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R C Bray

Publications and source records attributed to R C Bray.

At least 91 records · Page 5Linked to original sources
PubMed1990

Expression of a synthetic gene for horseradish peroxidase C in Escherichia coli and folding and activation of the recombinant enzyme with Ca2+ and heme.

A synthetic gene encoding horseradish peroxidase isoenzyme C (HRP C) has been synthesized and expressed in Escherichia coli. The nonglycosylated recombinant enzyme (HRP C*) was produced in inclusion bodies in an insoluble inactive form containing only traces of heme. HRP C* was solubilized and conditions under which it folded to give active enzyme were determined. Folding was shown to be critically dependent upon the concentrations of urea, Ca2+, and heme and on oxidation by oxidized glutathione. Purification of active HRP C* from the folding mixture gave a peroxidase, with about half the activity of HRP C. Glycosylation is thus not essential for correct folding and activity. The C-terminal and N-terminal extensions to HRP identified previously in cloned cDNA sequences are also not required for correct folding. However, Ca2+ appears to play a key role in folding to give the active enzyme. The overall yield of purified active enzyme was 2-3%, but this could be increased by reprocessing material that precipitated during folding.

Amino Acid Sequence↗
PubMed1990

Proton electron-nuclear double-resonance spectra of molybdenum(V) in different reduced forms of xanthine oxidase.

Electron-nuclear double-resonance (ENDOR) spectra of protons coupled to molybdenum(V) in reduced xanthine oxidase samples have been recorded. Under appropriate conditions these protons may be studied without interference from protons coupled to reduced iron-sulfur centers. Spectra have been obtained for the molybdenum(V) species known as Rapid, Slow, Inhibited, and Desulfo Inhibited. Resonances corresponding to at least nine protons or sets of protons are observed for all four species, with coupling constants in the range 0.08-4 MHz. Most of these protons do not exchange when 2H2O is used as solvent. Additional protons giving couplings up to 40 MHz are also detected. These correspond to EPR-detectable protons studied in earlier work. The strongly coupled protons may be replaced by 2H, through appropriate use of 2H2O or of 2H-substituted substrates, with consequent disappearance of the 1H resonances. In most cases the corresponding 2H ENDOR features have also been observed. The nature of the various coupled protons is briefly discussed. Results permit specific conclusions to be drawn about the structures of the Inhibited and Desulfo Inhibited species. In particular, the data indicate that the aldehyde residue of the Inhibited species has been oxidized and that the four protons derived from the ethylene glycol molecule in the Desulfo Inhibited species are not all equivalent. Recent assignments [Edmondson, D.E., & D'Ardenne, S.C. (1989) Biochemistry 28, 5924-5930] of the weakly coupled protons in the latter species appear not to be soundly based. The possibility of obtaining more detailed structural information from the spectra is briefly considered.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron Spin Resonance Spectroscopy↗
PubMed1990

Cytochemical evidence for a proteoglycan-associated filamentous network in ligament extracellular matrix.

The purpose of this investigation was to examine the extracellular matrix of rabbit ligament before and after digestion with glycosaminoglycan degrading enzymes. In order to preserve and enhance the visibility of negatively charged tissue components, particularly the glycosaminoglycan-containing proteoglycans, the cationic stains ruthenium red (RR) and ruthenium hexamine trichloride (RHT) were used. Cross-sections of the midsubstance of 10-month-old (mature) rabbit medial collateral ligaments fixed using conventional procedures revealed a sparse population of stellate-shaped cells that did not appear to be interconnected. Similar tissue fixed in either RR or RHT showed an extensive network of thin, electron-dense "seams" that interconnected cells and appeared to irregularly subdivide the extracellular matrix (ECM). These seams mainly consisted of a meshwork of microfilaments throughout which small granules were dispersed. Numerous 14-nm microfibrils, as well as mature elastic fibers were also present within the seams. The size and shape of the microfilaments, together with their threadlike, beaded appearance suggested that they could be Type VI collagen. The seam granules were easily removed with chondroitinase ABC, chondroitinase AC II, and mild (0.18 M) salt treatment. Only chondroitinase ABC succeeded in removing additional granules, tentatively identified as proteodermatan sulphate molecules, that were periodically located at d band sites along the Type I collagen fibrils. These results suggest that the seam granules are not dermatan sulphate containing proteoglycans, and further, that these proteoglycans may be sequestered into specific zones within the ECM through loose association with the seam microfilaments. While the functional significance of the seams remains unknown and their specific composition clearly requires further study, it is likely that they represent important functional (e.g., viscoelastic) or biological (e.g., nutritional) subdivisions of ligament substance.

