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M C Kennedy

Publications and source records attributed to M C Kennedy.

At least 37 records · Page 2Linked to original sources
PubMed1995

The 5'-untranslated region of human transferrin mRNA, which contains a putative iron-regulatory element, is bound by purified iron-regulatory protein in a sequence-specific manner.

Human transferrin mRNA contains a 5'-untranslated region that (1) has homology to an iron responsive element and (2) is implicated in translational iron regulation of human transferrin transgenes in transgenic mice. Ferritin mRNA contains a 5'-untranslated region iron-responsive element, but iron regulation of ferritin differs from that of human transferrin transgenes in both magnitude and direction. Structural differences between the ferritin iron-responsive element and the human transferrin putative iron-responsive element may influence their iron-regulatory protein interactions and direct the differing translational responses. This study examines human transferrin RNA nucleotide sequence requirements for binding of cytoplasmic proteins and purified iron-regulatory protein. Mutations of the putative transferrin iron-responsive element similarly affected binding of purified iron-regulatory protein and liver cytoplasmic proteins, providing evidence that the IRP is one of the liver cytoplasmic proteins that binds the human transferrin iron-regulatory element and suggesting that it may be involved in iron-regulation of transferrin.

Animals↗
PubMed1995

Novel production of interleukin-1 receptor antagonist peptides in normal human cornea.

Interleukin-1 receptor antagonist (IL-1ra) is an important modulator of IL-1 activity in a variety of tissues. IL-1ra is differentially produced by different cell types as a 22-26-kD secreted peptide (sIL-1ra) and/or a smaller 16- or 18-kD intracellular peptide (icIL-1ra). This study was undertaken to evaluate the production of IL-1ra in the human cornea. IL-1ra mRNA can be detected in early passage human corneal epithelial cells and corneal stromal fibroblasts and is significantly enhanced by IL-1. Corneal endothelial cells do not express IL-1ra mRNA. Immunohistochemical studies of cultured corneal cells and whole human cornea demonstrate IL-1ra protein production by both the epithelial and stromal cells but not the endothelial cells. Reverse transcriptase polymerase chain reaction, ELISA, and immunoprecipitation studies indicate that corneal epithelial cells are capable of producing both icIL-1ra and sIL-1ra forms of IL-1ra whereas the corneal stromal cells produce only icIL-1ra. In addition to the larger 18-kD icIL-1ra, both corneal epithelial and stromal cells are also capable of producing a smaller recently described 16-kD icIL-1ra. Thus, the differential production of IL-1ra in the human cornea is unique; whereas both epithelial and stromal cells produce icIL-1ra (type 1 and type 2), the epithelial cells appear to also produce sIL-1ra. It is proposed that these IL-1ra proteins may play an important role in regulating IL-1-induced corneal inflammation.

Adult↗
PubMed1994

Crystal structures of aconitase with trans-aconitate and nitrocitrate bound.

Crystal structures of mitochondrial aconitase with the inhibitors trans-aconitate and nitrocitrate bound to the [4Fe-4S] cluster have been solved and refined at 2.05 A resolution with R-factors of 0.168 and 0.172, respectively. Crystallization of aconitase with the substrates citrate and cis-aconitate has not been possible because the enzyme turns over and selects enzyme with isocitrate bound into the crystal lattice. Therefore we have analyzed crystal structures of the enzyme complexed with inhibitor analogs of these two substrates. The structure with nitrocitrate bound provides a model for citrate binding. The structure with trans-aconitate bound provides a model for cis-aconitate binding in two ways: Fe4 of the [4Fe-4S] cluster is five-coordinate and the carbon at the C beta position is trigonal. These results allow the model for the reaction mechanism to be extended to all three natural substrates of aconitase. The results support a model in which citrate and isocitrate form similar chelate structures related by 180 degrees rotation about the C alpha-C beta bond while the intermediate cis-aconitate binds in either of two ways (citrate mode or isocitrate mode). In both inhibitor complexes a H2O molecule is also bound to Fe4. In the structure with nitrocitrate bound, partial occupancy of sulfate in the active site is observed accompanied by hydroxyl binding to Fe4. Comparison of the structures with isocitrate, trans-aconitate, nitrocitrate and sulfate bound reveals preferred orientations for the three types of oxygens ligated to Fe4 (carboxyl, hydroxyl and H2O) supporting the proposed roles for His101, Asp165 and His167 in the catalytic mechanism.

Aconitate Hydratase↗
PubMed1994

Overexpression of iron-responsive element-binding protein and its analytical characterization as the RNA-binding form, devoid of an iron-sulfur cluster.

