Some reactions of carbon monoxide and oxygen with carbodi-imide-modified cytochrome c.
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The reactivity of carbodi-imide-modified tuna and horse heart cytochromes c with the ferrous ion ligands CO and O2 has been studied. Both modified cytochromes bind one molecule of CO. Stopped-flow and flash-photolysis experiments indicate the presence of three kinetic processes in the reaction of the cytochromes with CO. The second-order rate constants associated with all three kinetic process are pH-independent being 2.8 x 10(5) M-1.s-1, 3.8 x 10(4) M-1.s-1 and 4 x 10(3) M-1.s-1 under all conditions studied. The concentration-dependence of the contributions made by each of the processes to the overall absorbance change indicates that the fast and slow kinetic phases are associated with two forms of the cytochromes which are in equilibrium, whereas the intermediate phase arises from a separate cytochrome species. The quantum yield for the photodissociation of CO from the ferrous cytochromes is unusually low. Both modified cytochromes are capable of binding and reducing O2. In the presence of excess reductant, the modified cytochromes can catalytically reduce large molar excesses of O2. In the absence of excess reducing agent, the oxy complex initially formed undergoes a pH-dependent intramolecular electron-transfer process with half-life approx. 10 min. EDC [1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide]-promoted internal cross-linking is proposed to account for differences between the EDC-modified proteins and carboxymethylated cytochrome c. (+info)
Rotations of a few cross-bridges in muscle by confocal total internal reflection microscopy.
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In order to measure the cycling of a few ( approximately 6) myosin heads in contracting skeletal muscle, myofibrils were illuminated by Total Internal Reflection and observed through a confocal aperture. Myosin heads rotated at a rate approximately equal to the ATPase rate, suggesting that bulk ATPase of a whole muscle reflects the cycle frequency of individual heads. (+info)
A carboxylic residue at the high-affinity, Mn-binding site participates in the binding of iron cations that block the site.
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The role of carboxylic residues at the high-affinity, Mn-binding site in the ligation of iron cations blocking the site [Biochemistry 41 (2000) 5854] was studied, using a method developed to extract the iron cations blocking the site. We found that specifically bound Fe(III) cations can be extracted with citrate buffer at pH 3.0. Furthermore, citrate can also prevent the photooxidation of Fe(II) cations by YZ. Participation of a COOH group(s) in the ligation of Fe(III) at the high-affinity site was investigated using 1-ethyl-3-[(3-dimethylamino)propyl] carbodiimide (EDC), a chemical modifier of carboxylic amino acid residues. Modification of the COOH groups inhibits the light-induced oxidation of exogenous Mn(II) cations by Mn-depleted photosystem II (PSII[-Mn]) membranes. The rate of Mn(II) oxidation saturates at > or = 10 microM in PSII(-Mn) membranes and > or = 500 microM in EDC-treated PSII (-Mn) samples. Intact PSII(-Mn) membranes have only one site for Mn(II) oxidation via YZ (dissociation constant, Kd = 0.64 microM), while EDC-treated PSII(-Mn) samples have two sites (Kd = 1.52 and 22 microM; the latter is the low-affinity site). When PSII(-Mn) membranes were incubated with Fe(II) before modifier treatment (to block the high-affinity site) and the blocking iron cations were extracted with citrate (pH 3.0) after modification, the membranes contained only one site (Kd = 2.3 microM) for exogenous Mn(II) oxidation by Y(Z)() radical. In this case, the rate of electron donation via YZ saturated at a Mn(II) concentration > or = 15 microM. These results indicate that the carboxylic residue participating in Mn(II) coordination and the binding of oxidized manganese cations at the HAZ site is protected from the action of the modifier by the iron cations blocking the HAZ site. We concluded that the carboxylic residue (D1 Asp-170) participating in the coordination of the manganese cation at the HAZ site (Mn4 in the tetranuclear manganese cluster [Science 303 (2004) 1831]) is also involved in the ligation of the Fe cation(s) blocking the high-affinity Mn-binding site. (+info)
A simple, cross-linked collagen tissue substitute for corneal implantation.
