (CT); examples include abnormalities of the temporal lobes and posterior fossa, some diseases that show subtle or no abnormalities on CT (many inflammatory and demyelinating disorders) and cases in which detailed anatomic assessment is necessary (vascular malformation, congenital anomalies and many tumours). Although MRI of the brain has become quite standardized, recent developments such as improved gradient-echo imaging, new coil designs, magnetic resonance angiography and the use of contrast agents in MRI have resulted in improved speed and utility.

Brain↗

PubMed1990

Clinical evaluation of computer-based respiratory care algorithms.

A collection of computer-based respiratory care algorithms were implemented as a prototype computer-based patient advice system (COMPAS) within the existing HELP hospital information system. Detailed medical logic recommended ventilator adjustments for 5 different modes of ventilation: assist/control (A/C), intermittent mandatory ventilation (IMV), continuous positive airway pressure (CPAP), pressure controlled inverted ratio ventilation (PC-IRV), and extracorporeal carbon dioxide removal (ECCO2R). Suggestions for adjusting the mode of ventilation, fraction of inspired oxygen (FiO2), positive end-expiratory pressure (PEEP), peak inspiratory pressure, and several other therapeutic measures related to the treatment of severe arterial hypoxemia in adult respiratory distress syndrome (ARDS) patients were automatically presented to the clinical staff via bedside computer terminals. COMPAS was clinically evaluated for 624 hours of patient care on the first 5 ARDS patients in a randomized clinical trial. The clinical staff carried out 84% (320/379) of the computerized therapy suggestions. In response to a questionnaire distributed to clinical users of the system, 86% judged the system to be potentially valuable. Through implementation of COMPAS, a computer-based ventilatory therapy advice system, we have laid the groundwork for standardization of ventilator management of arterial hypoxemia in critically ill ARDS patients.

Algorithms↗

PubMed1990

Assessment of interpersonal problem-solving skills.

A new instrument for assessing social skills of schizophrenic patients, Assessment of Interpersonal Problem-Solving Skills (AIPSS), is a videotaped-based test with an examiner's administration and scoring manual. The test measures an examinee's ability to describe an interpersonal social problem, to derive a solution to the problem, and to enact a solution in a role-played simulation test. In a study using a sample of schizophrenic outpatients and a comparison group of nonpatients, we found that the test had adequate psychometric properties, and the patients demonstrated deficits on all scales relative to the nonpatients. The results of the study also provided partial support for the validity of an information-processing model of social skills, which was used as a basis for constructing the AIPSS. Thus, the AIPSS represents a departure from previous methods of assessing social skills.

Adult↗

PubMed1990

Effectiveness of the kinetic treatment table for preventing and treating pulmonary complications in severely head-injured patients.

The efficacy of using the Kinetic Treatment Table (KTT) to prevent or reduce pulmonary complications in severely head-injured patients is unclear. This study is a prospective, randomized trial using the KTT vs. conventional bed care in severely head-injured patients. Outcome measures were hospital length of stay (LOS), mortality, CNS morbidity at hospital discharge, and rate of improvement of pulmonary status as gauged by chest radiograph, arterial/alveolar PO2 ratio, patient temperature, WBC count, suctioning frequency, sputum volume, and days on ventilator. The KTT group (n = 23) and conventional bed care group (n = 26) were well matched for age, sex, severity of injury, and pulmonary status. There was no significant difference in mortality, CNS morbidity, LOS in ICU or hospital, or rate of pulmonary improvement between the groups. The efficacy of the KTT in reducing pulmonary complications in head-injured patients remains unclear.

Acute Disease↗

PubMed1989

Substitutions engineered by chemical synthesis at three conserved sites in mitochondrial cytochrome c. Thermodynamic and functional consequences.