The iron-responsive element-binding protein (IRE-BP) has been defined and identified as an RNA-binding protein found in iron-deprived eukaryotic cells. IRE-BP binds to stem-loop structures, iron-responsive elements (IREs), which are located in the untranslated regions of the mRNAs for several genes including ferritin, and the transferrin receptor. When bound, IRE-BP prevents ferritin translation and stabilizes the transferrin receptor transcript. When cells are iron replete, an iron-sulfur cluster is ligated to the IRE-BP, the protein loses RNA binding properties, and it acquires aconitase activity. Cytosolic aconitase from liver can be converted into the IRE-BP by oxidative removal of its Fe-S cluster. We describe here overexpression of IRE-BP in baculovirus-infected insect cells which yields IRE-BP devoid of an iron-sulfur cluster. We describe a one-step purification of the IRE-BP and a quantitative analysis of Fe, S2-, S0, protein, and enzyme activity on IRE-BP, as obtained in cell lysates, after purification, and after reconstitution to active aconitase. On the average not more than 3% of the over-expressed purified protein contained an intact Fe-S cluster, and it was demonstrated that that cluster was not lost during purification. Scatchard analysis of RNA-binding data was compatible with a single high-affinity RNA-binding form of the IRE-BP. Active aconitase could be reconstituted from the purified IRE-BP obtained from the expression system by addition of iron, thiol, and sulfide, and the characteristic epr spectrum of the 3Fe form of cytosolic aconitase was obtained after ferricyanide oxidation of the reconstituted material.

Animals↗
PubMed1993

Metallothionein reacts with Fe2+ and NO to form products with A g = 2.039 ESR signal.

The interaction of metallothionein, Fe2+, and nitric oxide has been examined by electron spin resonance spectroscopy. When either apoMT 2 or Zn-MT 2 from rabbit liver was mixed with Fe2+ and NO, paramagnetic products with g values of 2.013 and 2.039 were found. These are characteristic of iron nitrosyl thiolates with stoichiometry, Fe(NO)2(SR)2. This product was not generated when beef heart mitochondrial aconitase was used as the source of iron.

Animals↗
PubMed1993

Anabolic steroid abuse and cardiac death.

OBJECTIVE: To examine the relationship between anabolic steroid abuse and cardiac death. We report the first two cases in Australia. They are the only reported cases in which the anabolic steroid oxymesterone has been detected. This compound has never been approved for use in Australia. CLINICAL FEATURES: Two footballers, aged 18 and 24, sustained fatal cardiac arrests while at training sessions. Both were considered fit and healthy. OUTCOME: Autopsy revealed features of a hypertrophic cardiomyopathy in the 18-year-old; the 24-year-old had findings of a myocarditis. In both cases the coronary arteries were normal and there was no evidence of coronary thrombosis. Urine in both subjects contained the anabolic steroid oxymesterone. CONCLUSIONS: There are limited clinical uses for anabolic steroids but they are widely abused by athletes in attempts to alter lean body mass and strength. Acute non-fatal myocardial infarction was first reported in 1988 and fatal myocardial infarction in 1990. While a causal relationship is hard to prove, it is possible that the anabolic steroid contributed to the increase in cardiac size in the first subject and may have increased his responsiveness to catecholamines causing an arrhythmogenic event. In the second, the inflammatory changes could have provided the focus for an arrhythmia. It would appear that anabolic steroid abuse should be considered in any athlete presenting with an acute vascular event.

Adolescent↗
PubMed1993

Application of a simplified method to determine bioavailability of an oral dose of phenytoin.

The bioavailability of capsules of phenytoin was determined by two methods: a method involving the numerical integration of the Michaelis-Menten equation and an alternative method involving fitting the time course of plasma concentrations, following the administration of the reference intravenous dosage, to an empirical quadratic function of time. The latter procedure requires much simpler computations. The two methods yielded very similar estimates of the rate and extent of absorption of phenytoin. Total absorption was 0.90 +/- 0.05 and 0.89 +/- 0.05 (mean +/- SE, n = 6) using the methods of numerical integration and quadratic curve fitting, respectively. Both methods indicated that the rate of absorption of phenytoin was inconsistent and slow. Half the total absorption of phenytoin occurred over 2.5 +/- 0.3 hr but the remainder was absorbed very slowly over a period of about 30 hr. Empirical functions may be more generally useful in the determinations of the bioavailability of drugs, particularly if some aspects of the disposition are saturable.