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PURPOSE: To develop a simple corneal substitute from cross-linked collagen. METHODS: Porcine type I collagen (10%; pH 5), was mixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The final homogenous solution was molded to corneal dimensions, cured, and then implanted into rabbits and minipigs by lamellar keratoplasty. The implants were followed for up to 6 months after surgery. Clinical examinations of the cornea included detailed slit lamp biomicroscopy, in vivo confocal microscopy, topography and esthesiometry for nerve function. Histopathologic examinations were also performed on rabbit corneas harvested after 6 months. RESULTS: Cross-linked collagen (refractive index, 1.35) had optical clarity superior to human corneas. Implanted into rabbit and porcine corneas, only 1 of 24 of the surgical corneas showed a slight haze at 6 months after surgery. All other implants showed no adverse reactions and remained optically clear. Topography showed a smooth surface and a profile similar to that of the contralateral nonsurgical eye. The implanted matrices promoted regeneration of corneal cells, tear film, and nerves. Touch sensitivity was restored, indicating some restoration of function. The corneas with implants showed no significant loss of thickness and demonstrated stable host-graft integration. CONCLUSIONS: Collagen can be adequately stabilized, using water soluble carbodiimides as protein cross-linking reagents, in the fabrication of corneal matrix substitutes for implantation. The simple cross-linking methodology would allow for easy fabrication of matrices for transplantation in centers where there is a shortage of corneas, or where there is need for temporary patches to repair perforations in emergency situations. (+info)
Effect of curing time and concentration for a chemical treatment that improves surface gliding for extrasynovial tendon grafts in vitro.
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The purpose of this study was to evaluate whether treatment time and concentration of these reagents have an effect on the resulting gliding resistance. Forty peroneus longus (PL) tendons were used, from 20 adult mongrel dogs, along with the A2 pulley obtained from the ipsilateral hind paw. After the baseline gliding resistance was measured, the PL tendons were treated with one of three concentrations of hyaluronic acid (HA) and 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) or N-hydroxysuccinimide (NHS) mixed with 10% gelatin for various times (5, 30, and 60 min). Tendon friction was measured over 1000 cycles of simulated flexion/extension motion. Gliding resistance of the untreated PL tendons had no significant difference among the groups. After surface treatment with low concentration of HA and EDC/NHS for 5-min cure, the gliding resistance was similar to that of the untreated PL tendon and significantly higher than its 30- and 60-min treatment. For the rest of high concentration of HA and EDC/NHS groups, the gliding resistance was lower than that of untreated PL tendon. However, there was no significant difference among the timing points. It is possible to optimize the effect of surface treatment on friction and durability by regulating cure time and concentration of reagents in a canine extrasynovial tendon in vitro. (+info)
Surface treatment of flexor tendon autografts with carbodiimide-derivatized hyaluronic Acid. An in vivo canine model.
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BACKGROUND: Clinical and experimental studies have demonstrated that restrictive adhesions and poor digital motion are common complications after extrasynovial tendon grafting in an intrasynovial environment. The purpose of this study was to test the hypothesis that surface modification of an extrasynovial tendon with use of a carbodiimide-derivatized hyaluronic acid-gelatin polymer (cd-HA) improves gliding ability and digital function after tendon grafting in a canine model in vivo. METHODS: The peroneus longus tendons from both hindpaws of twenty-four dogs were harvested and transplanted to replace the flexor digitorum profundus tendons in the second and fifth digits of one forepaw. Prior to grafting, one of the peroneus longus tendons was coated with cd-HA, which consists of 1% hyaluronic acid, 10% gelatin, 0.25% 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and 0.25% N-hydroxysuccinimide (NHS), while the other was immersed in saline solution only. Eight dogs were killed at one, three, and six weeks. Digital normalized work of flexion, tendon gliding resistance, and hyaluronic acid quantification (with the hyaluronic acid-binding-protein staining technique) were the outcome measures. RESULTS: The normalized work of flexion of the tendons treated with cd-HA was significantly lower than that of the saline-solution-treated controls at each time-point (p < 0.05). The gliding resistance of the cd-HA group was significantly lower than that of the saline-solution group at three and six weeks (p < 0.05). The ratio between the intensity of staining of the cd-HA-treated tendons with that of the saline-solution-treated controls was significantly greater at time-0 than at three or six weeks (p < 0.05), but there was no significant difference between time-0 and one-week values. CONCLUSIONS: Treating the surface of an extrasynovial tendon autograft with a carbodiimide-derivatized hyaluronic acid-gelatin polymer decreases digital work of flexion and tendon gliding resistance in this flexor tendon graft model in vivo. CLINICAL RELEVANCE: cd-HA gelatin may provide surgeons with a new and useful method to improve the quality of tendon graft surgery. (+info)
Interaction of ferredoxin with carbon monoxide dehydrogenase from Clostridium thermoaceticum.
