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M C Kennedy

Publications and source records attributed to M C Kennedy.

At least 73 records · Page 4Linked to original sources
PubMed1983

The role of iron in the activation-inactivation of aconitase.

Methods are described for the convenient preparation of aconitase from beef heart mitochondria in its inactive [3Fe-4S] form and largely in its active [4Fe-4S] form. Inactive aconitase can be activated anaerobically by various reducing agents without addition of iron. Under these conditions, maximally 70-80% of the activity attainable in the presence of added iron can be reached. It is concluded that during activation without added iron, [4Fe-4S] clusters are built from [3Fe-4S] clusters at the expense of a fraction of the 3Fe clusters present. This explains the approximately 75% maximal activation observed and concomitant loss of approximately 25% of total clusters as quantitated by EPR. Time course plots of aconitase activation appear to be second order but are not amenable to simple kinetic analysis because of the requirements of both reduction and Fe2+ for activation. Activation of aconitase with 59Fe leads to rapid (minutes) incorporation of 1 iron atom/cluster, which on subsequent inactivation is readily lost again. With longer incubation times (hour), 59Fe is found in more than a single site/cluster. It is concluded that, in analogy to cluster loss during activation in absence of added iron, the appearance of 59Fe in more than one cluster site can be due to complete breakdown and rebuilding of clusters. However, exchange into intact clusters cannot be ruled out. Ferric iron can be bound nonspecifically to active and inactive aconitase but can be readily removed by chelating agents. Sulfide is not required for activation of aconitase in keeping with the proposal that inactive aconitase, as isolated, contains a [3Fe-4S] cluster. It is demonstrated that oxidation initiates the inactivation of aconitase with concomitant release of iron and formation of 3Fe clusters as determined by EPR.

Aconitate Hydratase↗
PubMed1983

Optical and EPR characterization of different species of active and inactive aconitase.

It has been shown by spectroscopic (Kent, T. A., Dreyer, J-L., Kennedy, M.C., Huynh, B.H., Emptage, M.H., Beinert, H., and Münck, E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1096-1100) and chemical (Kennedy, M.C., Emptage, M.H., Dryer, J-L., and Beinert, H. (1983) J. Biol. Chem. 258, 11098-11105) methods that interconversion of [3Fe-4S] and [4Fe-4S] clusters underlies activation and inactivation of aconitase. Since Fe-S clusters can assume different oxidation states, a number of different species of the enzyme can be expected to exist. Observations on activation-inactivation, as well as light absorption and EPR spectra, can be interpreted in terms of four species: [3Fe-4S]1+, the oxidized inactive enzyme as obtained on aerobic preparation from mitochondria; [3Fe-4S]0, the reduced inactive form as obtained on reduction in the presence of EDTA; [4Fe-4S]2+, the oxidized active form as obtained on reductive activation; and [4Fe-4S]1+, the reduced active form prepared by photoreduction of active aconitase. The light absorption spectra of each species are presented. Oxidized inactive aconitase shows EPR spectra typical of oxidized 3Fe clusters (g = 2.01), and reduced active enzyme shows spectra typical of reduced ferredoxins (g1,2,3 = 2.06, 1.93, 1.86). The EPR spectrum of the latter is drastically changed (g1,2,3 = 2.04, 1.85, 1.78) on addition of substrate. The active enzyme can be quantitatively converted to inactive enzyme by titration with ferricyanide in the presence of substrate. The correlation of EPR and optical spectra with enzymatic activity observed during titration demonstrates further that active aconitase requires an intact [4Fe-4S] cluster. A model of aconitase incorporating the four cluster species is presented, and explanations for some previous conflicting data concerning aconitase are offered.

Aconitate Hydratase↗
PubMed1983

Mössbauer and EPR studies of activated aconitase: development of a localized valence state at a subsite of the [4Fe-4S] cluster on binding of citrate.

