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R C Bray

Publications and source records attributed to R C Bray.

At least 73 records · Page 4Linked to original sources
PubMed1994

pH-dependent properties of a mutant horseradish peroxidase isoenzyme C in which Arg38 has been replaced with lysine.

Arg38 in the active site of horseradish peroxidase isoenzyme C (HRP-C) has been replaced with lysine by site-directed mutagenesis. As a preclude to a detailed kinetic analysis of this variant, the present study characterizes a pH-dependent cycle of reactions for recombinant horseradish peroxidase isoenzyme C with Arg38 replaced by lysine ([R38K]HRP-C*), which involves time-dependent changes in both specific activity and the electronic absorption spectrum of the enzyme. This pH-dependent cycle resembles that previously suggested for a cytochrome-c peroxidase variant in which Asp235 was replaced with asparagine. When the pH of a solution of resting [R38K]HRP-C* at pH 6.6 (form AH) is raised to pH 8.6, a rapid alkaline transition occurs. This results in spectral changes characteristic of a shift from a predominantly pentacoordinate to a completely hexacoordinate high-spin haem iron (form A-) with a pKa of 7.5. When the pH of a solution of form A- is raised from 8.7 to 12.0, no further spectral changes are observed. The reaction is reversible, but when the high-pH form of the enzyme (A-) is allowed to stand at pH 8.6, it slowly becomes converted into a third enzyme form (form I-) at a rate which is independent of pH (k = 0.56 h-1). When the pH of a sample of form I- is lowered from 8.6 to 6.6, the original low-pH form (AH) of the enzyme is recovered. Recovery of form AH from form I- does not occur via form A-, but via at least one further intermediate, form X. Following a downward pH jump, the rate constant for the formation of form X from form I- shows a small dependence on pH, changing from 48 s-1 at pH 6.8 to 39 s-1 at pH 7.4. The rate of formation of form AH from form X is also pH dependent and biphasic in nature, with measured rate constants ranging from 11.9-2.1 h-1. The possible structures of the different forms of [R38K]HRP-C* are discussed in the light of similar data in the literature for variants of cytochrome-c peroxidase. The properties may be indicative of a greater degree of conformational flexibility within the active site of this mutant caused by the smaller bulk of the lysine side-chain and the probable disruption of a part of the haem-linked hydrogen-bonding network in the distal haem pocket. The wild-type enzyme undergoes no such pH induced changes.

Arginine↗
PubMed1993

Ultrastructural immunolocalization of type-VI collagen and chondroitin sulphate in ligament.

Immunological methods were used to determine the identity of the major components comprising a network of electron-dense seams (described by the authors in a previous work) within the extracellular matrix of medial collateral ligament (MCL) from humans and rabbits. Tissue obtained from MCL midsubstance was subjected to pre-embedding labelling with colloidal gold at the electron microscopic level with monoclonal antibodies (MAbs) against type-VI collagen and chondroitin sulphate (CS), before and after digestion with chondroitinase ABC and testicular hyaluronidase. Tissue labelled with anti-type-VI MAbs showed gold conjugates attached to the microfilamentous component of the seams both before and after enzyme digestion, which confirmed the identity of the beaded microfilaments as type-VI collagen. Treatment of the tissue with anti-CS MAbs resulted in labelling of undigested tissue only. In these treatments, gold particles were found attached to granules that were interspersed throughout the network of type-VI microfilaments. Both the granules and gold labels were absent from the network following enzyme digestion. Thin nonbeaded microfilaments that did not label with anti-type-VI MAbs also were present within the seams. The loss of these nonbeaded microfilaments following enzyme digestion suggested that they might represent strands of hyaluronan. The codistribution and sequestering of type-VI collagen and CS within discrete seams or channels suggests that these regions of the MCL midsubstance may contain higher concentrations of water than the surrounding dense fibrillar matrix.

Animals↗
PubMed1993

The effect of the entry hole for an intramedullary nail on the strength of the proximal femur.

We used 15 pairs of femora from fresh human cadavers to study the effects of the size and location of the entry hole for an intramedullary nail on the strength of the femur. Right femora were used as controls. Left femora in group 1 had a 10 mm entry hole in the 'ideal' location in the piriformis fossa. Group 2 had a 14 mm entry hole in the same position and group 3 had a 14 mm entry hole anterior to this on the superior aspect of the femoral neck. Femora were equipped with strain gauges and loaded to failure in compression. There was reduced stiffness and load to failure in group 3 specimens, suggesting that the location of the hole was more important than its size.

Aged↗
PubMed1993

Abnormal joint mechanics and the proteoglycan composition of normal and healing rabbit medial collateral ligament.