Administration, Oral↗
PubMed1993

Aconitase, a two-faced protein: enzyme and iron regulatory factor.

In this brief survey, the path of development of our knowledge of the iron-sulfur enzyme aconitase [citrate(isocitrate)hydrolyase EC4.2.1.3.] is traced from its discovery in 1937. Particular emphasis is on developments in the past decade, when EPR, Mössbauer and electron nuclear double resonance spectroscopies, X-ray crystallography, and mutational analysis were applied to the problem. More recently discovered was the significant amino acid sequence identity between mitochondrial aconitase and the iron regulatory factor or iron-responsive element binding protein (IRE-BP). This has led to the realization that IRE-BP is an alternative form of cytosolic (not of mitochondrial) aconitase that is devoid of its cubane Fe-S cluster.

Aconitate Hydratase↗
PubMed1992

Purification and characterization of cytosolic aconitase from beef liver and its relationship to the iron-responsive element binding protein.

In recent reports attention has been drawn to the extensive amino acid homology between pig heart, yeast, and Escherichia coli aconitases (EC 4.2.1.3) and the iron-responsive element binding protein (IRE-BP) of mammalian cells [Rouault, T. A., Stout, C. D., Kaptain, S., Harford, J. B. & Klausner, R. D. (1991) Cell 64, 881-883.; Hentze, M. W. & Argos, P. (1991) Nucleic Acids Res. 19, 1739-1740.; Prodromou, C., Artymiuk, P. J. & Guest, J. R. (1992) Eur. J. Biochem. 204, 599-609]. Iron-responsive elements (IREs) are stem-loop structures located in the untranslated regions of mRNAs. IRE-BP is required in the posttranscriptional regulation of ferritin mRNA translation and stabilization of transferrin receptor mRNA. In spite of substantial homology between the amino acid sequences of mammalian mitochondrial aconitase and IRE-BP, the mitochondrial protein does not bind IREs. However, there is a second aconitase, found only in the cytosol of mammalian tissues, that might serve as an IRE-BP. To test this possibility, we have prepared sufficient quantities of the heretofore poorly characterized beef liver cytosolic aconitase. This enzyme is isolated largely in its active [4Fe-4S] form and has a turnover number similar to that of mitochondrial aconitase. The EPR spectra of the two enzymes are markedly different. The amino acid composition, molecular weight, isoelectric point, and the sequences of six random peptides clearly show that these physicochemical and structural characteristics are identical to those of IRE-BP, and that c-aconitase is distinctly different from m-aconitase. In addition, both cytosolic aconitase and IRE-BP can have aconitase activity or function as IRE-BPs, as shown in the following paper and elsewhere [Zheng, L. Kennedy, M. C., Blondin, G. A., Beinert, H. & Zalkin, H. (1992) Arch. Biochem. Biophys., in press]. This leads us to the conclusion that cytosolic aconitase is IRE-BP.

Aconitate Hydratase↗
PubMed1992

Cellular regulation of the iron-responsive element binding protein: disassembly of the cubane iron-sulfur cluster results in high-affinity RNA binding.

The translation of ferritin mRNA and degradation of transferrin receptor mRNA are regulated by the interaction of an RNA-binding protein, the iron-responsive element binding protein (IRE-BP), with RNA stem-loop structures known as iron-responsive elements (IREs) contained within these transcripts. IRE-BP produced in iron-replete cells has aconitase (EC 4.2.1.3) activity. The protein shows extensive sequence homology with mitochondrial aconitase, and sequences of peptides prepared from cytosolic aconitase are identical with peptides of IRE-BP. As an active aconitase, IRE-BP is expected to have an Fe-S cluster, in analogy to other aconitases. This Fe-S cluster has been implicated as the region of the protein that senses intracellular iron levels and accordingly modifies the ability of the IRE-BP to interact with IREs. Expression of the IRE-BP in cultured cells has revealed that the IRE-BP functions either as an active aconitase, when the cells are iron-replete, or as an active RNA-binding protein, when the cells are iron-depleted. We compare properties of purified authentic cytosolic aconitase from beef liver with those of IRE-BP from tissue culture cells and establish that characteristics of the physiologically relevant form of the protein from iron-depleted cells resemble those of cytosolic aconitase apoprotein. We demonstrate that loss of the labile fourth iron atom of the Fe-S cluster results in loss of aconitase activity, but that more extensive cluster alteration is required before the IRE-BP acquires the capacity to bind RNA with the affinity seen in vivo. These results are consistent with a model in which the cubane Fe-S cluster is disassembled when intracellular iron is depleted.