Analogues of the 39-residue CNBr fragment of horse cytochrome c (66-104) have been prepared by total chemical synthesis. Conformationally assisted ligation of these peptides with the native cytochrome c fragment 1-65 (homoserine lactone form) occurred in high yield. Semisynthetic protein molecules of the expected molecular weight were obtained that had folded structures similar to the native molecule as shown by spectral properties and by cross-reactivity with a panel of monoclonal antibodies sensitive to the three-dimensional integrity of cytochrome c. Point mutations were introduced into the horse sequence at three strongly conserved sites: Tyr67, Thr78, and Ala83. The contributions of these 3 residues to the stability of the heme crevice were estimated by titration of the 695 nm absorption due to coordination of ferric iron by the sixth ligand methionine sulfur. The roles of these residues in catalysis of electron transfer and in establishing the value of the redox potential of cytochrome c were also investigated. The hydroxyl group of Tyr67 modulates the spectral properties of the heme and has a profound influence on its redox properties, but hydrogen bonding involving this phenolic hydroxyl does not stabilize the heme crevice. In contrast, we find that Thr78 is strongly stabilizing and that asparagine is not an adequate substitute for this residue because of the greater entropic cost of burying its side chain. The low biological activity of analogues modified at this position, despite normal redox potentials, imply a role for Thr78 in the electron transfer mechanism. The replacement of Ala83 by proline induces a similar phenomenon. An involvement of this residue in the catalysis of electron transfer provides an explanation of the low reactivity of plant mitochondrial cytochromes c in mammalian redox systems.

Amino Acid Sequence↗

PubMed1989

Conformation-directed recombination of enzyme-activated peptide fragments: a simple and efficient means to protein engineering. Its use in the creation of cytochrome c analogues for structure-function studies.

Protein fragments have been activated by the addition of amino acid esters using proteolytic enzymes under conditions where the equilibria are shifted in the direction of synthesis. Because of the natural propensity of large protein fragments to form complexes approximating native conformation, these activated fragments have been induced to recombine by formation of the missing peptide bond. Although the incorporated ester is only weakly activating, the complex, mimicking an enzyme, provides proximity and orientation at the reacting termini, so that coupling yields are high. In other words, the protein catalyzes its own resynthesis. What distinguishes our technique from the rare natural examples of this phenomenon is that it operates at a variety of cleavage sites and with varying chain lengths. There seems to be no particular limitations on the amino acid esters that can be added, and serine proteases with a wide range of specificities can be used. It thus appears that we have a truly general method for the condensation of large fragments in protein synthesis, be they natural or the products of synthetic or genetic methods. This approach has the advantages over conventional methods of great specificity, high efficiency, and mild conditions of use. With our model protein, cytochrome c, we have used this approach to make analogues that illuminate structure-function relations. Both the highly conserved lysine 39 and the functionally invariant threonine 40 have been replaced by a range of substitutions. The results show how crucial these residues are to the structural and functional integrity of the bottom omega-loop of the protein.

Cyanogen Bromide↗

PubMed1989

The cytochrome c oxidase-cytochrome c complex: spectroscopic analysis of conformational changes in the protein-protein interaction domain.

Binding to cytochrome c oxidase induces a conformational change in the cytochrome c molecule. This conformational change has been characterized by comparing the binding of native cytochrome c and chemically modified cytochrome c derivatives to bovine cytochrome c oxidase by using absorption, circular dichroism (CD), and magnetic circular dichroism (MCD) spectroscopy. The following derivatives were analyzed: (i) cytochrome c modified at all 19 lysine residues to yield the (N epsilon-acetimidyl)19 cytochrome c, (N epsilon-isopropyl)19 cytochrome c, and (N epsilon,N epsilon-dimethyl)19 cytochrome c; (ii) cytochrome c in which Met65 and Met80 are converted to the methionine sulfoxide; (iii) cytochrome c with a single break in the polypeptide chain at Arg38 or Gly37. The derivatives bind to cytochrome c oxidase at a ratio of one heme c per heme aa3. The association constants are similar to that of native cytochrome c except for (N epsilon-isopropyl)19 and (N epsilon,N epsilon-dimethyl)19 cytochromes c, which bind respectively four times and six times less strongly. The derivatives are good substrates for the cytochrome c oxidase reaction. The spectral changes accompanying the binding of the modified cytochromes c to cytochrome c oxidase are quite different from the spectral changes observed with native cytochrome c. The different optical absorption and MCD changes are explained by a polarity change around the exposed heme edge in the cytochrome c-cytochrome c oxidase complex. The CD changes indicate a conformational rearrangement restricted to the surface area surrounding the exposed heme edge. The rearrangement may involve a movement of the evolutionarily conserved Phe82 out of the vicinity of the heme.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Monoclonal antibodies as probes of conformational changes in protein-engineered cytochrome c.