OBJECTIVE: This study investigated the influence that abnormal joint mechanics may have upon the biochemical composition of the joint's own soft tissue holding elements. DESIGN: The investigation used an animal model of ligament injury, the rabbit medial collateral ligament (MCL). The proteoglycan component of the ligament extracellular matrix was extracted, purified and characterized. INTERVENTIONS: The experimental groups consisted of: a) a control group consisting of the MCL from both right and left knees of six animals that had not undergone surgery; b) a group (healing gap injury) of six MCL from right knees in which a segment of tissue had been excised from the anterior cruciate and the MCL of the right knee 3 wk prior to sacrifice; and c) a third group (contralateral gap injury) comprised of the MCL from the six left knees of the same gap injury animals. OUTCOME MEASURES: The MCL water content, total proteoglycan content, hexose and hexuronate-containing proteoglycan and proteoglycan electrophoretic mobility were determined for each group studied. RESULTS: The healing gap injury MCL was found to have a higher water content, a higher total proteoglycan content and a higher proportion of aggregating proteoglycan than MCL from control animals. The nonaggregating proteoglycan fraction from the contralateral MCL (group 3) had a greater electrophoretic mobility and probably, therefore, a smaller molecular weight than that found in the MCL from the same knee of control animals. CONCLUSIONS: Since MCL healing took place in an abnormal mechanical environment, these results suggest that joint biomechanics may be an important factor in mediating connective tissue proteoglycan composition.

Animals↗
PubMed1992

Use of rosy mutant strains of Drosophila melanogaster to probe the structure and function of xanthine dehydrogenase.

The usefulness in structure/function studies of molybdenum-containing hydroxylases in work with rosy mutant strains of Drosophila melanogaster has been investigated. At least 23 such strains are available, each corresponding to a single known amino acid change in the xanthine dehydrogenase sequence. Sequence comparisons permit identification, with some certainty, of regions associated with the iron-sulphur centres and the pterin molybdenum cofactor of the enzyme. Procedures have been developed and rigorously tested for the assay in gel-filtered extracts of the flies, of different catalytic activities of xanthine dehydrogenase by the use of various oxidizing and reducing substrates. These methods have been applied to 11 different rosy mutant strains that map to different regions of the sequence. All the mutations studied cause characteristic activity changes in the enzyme. In general these are consistent with the accepted assignment of the cofactors to the different domains and with the known reactivities of the molybdenum, flavin and iron-sulphur centres. Most results are interpretable in terms of the mutation affecting electron transfer to or from one redox centre only. The activity data provide evidence that FAD and the NAD+/NADH binding sites are retained in mutants mapping to the flavin domain. Therefore, despite some indications from sequence comparisons, it is concluded that the structure of this domain of xanthine dehydrogenase cannot be directly related to that of other flavoproteins for which structural data are available. The data also indicate that the artificial electron acceptor phenazine methosulphate acts at the iron-sulphur centres and suggest that these centres may not be essential for electron transfer between molybdenum and flavin. The work emphasizes the importance of combined genetic and biochemical study of rosy mutant xanthine dehydrogenase variants in probing the structure and function of enzymes of this class.

Amino Acid Sequence↗
PubMed1992

Structural studies by proton-NMR spectroscopy of plant horseradish peroxidase C, the wild-type recombinant protein from Escherichia coli and two protein variants, Phe41----Val and Arg38----Lys.

Wild-type recombinant horseradish peroxidase isoenzyme C and two protein variants, Phe41----Val and Arg38----Lys, have been characterised using both one- and two-dimensional NMR spectroscopy. Proton NMR spectra recorded in both resting and cyanide-ligated states of the proteins were compared with those of the corresponding plant peroxidase. The latter contains 18% carbohydrate in eight N-linked oligosaccharide side chains whereas the recombinant proteins are expressed in nonglycosylated form. The spectra of the plant enzyme and refolded recombinant protein are essentially identical with the exception of carbohydrate-linked resonances in the former, indicating that their solution structures are highly similar. This comparison also identifies classes of carbohydrate resonances in the plant enzyme which provides new information on the local environment and mobility of the oligosaccharide side chains. Comparison of the spectra of the cyanide-ligated states of the two variants and those of plant horseradish peroxidase C indicated that there were significant differences with respect to haem and haem-linked resonances. These could not be rationalised simply on the basis of the local perturbation expected from a single-site substitution. The two substitutions made to residues on the distal side of the haem apparently influenced the degree of imidazolate character of the proximal His170 imidazole ring thus perturbing the magnetic environment of the haem group. Inspection of the spectra of the Phe41----Val variant also showed that the resonances of a phenylalanine residue in the haem pocket had been incorrectly assigned to Phe41 in a previous study. A new assignment, based on additional information from two-dimensional nuclear Overhauser enhancement spectroscopy, was made to Phe152. The assignments made for the Phe41----Val variant were also used as a basis to investigate the structure of the complex formed with the aromatic donor molecule, benzhydroxamic acid.