Aconitate Hydratase↗
PubMed1992

Mutational analysis of active site residues in pig heart aconitase.

A cDNA encoding mature porcine heart aconitase was over-expressed in Escherichia coli under the control of a phage T7 promoter. Recombinant aconitase purified from E. coli was identical to the enzyme from pig and beef heart in size, [3Fe-4S] and [4Fe-4S] cluster structure and enzymatic activity. Nine amino acid residues in close proximity to the Fe-S cluster and bound substrate (Lauble, H., Kennedy, M.C., Beinert, H., and Stout, C.D. (1992) Biochemistry, in press) were replaced by site-directed mutagenesis. Fe-S cluster environment as indicated by the EPR spectrum, tight binding of substrate, and enzymatic activity were compared for the mutant and wild type enzymes. Significant perturbations were detected for all of the mutant enzymes. Replacements for Asp100, His101, Asp165, Arg580, and Ser642 result in a 10(3)-10(5)-fold drop in activity, which suggests that these residues are involved in critical aspects of the reaction. Arg580 appears to be a key residue for substrate binding, as shown by a 30-fold increased Km and loss of tight substrate binding. Results of mutagenesis support the interpretation of the x-ray model, namely that Asp100 and His101 form an ion pair for elimination of the substrate hydroxyl and Ser642 may function as a general base for proton abstraction from citrate or isocitrate in the dehydration step and protonation of cis-aconitate in the hydration step. Asp165 appears to play a critical role in the interaction of Fea with substrate.

Aconitate Hydratase↗
PubMed1992

Crystal structures of aconitase with isocitrate and nitroisocitrate bound.

The crystal structures of mitochondrial aconitase with isocitrate and nitroisocitrate bound have been solved and refined to R factors of 0.179 and 0.161, respectively, for all observed data in the range 8.0-2.1 A. Porcine heart enzyme was used for determining the structure with isocitrate bound. The presence of isocitrate in the crystals was corroborated by Mössbauer spectroscopy. Bovine heart enzyme was used for determining the structure with the reaction intermediate analogue nitroisocitrate bound. The inhibitor binds to the enzyme in a manner virtually identical to that of isocitrate. Both compounds bind to the unique Fe atom of the [4Fe-4S] cluster via a hydroxyl oxygen and one carboxyl oxygen. A H2O molecule is also bound, making Fe six-coordinate. The unique Fe is pulled away approximately 0.2 A from the corner of the cubane compared to the position it would occupy in a symmetrically ligated [4Fe-4S] cluster. At least 23 residues from all four domains of aconitase contribute to the active site. These residues participate in substrate recognition (Arg447, Arg452, Arg580, Arg644, Gln72, Ser166, Ser643), cluster ligation and interaction (Cys358, Cys421, Cys424, Asn258, Asn446), and hydrogen bonds supporting active site side chains (Ala74, Asp568, Ser571, Thr567). Residues implicated in catalysis are Ser642 and three histidine-carboxylate pairs (Asp100-His101, Asp165-His147, Glu262-His167). The base necessary for proton abstraction from C beta of isocitrate appears to be Ser642; the O gamma atom is proximal to the calculated hydrogen position, while the environment of O gamma suggests stabilization of an alkoxide (an oxyanion hole formed by the amide and side chain of Arg644). The histidine-carboxylate pairs appear to be required for proton transfer reactions involving two oxygens bound to Fe, one derived from solvent (bound H2O) and one derived from substrate hydroxyl. Each oxygen is in contact with a histidine, and both are in contact with the side chain of Asp165, which bridges the two sites on the six-coordinate Fe.

Aconitate Hydratase↗
PubMed1992

14,15N, 13C, 57Fe, and 1,2H Q-band ENDOR study of Fe-S proteins with clusters that have endogenous sulfur ligands.