Determination of the nature of the antigen-antibody complex has always been the ultimate goal of three-dimensional epitope mapping studies. Various strategies for epitope mapping have been employed which include comparative binding studies with peptide fragments of antigens, binding studies with evolutionarily related proteins, chemical modifications of epitopes, and protection of epitopes from chemical modification or proteolysis by antibody shielding. In this study we report the use of protein engineering to modify residues in horse cytochrome c that are in or near the epitopes of four monoclonal antibodies specific for this protein. The results demonstrate not only that site-specific changes in the antigen binding site dramatically affect antibody binding, but, more importantly, that some of the site-specific changes cause local and long-range perturbations in structure that are detected by monoclonal antibody binding at other surfaces of the antigen. These findings emphasize the role of native conformation in the stabilization of the interaction between protein antigens and high affinity monoclonal antibodies. Furthermore, the results demonstrate that monoclonal antibodies are more sensitive probes of changes in conformation brought about by protein engineering than low resolution spectroscopic methods such as circular dichroism, where similar spectra are observed for all the analogues. These findings suggest a role for monoclonal antibodies in detecting conformational changes invoked by nonconservative amino acid substitutions or substitutions of evolutionarily conserved residues in protein-engineered or recombinant proteins.

Animals↗

PubMed1988

The oxidation-state-dependent ATP-binding site of cytochrome c. Implication of an essential arginine residue and the effect of occupancy on the oxidation-reduction potential.

Arg-91 is not part of the active site of cytochrome c that mediates binding and electron transfer, yet it is absolutely conserved in eukaryotic cytochromes c, indicating a special function. The physicochemical properties of analogues are unaffected by the modification of this residue, so they can be used with confidence to study the role of Arg-91. We have established limiting conditions under which this residue alone is specifically modified by cyclohexane-1,2-dione, and have subsequently shown that ATP, and to a lesser extent ADP or Pi, protects it from the action of the reagent in an oxidation-state-dependent manner. These observations strongly support the idea that this site exerts a controlling influence on cytochrome c activity in the electron transport or other cellular redox systems, and we have commenced a study of how that influence might operate. We find that the redox potentials of both cytochrome c and analogue are little affected by changing ATP or Pi concentrations.

Adenosine Triphosphate↗

PubMed1988

Enzyme-assisted semisynthesis of polypeptide active esters and their use.

A method is described for the preparation of polypeptides activated uniquely at the C-terminus. The polypeptide is incubated in a concentrated solution of an amino acid active ester, the latter having its amino group free but adequately protected by protonation. The amino acid ester is coupled via its amino group to the C-terminus of the polypeptide by enzymic catalysis (reverse proteolysis). The resulting polypeptide C-terminal active ester is then isolated and coupled to a suitable amino component (generally a polypeptide) in a subsequent chemical coupling. The method appears to be generally applicable; fragments of horse heart cytochrome c, and porcine insulin, are used as examples. Two new analogues of cytochrome c have been prepared by using this method, with yields of up to 60% in the final coupling. Scope and limitations of the method are discussed.

Alanine↗

PubMed1988

Novel Ca2+-activated neutral protease from an aquatic fungus, Allomyces arbuscula.

A Ca2+-activated neutral protease was purified to homogeneity from an aquatic Phycomycete fungus, Allomyces arbuscula. It requires millimolar concentrations of Ca2+ for activation (1.8 to 2 mM for 50% activation). Sr2+ can replace Ca2+ but at higher concentrations (4 mM for 50% activation). The enzyme is a dimer of 40-kilodalton subunits and contains six cysteine residues, three of which are revealed only after the addition of micromolar concentrations of Ca2+; the other three are free. Enzyme activity is strongly inhibited by SH-group inhibitors and some trypsin inhibitors (leupeptin and alpha-N-tosyl-L-lysine chloromethyl ketone). The enzyme lacks general trypsinlike specificity, since substrates containing tryptic cleavage sites are not cleaved nor is enzyme activity inhibited by other trypsin inhibitors. The enzyme has many functional similarities to the extensively characterized mammalian and avian Ca2+-activated neutral proteases but differs in its substrate specificity, inhibition by alpha-N-tosyl-L-phenylalanine chloromethyl ketone, and subunit structure. It is, nevertheless, presumed that this enzyme has a similar high order of specificity and is involved in the regulation of a specific growth function.

Calcium↗

PubMed1987

Alkylamine derivatives of cytochrome c. Comparison with other lysine-modified analogues illuminates structure/function relations in the protein.

