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J K Grady

Publications and source records attributed to J K Grady.

12 recordsLinked to original sources
PubMed1998

Mutations at nonliganding residues Tyr-85 and Glu-83 in the N-lobe of human serum transferrin. Functional second shell effects.

The x-ray crystal structure of the N-lobe of human serum transferrin has shown that there is a hydrogen bond network, the so-called "second shell," around the transferrin iron binding site. Tyrosine at position 85 and glutamic acid at position 83 are two nonliganding residues in this network in the human serum transferrin N-lobe (hTF/2N). Mutation of each of these two amino acids has a profound effect on the metal binding properties of hTF/2N. When Tyr-85 is mutated to phenylalanine, iron release from the resulting mutant Y85F is much more facile than from the parent protein. Elimination of the hydrogen bond between Tyr-85 and Lys-296 appears to interfere with the "di-lysine (Lys-206-Lys-296) trigger," which affects the iron binding stability of the protein. Surprisingly, mutation of Glu-83 to alanine leads to the absence of one of the normal iron binding ligands; introduction of a monovalent anion is able to restore the normal first coordination sphere. The missing ligand appears to be His-249, as revealed by comparison of the metal binding behaviors of mutants H249Q and E83A and structural analysis. Glu-83 has a strong H bond linkage with His-249 in apo-hTF/2N, which helps to hold the His-249 in the proper position for iron binding. Disabling Glu-83 by mutation to an alanine seriously disturbs the H bond network, allowing His-249 to move away. A monovalent anion can help reestablish the normal network by providing a negative charge near the position of Glu-83 to reach charge balance, so that ligand His-249 is available again for iron binding.

Base Sequence↗
PubMed1997

Inequivalence of the two tyrosine ligands in the N-lobe of human serum transferrin.

Human serum transferrin N-lobe (hTF/2N) has four iron-binding ligands, including one histidine, one aspartate, and two tyrosines. The present report elucidates the inequivalence of the two tyrosine ligands (Tyr 95 and Tyr 188) on the metal-binding properties of hTF/2N by means of site-directed mutagenesis, metal release kinetics, and absorption and electron paramagnetic resonance (EPR) spectroscopies. When the liganding tyrosines were mutated individually to phenylalanine, the resulting mutant Y95F showed a weak binding affinity for iron and no affinity for copper, whereas, mutant Y188F completely lost the ability to bind iron but formed a stable complex with copper. Since other studies have demonstrated that mutations of the other two ligands, histidine and aspartate, did not completely abolish iron binding, the present findings suggest that the tyrosine ligand at position 188 is essential for binding of iron to occur. Replacement of Tyr 188 with phenylalanine created a favorable chemical environment for copper coordination but a fatal situation for iron binding. The positions of the two liganding tyrosines in the metal-binding cleft suggest a reason for the inequivalence.

Aspartic Acid↗
PubMed1995

The effect of salt and site-directed mutations on the iron(III)-binding site of human serum transferrin as probed by EPR spectroscopy.

The effects of site-directed mutation and salt on the iron(III)-binding site of the recombinant half-molecule of the N-terminal lobe (hTf/2N) of human transferrin was studied by EPR spectroscopy. Changes were observed in the EPR spectra of all variants investigated (D63S, D63C, G65R, K206Q, H207E, H249E, H249Q, K296E and K296Q) compared with that of the wild-type protein. The most pronounced changes in the metal site were caused by replacement of the coordinating residues, Asp-63 and His-249, and the non-coordinating residue Lys-296, which is located in the hinge region of the iron-binding cleft. The EPR spectral changes from replacement of other non-coordinating residues were more subtle, indicating small changes in Fe3+ coordination to the protein. The EPR spectrum of variant G65R suggests that it adopts two distinct conformations in solution, one in which the two domains forming the iron-binding cleft are closed and one in which they are open; in the latter instance Asp-63 is no longer coordinated to the Fe3+. Chloride-binding studies on hTf/2N, K206Q, H207E, K296Q and K296E showed similar binding isotherms, indicating that none of the hinge region residues replaced, i.e. Lys-206, His-207 or Lys-296, are the sites of chloride binding. The results show that the coordination environment of the Fe3+ is sensitive to structural changes from site-directed mutation of both remote and coordinated residues and also to chloride-binding and ionic strength effects.

Binding Sites↗
PubMed1994

Salt effects on the physical properties of the transferrins.

Salts are known to have a pronounced effect on the spectroscopic, thermodynamic and kinetic properties of human serum transferrin. The present study was undertaken to examine the effect of NaCl on the related proteins ovotransferrin and lactoferrin. EPR difference spectroscopy was used to probe changes in the metal site of these proteins. Sodium chloride was found to perturb the g' = 4.3 EPR spectra of both ovotransferrin and lactoferrin but in different ways. The spectrum of ovotransferrin is reduced in amplitude with a broad feature appearing at g' = 4.8 whereas there is a loss of resolution of the doublet feature at the peak of the EPR derivative spectrum for lactoferrin. The increase in the amplitude of the ovotransferrin EPR difference spectrum (spectrum without NaCl minus spectrum with NaCl) as a function of NaCl concentration is suggestive of saturation binding. A Hill plot binding isotherm gave n = 1.87 +/- 0.32 and log K = 1.49 +/- 0.03 for ovotransferrin, where n is the number of C1- ions binding to either one or both iron containing lobes of the protein and K is the overall association constant. Preliminary measurements with lactoferrin gave n = 1.95 +/- 0.34 and log K = 1.41 +/- 0.06. These results are similar to those previously reported for serum transferrin and suggest that Cl- binds to all the transferrins with strong pairwise cooperativity. This binding may reflect a functional role for chloride and other physiological anions in the uptake and release of iron by the transferrins.