The benefits of performing ENDOR experiments at higher microwave frequency are demonstrated in a Q-band (35 GHz) ENDOR investigation of a number of proteins with [nFe-mS] clusters, n = 2, 3, 4. Each protein displays several resonances in the frequency range of 0-20 MHz. In all instances, features are seen near v approximately 13 and 8 MHz that can be assigned, respectively, to "distant ENDOR" from 13C in natural-abundance (1.1%) and from 14N (the delta m1 = +/- 2 transitions); the nuclei involved in this phenomenon are remote from and have negligible hyperfine couplings to the cluster. In addition, a number of proteins show local 13C ENDOR signals with resolved hyperfine interactions; these are assigned to the beta carbons of cysteines bound to the cluster [A(13C) approximately 1.0 MHz]. Five proteins show resolved, local delta m1 = +/- 2 ENDOR signals from 14N with an isotropic hyperfine coupling, 0.4 less than or equal to A(14N) less than or equal to 1.0, similar to those seen in ESEEM studies; these most likely are associated with N-H...S hydrogen bonds to the cluster. Anabaena ferredoxin further shows a signal corresponding to A(14N) approximately 4 MHz. Quadrupole coupling constants are derived for both local and distant 14N signals. The interpretation of the data is supported by studies on 15N- and 13C-enriched ferredoxin (Fd) from Anabaena 7120, where the 15N signals can be clearly correlated with the corresponding 14N signals and where the 13C signals are strongly enhanced. Thus, the observation of 14N delta m1 = +/- 2 signals at Q-band provides a new technique for examining weak interactions with a cluster.(ABSTRACT TRUNCATED AT 250 WORDS)

Aconitate Hydratase↗
PubMed1992

Binding of cytosolic aconitase to the iron responsive element of porcine mitochondrial aconitase mRNA.

The 5' end of porcine mitochondrial aconitase mRNA contains an iron responsive element (IRE)-like secondary structure (T. Dandekar, R. Stripecke, N. K. Gray, B. Goosen, A. Constable, H. E. Johansson, and M. W. Hentze (1991) EMBO J. 10, 1903-1909). A protein from a liver extract binds to a mitochondrial aconitase RNA probe and supports the identification of this sequence as an IRE. Purified cytosolic aconitase but not the mitochondrial enzyme binds to this IRE as well as to a ferritin IRE. All forms of cytosolic aconitase, [4Fe-4S] enzyme, [3Fe-4S] enzyme and apoenzyme bind with similar affinity. A Kd of 0.25 nM was calculated for the apoaconitase-IRE interaction from Scatchard analysis. These results support the conclusion that cytosolic aconitase is an IRE-binding protein which may regulate translation of mitochondrial aconitase mRNA.

Aconitate Hydratase↗
PubMed1991

Investigations on microbial sulfur respiration. Isolation, purification, and characterization of cellular components from Spirillum 5175.

The sulfur-reducing bacterium Spirillum 5175 was investigated with regard to membrane constituents that might be part of the sulfur oxidoreductase which converts elemental sulfur to hydrogen sulfide. Regardless of the electron acceptor used for cultivation of the bacteria, i.e. elemental sulfur, fumarate, or nitrate (Sp. 5175S,F,N), the qualitative pattern of cytochromes and Fe-S proteins did not change significantly, as documented by ultraviolet/visible and electron paramagnetic resonance spectroscopy of oxidized (as isolated) and reduced (dithionite) samples. With elemental sulfur the prominent cytochrome exhibited absorption maxima at 553, 522.5 and 426 nm in the reduced state. In fumarate-grown cells two prominent cytochromes were found with maxima at 561, 551, 530, 521 and 430 nm. Two b-type cytochromes with Em at -198 mV and -20 mV vs the standard hydrogen electrode were identified in the membrane fraction of Sp. 5175F. A yellow pigment was extracted and identified as a flexirubin-type pigment. Although present in large quantities, it seemed not to be involved in the reduction of elemental sulfur. Menaquinone, MK 6 (Mr 580) was the prominent quinone identified in Sp. 5175. Characterization of a second quinone was not attempted because of its much lower concentration. The membrane constituents of Sp. 5175 were solubilized by a variety of detergents and detergent mixtures. A colorimetric procedure with photochemically reduced phenosafranin as the electron donor and cysteamine trisulfide (RS-S-SR, R = -CH2CH2NH2) as the electron acceptor was used to detect sulfur oxidoreductase activity. Three membrane proteins of Sp. 5175 were purified: (1) an [NiFe] hydrogenase, homogeneous by SDS/polyacrylamide gel electrophoresis, with electron paramagnetic resonance signals as isolated at gx,y,z = 2.01, 2.16, 2.33 (100 K), and a strong signal at g = 2.02 below 20 K; (2) a cytochrome b, Fe-S-dependent fumarate reductase, and (3) a protein apparently linked to the sulfur oxidoreductase activity. In contrast to fumarate reductase, no b-type cytochrome was present in the fractions exhibiting sulfur oxidoreductase activity. The presence of Fe-S centers was demonstrated by electron paramagnetic resonance spectroscopy at 10 K. It is not clear whether the c-type cytochrome in the same fractions is part of the sulfur-reducing apparatus of Sp. 5175.

Cell Membrane↗