Animals↗
PubMed1990

Fine vascular anatomy of adult rabbit knee ligaments.

The microvascular anatomy of discrete knee ligaments in adult rabbits is described. Epiligamentous plexuses give rise to a limited number of vessels which penetrate deeply into ligament substance. Intraligamentous vessels are usually longitudinally orientated, widely separated linear anastomoses but occasionally complex glomus-like configurations are present. The significance of these findings is discussed in relation to other articular connective tissues and to the possible roles of the intraligamentous microvasculature.

Animals↗
PubMed1989

Isolation, in the intact state, of the pterin molybdenum cofactor from xanthine oxidase.

A procedure is described for isolation of the pterin molybdenum cofactor, in the active molybdenum-containing state, starting from purified milk xanthine oxidase. The method depends on the use of anaerobic-glove-cabinet techniques and on working in aqueous solution, in the presence of 1 mM-Na2S2O4. SDS was used to denature the protein, followed by ion-exchange chromatography and gel filtration. The cofactor, obtained at concentrations up to 0.5-1.0 mM, was fully active in the nit-1 assay [Hawkes & Bray (1984) Biochem. J. 214, 481-493], with a specific activity of 22 nmol of NO2-/min per pg-atom of Mo (with 15% molybdate-dependence). The Mr, determined by gel filtration, was about 610, consistent with the structure proposed by Kramer, Johnson, Ribeiro, Millington & Rajagopalan [(1987) J. Biol. Chem. 262, 16357-16363]. At pH 5.9, under anaerobic conditions, the cofactor was stable for at least 300 h at 20-25 degrees C.

Animals↗
PubMed1989

Information from e.x.a.f.s. spectroscopy on the structures of different forms of molybdenum in xanthine oxidase and the catalytic mechanism of the enzyme.

X-ray spectroscopy was used to provide further information on the structure of the molybdenum centre of xanthine oxidase. Earlier work was confirmed and two states of the enzyme, not reported on by previous workers, were studied. One of these was the complex of the enzyme with pyridine-3-carboxaldehyde, in which most of the metal is in the Mo(V) state, giving the e.p.r. signal known as Inhibited. The other was the complex with the inhibitor alloxanthine, with the metal as Mo(IV). For both complexes clear evidence was obtained that an oxo ligand of molybdenum was present, but not a sulphido ligand. This information complements structural information on these complexes already available from e.p.r. spectroscopy, and has permitted us to revise and refine the structures previously proposed. The mechanism of action of the enzyme is discussed in the light of the present findings on the persistence of the oxo group in the reduced enzyme complexes, as well as of related evidence [George & Bray (1988) Biochemistry 27, 3603-3609] for an oxo group in the catalytic intermediate that gives the Mo(V) e.p.r. signal known as Very Rapid.

Catalysis↗
PubMed1989

X-ray-absorption and electron-paramagnetic-resonance spectroscopic studies of the environment of molybdenum in high-pH and low-pH forms of Escherichia coli nitrate reductase.

Previous e.p.r. work [George, Bray, Morpeth & Boxer (1985) Biochem. J. 227, 925-931] has provided evidence for a pH- and anion-dependent transition in the structure of the Mo(V) centre of Escherichia coli nitrate reductase, with the low-pH form bearing both an anion and probably a hydroxy-group ligand. Initial e.x.a.f.s. measurements [Cramer, Solomonson, Adams & Mortenson (1984) J. Am. Chem. Soc. 106, 1467-1471] demonstrated the presence of sulphur (or chloride) ligands in the Mo(IV) and Mo(VI) oxidation states, as well as a variable number of terminal oxo (Mo = O) groups. To synthesize the e.p.r. and e.x.a.f.s. results better, we have conducted new e.p.r. experiments and complementary e.x.a.f.s. measurements under redox and buffer conditions designed to give homogeneous molybdenum species. In contrast with results on other molybdoenzymes, attempts to substitute the enzyme with 17O by dissolving in isotopically enriched water revealed only very weak hyperfine coupling to 17O. The significance of this finding is discussed. Experiments with different buffers indicated that buffer ions (e.g. Hepes) could replace the Cl- ligand in the low-pH Mo(V) enzyme form, with only a small change in e.p.r. parameters. E.x.a.f.s. studies of the oxidized and the fully reduced enzyme were consistent with the e.p.r. work in indicating a pH- and anion-dependent change in structure. However, in certain cases non-stoichiometric numbers of Mo = O interactions were determined, complicating the interpretation of the e.x.a.f.s. Uniquely for a molybdenum cofactor enzyme, a substantial proportion of the molecules in a number of enzyme samples appeared to contain no oxo groups. No evidence was found in our samples for the distant 'heavy' ligand atom reported in the previous e.x.a.f.s. study. The nature of the high-pH-low-pH transition is briefly discussed.