During activation of aconitase a ferrous ion is incorporated into a [3Fe-4S] cluster to yield a structure with a [4Fe-4S] core. Using 57Fe or 56Fe for activation we have studied with Mössbauer spectroscopy the beef heart enzyme in the presence of citrate. Our studies show that the environment of one iron site (Fea) of the [4Fe-4S] cluster is drastically altered in the presence of citrate. Fea is the iron acquired during activation of aconitase. In the oxidized [4Fe-4S]2+ state two species with enzyme-bound substrate are observed, whereas only one is observed in the reduced [4Fe-4S]+ state. The Mössbauer parameters of Fea reveal that the site has acquired substantial high-spin ferrous character. This is most pronounced in the 1+ state where at Fea the cluster exhibits a localized valence state. The dramatic increase of the isomer shift upon substrate binding strongly suggests that the ligand environment of Fea has become at least five-coordinate and that the cluster may function as a Lewis acid. In the absence of citrate the EPR spectra of the active [4Fe-4S]+ enzyme (g1,2,3 = 2.06, 1.93, 1.86) show no hyperfine broadening in the presence of H2 17O. However, in the presence of citrate (g1,2,3 = 2.04, 1.85, 1.78) sizable transferred hyperfine interactions are observed; under the experimental conditions the hydroxyl groups of citrate and isocitrate as well as water are labeled with 17O. We did not detect broadening by 17O-labeled carboxyl groups of citrate in H2 16O. Implications for the mechanism of aconitase are discussed.

Aconitate Hydratase↗
PubMed1982

Mössbauer studies of beef heart aconitase: evidence for facile interconversions of iron-sulfur clusters.

Beef heart aconitase, isolated under aerobic conditions, has been studied with Mössbauer and EPR spectroscopy. In the oxidized state, the enzyme exhibits an EPR signal at g = 2.01. The Mössbauer data show that this signal is associated with a 3Fe cluster. In dithionite-reduced aconitase, the 3Fe cluster, probably of the [3Fe-3S] type, is in a paramagnetic state of interger electronic spin (S = 2); the Mössbauer spectra exhibit al the unique features reported for proteins with 3Fe clusters. On activation of aconitase with ferrous ion, the paramagnetic 3Fe cluster of dithionite-reduced enzyme is converted into a diamagnetic (S = 0) form. Activation studies with iron enriched in either 27 Fe or 56 Fe suggest that activation transforms the 3Fe cluster into a center that has a [4Fe-4S] core. This conclusion is supported by the observation that EPR signals characteristic of reduced [4Fe-4S] clusters can be elicited under appropriate conditions. It has frequently been assumed that the activation of aconitase with Fe2+ produces an active site containing a single ferrous ion. The data reported here suggest that a ferrous ion is used to rebuild a [4Fe-4S] cluster.

Aconitate Hydratase↗
PubMed1980

Cimetidine and acute upper gastrointestinal bleeding: a double-blind controlled trial.

Patients presenting with acute upper gastrointestinal bleeding from a variety of lesions were admitted to a prospective double-blind controlled trial to determine if cimetidine reduces the severity of bleeding and/or the incidence of rebleeding. During the first 48 hours, the patients received intravenous cimetidine (200 mg four-hourly) or placebo, and for the following ten days, oral cimetidine (1 g/24 hr) or placebo. Eight-eight patients entered the trial of whom 45 (51%) were in the cimetidine-treated group. Six of the seven patients requiring surgery for life-threatening bleeding and four of the six patients who rebled were in the cimetidine-treated group. This study failed to demonstrate any advantage of using cimetidine routinely in the treatment of acute upper gastrointestinal bleeding.

Adult↗
PubMed1979

Phenytoin absorption in patients with ileojejunal bypass.

1 The absorption and elimination of phenytoin was studied after a 200 mg oral dose was administered to seven patients with ileojejunal bypass and nine control patients. 2 Analysis of the area under the plasma concentration time curve showed the absorption of phenytoin was decreased in the subjects with ileojejunal bypass (P less than 0.005). 3 The half life of elimination of the drug was shorter in the bypass group (15.1 h) than in the controls (17.8 h, P less than 0.05). 4 Subjects with an ileojejunal bypass receiving phenytoin are likely to require increased oral dosages to achieve an optimal plasma concentration. There is probably malabsorption of other poorly soluble drugs and parenteral therapy may be necessary to ensure adequate bioavailability.