Arginine↗
PubMed1992

The early effects of joint immobilization on medial collateral ligament healing in an ACL-deficient knee: a gross anatomic and biomechanical investigation in the adult rabbit model.

In this study, the short-term effects of immobilization on joint damage and medial collateral ligament (MCL) healing were investigated in unstable, anterior cruciate ligament (ACL)-deficient knees in rabbits. Forty-six 12-month-old female New Zealand white rabbits were separated into three groups. Animals from each group had surgery on their right knees: group I, sham controls (n = 9); group II, complete transection of the ACL and removal of a 4 mm segment (gap injury) of MCL midsubstance with no immobilization of the limb (n = 19); and group III, same injuries to the ACL and MCL (as group II) but with immobilization of the limb (n = 18). No surgical repair of disrupted ligaments was performed. Left knees served as unoperated contralateral controls. All animals were allowed unrestricted cage activity until sacrifice in subgroups at 3, 6, and 14 weeks of healing when biomechanical properties of all MCLs were measured. All knee joints were systematically examined for gross evidence of damage to articular cartilage, menisci, and periarticular soft tissues. To monitor relative in vivo loads on injured limbs during healing, hindlimb weight bearing was assessed at biweekly intervals. Results indicated that animals in both groups II and III bore relatively lower loads (compared to preinjury values) on their injured hindlimbs. Mechanical testing of MCLs showed only minor changes in sham controls, while group II and III healing MCLs demonstrated significantly lower force and stress at MCL complex failure compared to contralateral controls. In specific comparisons of group III to group II animals, we noted that immobilization prevented joint damage over the early intervals studied. In addition, immobilization resulted in MCL laxity similar to contralateral control values but inhibited development of structural strength and stiffness in healing MCLs. These results suggest that in the rabbit, short-term immobilization of an ACL-deficient knee offers some advantages to the joint and to certain low load behaviors of the healing MCL, but it also results in a smaller quantity of scar tissue that is less able to resist higher loads. Longer-term studies involving remobilization are necessary before the effects of brief immobilization on joint damage and MCL healing in this ACL-deficient model can be fully defined.

Animals↗
PubMed1992

Quantitative analysis of the fine vascular anatomy of articular ligaments.

An image analysis technique has been developed to quantitatively describe the fine vascular patterns observed in ligament tissue. The longitudinal orientational distribution and total vessel volume of India-ink-perfused blood vessel segments in normal and healing ligaments were determined. The methods involved special vascular preparation of adult rabbit knee medial collateral ligaments (MCL) by India-ink perfusion. Black and white microscope images of ink-perfused tissue sections were subjected to a thresholding procedure to binarize digitized ligament images, which were then skeletonized and analyzed for directional distribution based on the least-squares technique. Analysis of medial collateral ligaments in New Zealand White rabbits using this method has shown that scarred tissue is more vascular and has a more chaotic angular distribution of blood-vessel segments than normal ligament tissue.

Algorithms↗
PubMed1991

Information from e.p.r. spectroscopy on the iron-sulphur centres of the iron-molybdenum protein (aldehyde oxidoreductase) of Desulfovibrio gigas.

E.p.r. spectra of reduced iron-sulphur centres of the aldehyde oxidoreductase (iron-molybdenum protein) of Desulfovibrio gigas were recorded at X-band and Q-band frequencies and simulated. Results are consistent with the view that only two types of [2Fe-2S] clusters are present, as in eukaryotic molybdenum-containing hydroxylases. The data indicate the Fe/SI centre to be very similar, and the Fe/SII centre somewhat similar, to these centres in the eukaryotic enzymes.

Aldehyde Oxidoreductases↗
PubMed1991

31P ENDOR studies of xanthine oxidase: coupling of phosphorus of the pterin cofactor to molybdenum (V).

31P ENDOR spectra are described for three different molybdenum(V) species in reduced xanthine oxidase samples. The spectra were not affected by removing the FAD from the enzyme, implying that this is located at some distance from molybdenum. Furthermore, in confirmation of the work of J. L. Johnson, R. E. London, and K. V. Rajagopalan [(1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6493-6497], NMR and chemical analysis of the phosphate content of highly purified xanthine oxidase showed there are only three phosphate residues per subunit of the enzyme. It is concluded that the ENDOR features are due to hyperfine coupling of the phosphate group of the pterin cofactor to the molybdenum atom. Evaluation of the dipolar component of the coupling has permitted estimation of the molybdenum-phosphorus distances as 7-12 A. This implies that the cofactor is in an extended conformation in the enzyme molecule. Less detailed 31P ENDOR data on sulfite oxidase are consistent with a similar conformation for the cofactor in this enzyme.

Animals↗