Chlorides↗
PubMed1993

Characterization of the non-heme iron center of human 5-lipoxygenase by electron paramagnetic resonance, fluorescence, and ultraviolet-visible spectroscopy: redox cycling between ferrous and ferric states.

Purified human 5-lipoxygenase, a non-heme iron containing enzyme, has been characterized by electron paramagnetic resonance, (EPR), ultraviolet (UV)-visible and fluorescence spectroscopy. As isolated, the enzyme is largely in the ferrous state and shows a weak X-band EPR signal extending from 0 to 700 G at 15 K, tentatively ascribed to integer spin Fe(II). Titration of the protein with 13-HPOD (13-hydroperoxyoctadecadienoic acid) generates a strong multicomponent EPR signal in the g' approximately 6 region, a yellow color associated with an increased absorption between 310 and 450 nm (epsilon 330nm = 2400 M-1 cm-1), and a 17% decrease in the intrinsic protein fluorescence. The multiple component nature of the g' approximately 6 signal indicates that the metal center in its oxidized state exists in more than one but related forms. The g' approximately 6 EPR signal and the yellow color reach a maximum when approximately 1 mol of 13-HPOD is added/mol of iron; the resultant EPR spectrum accounts quantitatively for all of the iron in the protein with a signal at g' = 4.3 representing less than 3% of the total iron in the majority of samples. Addition of a hydroxyurea reducing agent abolished the g' approximately 6 signal and yellow color of the protein and also reversed the decrease in fluorescence caused by the oxidant 13-HPOD. The results indicate that the g' approximately 6 EPR signal, the yellow color, and the decreased fluorescence are associated with the formation of the Fe(III) form of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Arachidonate 5-Lipoxygenase↗
PubMed1991

Fluorescence and kinetic properties of Ru(III) (NH3)5 modified transferrin.

Diferric transferrin was modified using aquopentaammine ruthenium(II), a reagent for surface-accessible uncoordinated histidines. Introduction of the cationic Ru(III) (NH3)3 + 5 group on the imidazole of only 5.5 of the 17 uncoordinated histidines enhances the rates of pyrophosphate-assisted iron removal from the N-terminal and C-terminal binding sites by 16- and 2-fold, respectively. This differential effect on the kinetics of the two sites may partially explain why in the native protein the N-terminal site is more labile than the C-terminal site in acidic solutions where histidine residues become positively charged through protonation. The distance between the metal site and nearby uncoordinated histidines was estimated from fluorescence energy transfer measurements using Tb (III) as the donor and pentaammine ruthenium(III)-labeled imidazole of histidine as the acceptor chromophore. A Tsou Chen-Lu statistical analysis of the fluorescence quenching data suggest that two residues in each lobe of the protein are involved in quenching the fluorescence. By using estimates for the index of refraction and the quantum yield and assuming the energy transfer follows parallel first-order kinetics, an upper limit for the donor-acceptor distance of about 1.4 nm was obtained, assuming two uncoordinated histidine residues equidistant from the metal. His-207 and His-242 in the N-terminal lobe of transferrin and His-535 and His-577 in the C-terminal lobe are within this distance, based on information from the lactoferrin crystal structure. It is postulated that His-207 in the N-terminal lobe and His-535 in the C-terminal lobe are the uncoordinated residues that, when protonated or modified with Ru(III) (NH3)3 + 5, lead to accelerated loss of iron from the two binding sites of the protein.

Binding Sites↗
PubMed1989

Hydroxyl radical production during oxidative deposition of iron in ferritin.

The chemistry of oxidative deposition of iron(III) in ferritin and apoferritin is poorly understood. This study was undertaken to look for radicals formed as the hydrous ferric oxide core is developed from Fe(II) and O2. Radicals were observed indirectly by using the spin-trapping reagent N-tert-butyl-alpha-phenylnitrone (PBN) at room temperature and directly by measuring ESR spectra of frozen solutions at 77 K. In both instances, radical production was inhibited by the hydroxyl radical scavenging agents dimethyl sulfoxide, thiourea, and mannitol and enhanced by the addition of hydrogen peroxide. These findings strongly suggest that hydroxyl radical, produced from the iron-catalyzed Haber-Weiss reaction, is a by-product of core formation in ferritin and is a precursor to the observed radicals. The yield of ESR-observable and spin-trapped radicals is quite low, being at the micromolar level when millimolar concentrations of ferrous ion are employed. Furthermore, radical production appears to be confined to the interior of the ferritin molecule, where cellular components would be protected from the oxygen-derived toxic effects of iron. It is postulated that hydroxyl radical-medicated oxidative damage to the protein, a process that may contribute to the formation of hemosiderin from ferritin, leads to the observed radicals. By serving as a sink for hydroxyl radical, the protein shell may therefore efficiently minimize damage to other biomolecules in the cell.