Adult↗
PubMed1977

Sport: assessing the risk.

Studies on the incidence of sports injuries are an integral part of sports medicine. A survey of three sports medicine journals showed a paucity of information in this area. In view of the need for well designed studies, a series of guidelines for studies is presented.

Athletic Injuries↗
PubMed1977

Retinal projections in larval, transforming and adult sea lamprey, Petromyzon marinus.

Unilateral enucleations were performed on larval, transforming and adult sea lampreys. Following 5 to 11 days survival, the animals were sacrificed and the brains were processed using a modified Fink-Heimer technique. In larvae, contralateral optic projections were found to the posterior one-third of the dorsal thalamus, the pretectum, and the optic tectum. No ipsilateral projections were present in the larvae. In enucleated transforming and adult lampreys, degenerating axons were observed in the optic chiasm and bilaterally in the optic tracts. Retinal efferents projected bilaterally to a lateral neuropil region ("tractus opticus") in the posterior one-half of the dorsal thalamus. Contralaterally, a conspicuous dorsomedial cell group (lateral geniculate nucleus) also received a projection. Contralateral projections to the superficial layers of the pretectum and optic tectum were observed. Ipsilateral retinal projections to the pretectum and optic tectum in transforming and adult lampreys were restricted to a small zone at the ventrolateral margins of the pretectum and tectum. The changes in distribution of retinofugal projections during transformation appear to be occurring at the same time that the eye differentiates into its adult form.

Animals↗
PubMed1976

Absence of glomerular renal tubular epithelial antigen in membranous glomerulonephritis.

Renal biopsies from 24 patients with membranous glomerulonephritis were examined by indirect immunofluorescence for the presence of autologous renal tubular epithelial antigen (RTE). Staining of the luminal layer of proximal tubular epithelium was found in all cases, as with normal human kidney, but granular staining along glomerular capillary walls was not observed. The role of RTE in the genesis of human membranous glomerulonephritis has not been confirmed.

Adult↗
PubMed1976

IgA and glomerular disease.

Serum IgA levels are raised in a proportion of children with Henoch-Schönlein disease (H. S. P.), patients with Berger's mesangial IgA disease, and in patients with glomerular lesions in association with hepatic cirrhosis. These conditions are all characterized by mesangial staining with anti-IgA antisera on immunofluorescence. It has been suggested that elevated serum IgA levels are of value in the diagnosis of these diseases. We have measured serum IgA levels by radial immunodiffusion in a variety of primary and secondary glomerular diseases. High serum IgA levels were confined to patients with glomerular IgA deposition, but were not restricted to H. S. P. and Berger's disease, nor to patients with deposition of IgA only in a mesangial pattern. Elevated salivary IgA levels were found in 3/4 patients with high serum IgA levels but we found no evidence for involvement of the secretory system in glomerular mesangial IgA deposition.

Adolescent↗
PubMed1975

New bronchodilator aerosol, salmefamol, in asthma.

Twenty-five asthmatics were tested with salmefamol aerosol (200 mug q.d.s.) for a period of 3 months. The ventilatory capacity before and after adrenaline was measured weekly and symptoms were assessed daily using a scoring system over a period of 9 months. The results during the 3 months' treatment with salmefamol were compared with the preceding and succeeding 3-month-periods when patients were receiving either salbutamol aerosol or orciprenaline aerosol. Statistically significant improvements were seen in ventilatory capacity before adrenaline inhalation and in symptom scores while on salmefamol. Ventilatory capacity after adrenaline inhalation remained unchanged throughout the study: thus there was no evidence of tachyphylaxis. A significantly greater number of patients preferred the new drug. Four patients developed slight muscle tremor in the first few days of salmefamol therapy, but there were no changes in haematological or biochemical values after 3 months' therapy. Thus, salmefamol seems to have marked efficacy with low toxicity and is generally well tolerated.

Adolescent↗