Chlorides↗
PubMed1989

Meniscal lesions and chronic anterior cruciate ligament deficiency. Meniscal tears occurring before and after reconstruction.

We examined the menisci in 47 patients at the time of anterior cruciate ligament reconstruction. Twenty-one patients had abnormal menisci at a mean of 34 months after injury, but there was no difference between the Lysholm scores of patients with intact or damaged menisci. Eleven patients had a new meniscal injury between reconstruction and review at a mean of six years later; only 15 patients had both menisci intact nine years after injury. If pivot shift had been cured, the incidence of meniscal injury was reduced, but remained higher than normal. If pivot shift returned after reconstruction there was a significantly higher incidence of meniscal injury. Meniscal lesions appear to be the result of instability and not the cause.

Cartilage, Articular↗
PubMed1988

The isolation of demolybdo xanthine oxidase from bovine milk.

It was deduced many years ago from indirect evidence that demolybdo xanthine oxidase is present in normal bovine milk. This has now been confirmed by isolation of this enzyme form by a method based on the folate-gel affinity-chromatography procedure described Nishino & Tsushima [(1986) J. Biol. Chem. 261, 11242-11246]. Enzymic and spectroscopic properties of demolybdo xanthine oxidase, which retains flavin and iron-sulphur centres, are generally in accordance with expectations. Like the normal enzyme, it yields on denaturation material fluorescing at 460 nm. Molybdenum cofactor activity measured by the Neurospora crassa nit-1 assay in the presence of added molybdate was 33% of that of the normal enzyme. The absorption spectrum in the near-u.v. region differs slightly, but significantly, from that of the active and desulpho forms of the enzyme. It is concluded that the molybdenum cofactor site contains a pterin-like material not identical with that in the normal enzyme. The significance of the occurrence of demolybdo xanthine oxidase in milk is discussed, and evidence in the literature for demolybdo forms of other molybdoenzymes is briefly reviewed. Additional studies on the use of the affinity procedure for large-scale preparation of high-activity xanthine oxidase are described. In agreement with our ability to isolate the demolybdo enzyme, the procedure appears less effective in eliminating the demolybdo than the desulpho enzyme.

Animals↗
PubMed1988

Investigation by electron paramagnetic resonance spectroscopy of the molybdenum centre of respiratory nitrate reductase from Paracoccus denitrificans.

The molybdenum centre of respiratory nitrate reductase from Paracoccus denitrificans has been investigated by e.p.r. spectroscopy of Mo(V). In common with the centres of the analogous enzymes from Escherichia coli and Pseudomonas aeruginosa, it undergoes a pH- and anion-dependent transition between two different e.p.r. signal-giving species. Comparison of the relevant e.p.r. parameters extracted with the help of computer simulations reveals ligation of the metal in the active centres of the three enzymes to be identical.

Electron Spin Resonance Spectroscopy↗
PubMed1988

Studies by electron paramagnetic resonance spectroscopy of xanthine oxidase enriched with molybdenum-95 and with molybdenum-97.

Investigations have been carried out on the nature of the species from the enzyme xanthine oxidase that give rise to two molybdenum (V) electron paramagnetic resonance (EPR) signals. Isotopic enrichment with 95Mo, 97Mo, 33S, and 17O was employed. Computer simulations of the EPR spectra recorded at 9- and 35-GHz microwave frequencies were used to evaluate the various hyperfine couplings and angular relations between the principal axes of g and A, as well as the nuclear electric quadrupole interaction for 97Mo. The results support the presence of an oxo ligand in the Rapid and of both an oxo and a sulfido ligand in the Very Rapid signal-giving species.

Animals↗
PubMed1988

Reconstruction for chronic anterior cruciate instability. A comparison of two methods after six years.