Apoferritins↗
PubMed1988

Radicals from "Good's" buffers.

Oxidative deposition of iron in ferritin or the autoxidation of iron in the absence of protein produces radicals from Good's buffers. Radical species are formed from the piperazine ring-based buffers Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Epps 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, and Pipes 1,4-piperazinediethanesulfonic acid, but not from Mes (4-morpholineethanesulfonic acid) which contains a morpholine ring. The radicals all have half-lives around 10 min and display very similar electron paramagnetic resonance spectra consisting of at least 30 lines. The Hepes radical can be formed by the addition of potassium superoxide directly to the buffer and its production during iron(II) autoxidation is inhibited by superoxide dismutase (EC 1.15.1.1). Catalase (EC 1.11.1.6) accelerates the decay of the EPR spectrum. Thus the buffer radicals are secondary radical species produced from oxygen radicals formed during the iron catalyzed Haber-Weiss process. The deoxyribose/thiobarbituric acid assay for hydroxyl radical production shows that Hepes is an effective hydroxyl radical scavenging agent. The Hepes radical can also be formed electrolytically at a potential of +0.8 V (vs standard hydrogen electrode). Oxidation of Hepes at pH 10 during the autoxidation of iron(II) or by the addition of hydrogen peroxide produces a nitroxide radical. These results indicate that piperazine ring Good buffers should be avoided in studies of redox processes in biochemistry.

Buffers↗
PubMed1986

Vanadium complexes of transferrin and ferritin in the rat.

Vanadium associates with serum transferrin of rats administered vanadyl(IV) sulfate or ammonium metavanadate(V) by gastric intubation. Low molecular weight species account for only 3% of the vanadium present in plasma. The element distributes between the two major isotransferrins in proportion to their concentrations. Rat apotransferrin binds both vanadium(IV) and vanadium(V), forming 2:1 metal-protein complexes in both instances. Although the two isotransferrins apparently differ in their physiological properties, they exhibit identical vanadyl(IV) (VO2+) EPR spectra, indicating identical or very similar metal binding sites for both proteins. In contrast to other transferrins, the two sites of the rat protein are spectroscopically indistinguishable and exhibit a VO2+ EPR spectrum similar to that of the C-terminal metal binding site of human serum transferrin. VO2+ EPR signals are observed with liver, spleen, and kidney tissue samples from animals maintained on a vanadium-supplemented diet. These signals arise from a specific intracellular VO2+ complex with the iron storage protein ferritin.

Animals↗
PubMed1986

The influence of uncoordinated histidines on iron release from transferrin. A chemical modification study.

Histidine residues that influence the chelate-mediated removal of iron from transferrin have been investigated. Diferric human serum transferrin was chemically modified to various extents using ethoxyformic anhydride, a reagent for histidines. A kinetic analysis of the modification reaction revealed the presence of a fast reacting pool of 9 +/- .8 histidine residues and a slow reacting pool of 5.8 +/- .6 residues. There are 18 histidine residues in transferrin. The rates of modification of the two pools differed by a factor of 5. The pyrophosphate-mediated removal of iron from the two binding sites of native and partially modified transferrins was studied at pH 6.9 using desferrioximine B as a terminal iron acceptor. Under these conditions, the rate of iron removal from the NH2-terminal site was about six times faster than from the COOH-terminal site. Both rates were significantly reduced, i.e. by a factor of approximately 6-8, upon complete ethoxyformylation of all reactive histidines on the protein. The kinetic data of partially modified transferrins were analyzed by the Tsou Chen-Lu statistical method; the results are consistent with the hypothesis that modification of a single uncoordinated histidine in each of the two iron binding domains stabilizes the protein kinetically against loss of iron. The dependence of the iron removal reaction on pH is consistent with such an interpretation. The putative histidines, although not ligands, may be close to the metal in both binding sites, thus influencing the rate of iron removal by pyrophosphate. These histidines belong to the pool of rapidly modified residues and thus are readily accessible to solvent and chelators.

Diethyl Pyrocarbonate↗
PubMed1976

Neuroparallelogram. A cerebral angiographic system allowing for isocentric magnification.

The Neuroparallelogram, a radiographic system with an opposing tube-serial film changer provides for isoentric 2:1 magnification, stero radiography, a.p. or p.a. projections, more accurately positioned bi-plane angiography and the capacity for angiotomography. Angiographic examinations are expedited not only by the ease of set-up for different projections, but also by the capacity to accommodate for head rotation without repositioning the patient. The tube and film changer can be uncoupled so that routine films may be obtained. The improved detail of magnification radiography and the simplicity of positioning can also be used to perform routine skull radiography.

Cerebral Angiography↗