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Acetogenic bacteria, as determined with Clostridium thermoaceticum, synthesize acetate by the acetyl-CoA pathway which involves the reduction of CO2 to a methyl group and then combination of the methyl with CoA and a carbonyl group formed from CO or CO2 (Wood, H.G., Ragsdale, S.W., and Pezacka, E. (1986) Trends Biochem. Sci. 11, 14-18). Carbon monoxide dehydrogenase (CODH), the key enzyme in this pathway not only catalyzes the oxidation of CO to CO2 but also the final step, the synthesis of acetyl-CoA from a methyl group, CO, and CoA. Previously, it has been shown that ferredoxin can stimulate exchange of CO with CH3 14COSCoA (Ragsdale, S.W., and Wood, H.G. (1985) J. Biol. Chem. 260, 3970-3977). In the present study, it has been observed that ferredoxin and CODH can form an electrostatically stabilized complex. In order to identify the ferredoxin binding region on CODH, the ferredoxin and CODH were cross-linked by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The cross-linked CODH-ferredoxin adduct was enzymatically as active as the uncross-linked complex. The native CODH and cross-linked CODH-ferredoxin complex were subjected to cyanogen bromide cleavage. By comparison of the high-performance liquid chromatography peptide profiles, it was observed that the mobility of at least one peptide is altered in the CODH-ferredoxin cross-linked complex. The peptide was identified with residues 229-239 of the alpha-subunit of CODH. (+info)
Identification of specific carboxyl groups on Anabaena PCC 7119 flavodoxin which are involved in the interaction with ferredoxin-NADP+ reductase.
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Flavodoxin from the nitrogen-fixing cyanobacteria Anabaena PCC 7119 forms an electron-transfer complex with ferredoxin--NADP+ reductase (FNR) from the same organism. The complex is mainly governed by electrostatic interactions between side-chain amino groups of the reductase and carboxyl residues of flavodoxin. In order to localize the binding site on flavodoxin, chemical modification of its carboxyl groups has been carried out. Treatment of flavodoxin with a water-soluble carbodiimide, N-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), in the presence of a nucleophile, glycine ethyl ester, caused a time-dependent modification of the protein that is responsible for the loss of its ability to participate as electron carrier in the photoreduction of NADP+ by chloroplast membranes, and also in NADPH--cytochrome-c reductase activity, by about 85%. Nevertheless, the ability of flavodoxin to receive electrons from the reducing side of photosystem I was much less affected. The inhibition was enhanced at low pH, suggesting that carboxylic acid groups were the target of chemical modification. Treated flavodoxin failed to form covalent complexes with FNR and the dissociation constant for the non-covalent complex with FNR was fourfold higher. After tryptic digestion of a sample of flavodoxin modified by EDC in the presence of [1-14C]glycine ethyl ester, two major radioactive peptides were isolated. The first protein fragment contained three carboxylic residues (Asp123, Asp126 and Asp129), corresponding to the region where long-chain flavodoxins show an insert compared to short-chain flavodoxins. The second peptide corresponded to a similar region, either in the amino acid sequence or in the three-dimensional structure of the protein and also containing three carboxyl groups (Asp144, Glu145 and Asp146). Four of these carboxyl groups (Asp123, Asp126, Asp144 and Asp146) are highly conserved in all long-chain flavodoxins, suggesting that they could play an essential role in substrate recognition. (+info)