Forty-seven patients with disabling instability due to isolated anterior cruciate deficiency are described. None had responded to conservative measures or to attempted correction of internal derangements. Eighteen patients were treated by extra-articular MacIntosh lateral substitution alone, the other 29 were treated by the same procedure combined with carbon-fibre replacement of the anterior cruciate ligament. No statistically significant difference was found between the two groups at six years. A satisfactory outcome was found in 44% of the extra-articular group and 55% of the carbon-fibre group at last review; however, the latter group had more complications. There was a marked deterioration in the quality of results between three and six years in both groups.

Adolescent↗
PubMed1987

The molybdenum iron-sulphur protein from Desulfovibrio gigas as a form of aldehyde oxidase.

The molybdenum iron-sulphur protein originally isolated from Desulfovibrio gigas by Moura, Xavier, Bruschi, Le Gall, Hall & Cammack [(1976) Biochem. Biophys. Res. Commun. 72, 782-789] has been further investigated by e.p.r. spectroscopy of molybdenum(V). The signal obtained on extended reduction of the protein with sodium dithionite has been shown, by studies at 9 and 35 HGz in 1H2O and 2H2O and computer simulations, to have parameters corresponding to those of the Slow signal from the inactive desulpho form of various molybdenum-containing hydroxylases. Another signal obtained on brief reduction of the protein with small amounts of dithionite was shown by e.p.r. difference techniques to be a Rapid type 2 signal, like that from the active form of such enzymes. In confirmation that the protein is a molybdenum-containing hydroxylase, activity measurements revealed that it had aldehyde:2,6-dichlorophenol-indophenol oxidoreductase activity. No such activity towards xanthine or purine was observed. Salicylaldehyde was a particularly good substrate, and treatment of the protein with it also gave rise to the Rapid signal. Molybdenum cofactor liberated from the protein was active in the nit-1 Neurospora crassa nitrate reductase assay. It is concluded that the protein is a form of an aldehyde oxidase or dehydrogenase. From the intensity of the e.p.r. signals and from enzyme activity measurements, 10-30% of the protein in the sample examined appeared to be in the functional form. The evolutionary significance of the protein, which may represent a primitive form of the enzyme rather than a degradation product, is discussed briefly.

Aldehyde Oxidase↗
PubMed1987

Comparison of arthroscopic and open techniques in carbon fibre reconstruction of the anterior cruciate ligament: long-term follow-up after 5 years.

Two groups of patients with chronic, symptomatic anterior cruciate ligament deficiency were reconstructed with carbon fibre. In 15 patients, the prosthesis was inserted through an anterolateral arthrotomy; in 14, it was inserted arthroscopically. Both groups also received a MacIntosh extraarticular lateral substitution. Assessment included subjective functional rating and objective clinical examination for instability. No significant difference in subjective functional rating was present after a minimum of 5 years; however, a reduced complication rate and diminished return of clinical instability was evident in the arthroscopic group during this period. Reconstruction was satisfactory in 47% of the arthrotomy group and 64% of the arthroscopy group when subjective and objective criteria were combined to assess the final outcome. This study supports the proposition that, in combination with extraarticular lateral substitution, arthroscopic insertion of cruciate prostheses compares favourably with conventional "open" techniques.

Adolescent↗
PubMed1987

Arthroscopic lateral release for anterior knee pain: a study comparing patients who are claiming worker's compensation with those who are not.

The purpose of this retrospective study was to assess the outcome of arthroscopic lateral retinacular release comparing patients who are claiming worker's compensation with those who are not. Twenty-three consecutive patients with anterior knee pain caused by injuries on the job were matched with an equal number of patients with other kinds of injuries on the basis of age, sex, and medical history. A numeric scoring scale, physical examination, isokinetic strength analysis, performance in a one-leg hop test, and radiography of the knee formed the data base. Evaluation of the outcome was based on subjective and objective criteria developed from this data base. Results demonstrated that "compensation" patients had significantly poorer overall subjective outcome (50% good or excellent) compared with "noncompensation" controls (58% good or excellent). When objective criteria were applied, only 46% of patients in each group demonstrated a satisfactory outcome. Subjective variables and poor quadriceps function had a significant effect on final outcome. The extent of patellar cartilage pathology, radiographic incongruity, and closure of the release site demonstrated no effect on final outcome. "Compensation" patients with anterior knee pain were less likely to believe that they had improved following surgery when compared with control "noncompensation" patients.

Adolescent↗