For investigations of the functional roles of the lysine residues of cytochrome c, analogues in which these residues are modified without charge loss are highly desirable. The 19 lysine residues of the horse heart protein have been modified by reductive alkylation. Two analogues were prepared, using formaldehyde and acetone as the dialkylating and monoalkylating reagent respectively. Modification was shown to be clean and quantitative. Characterisation of the alkylamine derivatives by physicochemical measurements and biological activity determinations was carried out. The potential of these analogues in structure/function studies of cytochrome c is discussed. It is illustrated by their use, in conjunction with other lysine-modified derivatives, to investigate the extent to which surface charge determines redox potential, and to study the physicochemical changes that accompany rising pH. In the latter case the observed phenomena are not as closely correlated as previously thought, suggesting that there is a complex set of rearrangements of structure underlying the functional changes. The data confirm that modification of the lysine residues influences these changes. These residues participate in numerous surface intramolecular links, so the lack of correlation may be explained if each of the changing parameters were under the influence of a different set of residues. However, neither a lysine residue, nor a histidine residue directly displaces methionine from the sixth coordination position of the haem iron at alkaline pH.

Alkylation↗

PubMed1987

Semisynthesis of cytochrome c analogues. The effect of modifying the conserved residues 38 and 39.

We have used chemical and enzymic protein engineering techniques to create analogues of the semisynthetic two-fragment complex (1-37).(38-104) of mitochondrial cytochrome c. This complex, unlike the natural product of specific tryptic cleavage, (1-38).(39-104), from which it is prepared, quite closely resembles the parent protein in functional characteristics and is thus a suitable substrate for modifications designed to study structure-function relations. We have replaced the invariant Arg-38 and the conserved Lys-39 with a range of alternative amino acids and have studied the effects on the principal functional parameters. The hydrogen-bonding capacity of Arg-38 is crucial to the stabilization of the bottom omega-loop, while the positive charge of Lys-39 helps maintain the high redox potential by electrostatic effects at the haem iron.

Amino Acids↗

PubMed1987

Functional consequences of the excision of an omega loop, residues 40-55, from mitochondrial cytochrome c.

A novel technique for protein semisynthesis, enzymic activation, has been used to create a mitochondrial cytochrome c analogue in which the conventional bottom loop has been deleted. The resulting structure resembles that of cytochrome c555 from a primitive photosynthetic sulfur bacteria. Comparisons of this analogue with natural cytochromes show which of the functional differences between cytochromes c and c555 may be related directly to the incorporation of the loop. The structure is an example of an omega loop, recently defined as a discrete category of protein secondary structure. The analogue maintains the overall structure of the parent protein, but a significant change in redox potential has been engineered. It provides support for the prediction that omega loops act as independent modules in folding, function, and evolution. The rapidity of the synthesis and the high yield of product show that this technique for protein engineering is both competitive with, and complementary to, genetic methods.

Animals↗

PubMed1987

On the relationship between oxidation-reduction potential and biological activity in cytochrome c analogues. Results from four novel two-fragment complexes.

We have confirmed the propensity of fragments of cytochrome c to form complexes that reproduce the structure and, in part, the functionality, of the native protein by preparing four novel complexes. We have used trypsin under three different sets of conditions in sequence to prepare a contiguous two-fragment complex (1-55).(56-104). One of the intermediates is a stable overlapping complex (1-65).(56-104). Conditions for limited acid hydrolysis of peptide bonds in cytochrome c have been developed that optimize the yield of fragments (1-50) and (51-104). These two fragments also form a stable association, as do (1-50) and (56-104). These complexes are potentially useful for the semisynthesis of analogues modified in the region of the cleavage sites, which include a number of highly conserved amino acid residues, and are being used for studies of protein folding, interactions with oxidase, cytochrome c immunogenicity and of artificially induced spontaneous resyntheses between complexing fragments. Like other known two-fragment complexes of cytochrome c, they exhibit normal visible spectra, including the presence of the 695 nm band, indicative of a functional haem crevice. Studies of their biological activities and redox potentials lead to a number of conclusions on structure-function relationships in cytochrome c. Most significantly there is a linear relationship between the logarithm of electron-transfer rates from cytochrome c reductase and redox potential in this series of analogues, indicating that such transfer is thermodynamically controlled. This discovery contributes to our understanding of the interaction of cytochrome and reductase. Since the relationship is obeyed by other types of analogues, except for those that involve modification of the active site of cytochrome c, we have a useful diagnostic for those residues that participate directly in electron transfer.

Chromatography, Ion Exchange↗