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Biomedical subjects

C E Wolff

Publications and source records attributed to C E Wolff.

11 recordsLinked to original sources
PubMed1996

Rhodobacter sphaeroides phosphoribulokinase: binary and ternary complexes with nucleotide substrate analogs and effectors.

Rhodobacter sphaeroides phosphoribulokinase (PRK) binds ATP substrate, as well as spectroscopically active ATP analogs (trinitrophenyl-ATP and ATP gamma S-acetamidoproxyl), to form stable binary complexes. Stoichiometric binding of these nucleotide triphosphates in PRK's substrate site is observed not only with wild-type enzyme but also with D42A and D169A mutants. The demonstration that these mutants contain a full complement of functional substrate binding sites indicates their substantial structural integrity and underscores the significance of their markedly diminished catalytic activity [Charlier et al. (1994) Biochemistry 33, 9343-9350]. Similarly, PRK forms a stable binary complex with the allosteric activator NADH. The negative allosteric effector AMP displaces activator NADH but not substrate from their respective binary complexes with enzyme. When trinitrophenyl-ATP, a fluorescent nucleotide triphosphate that functions as an alternative PRK substrate, forms a binary complex with enzyme, its fluorescence emission is enhanced and lambda max shifted from approximately 557 to 545 nm. Upon formation of a binary PRK-NADH complex, the fluorescence emission of the dinucleotide effector is also enhanced and the lambda max shifted from approximately 460 to 440 nm. PRK forms stable ternary complexes containing NADH and either ATP or trinitrophenyl-ATP. Due to energy transfer, NADH fluorescence in the ternary complex with trinitrophenyl-ATP is markedly quenched, allowing an estimation of the spatial separation between this novel donor/acceptor pair.

Adenosine Triphosphate↗
PubMed1993

1H NMR-based determination of the three-dimensional structure of the human plasma fibronectin fragment containing inter-chain disulfide bonds.

Human plasma fibronectin is a plasma glycoprotein that plays an important role in many biological processes. It consists of two identical 230-250-kDa subunits that are joined by two disulfide bonds near their carboxyl termini. Each subunit contains various binding domains composed of three types of homologous repeats. Recent work has determined the three-dimensional structures of various repeat fragments, but little is known about the three-dimensional structure of the carboxyl-terminal region. A recent NMR study of a plasmin-digested carboxyl-terminal inter-chain disulfide-linked heptapeptide dimer has proposed that the two subunits are arranged in an antiparallel fashion (An et al. (1992) Biochemistry 31, 9927-9933). We have now determined the three-dimensional structure for a substantial portion of a trypsin-digested interchain disulfide-linked 52-residue (6 kDa) fragment of the carboxyl-terminal of human plasma fibronectin (which includes the above-mentioned heptapeptide dimer) using two-dimensional NMR methods and a new strategy for NMR-based protein structure determination. The NMR data requires that the two chains in the dimer be linked in a symmetric, antiparallel arrangement. The resulting monomer conformation consists of two twisted or coiled segments, Thr3-Asn6 and Ile9-Phe12, connected by the Cys7-Pro8 residues in extended conformations, with the two monomer chains cross-linked at residues Cys7 and Cys11. The conformation of the heptapeptide dimer region differs substantially from the conformation proposed by An et al.

Amino Acid Sequence↗
PubMed1993

Solution structure of human plasma fibronectin under different solvent conditions. Fluorescence energy transfer, circular dichroism and light-scattering studies.

Human plasma fibronectin is a high molecular weight (530,000), multi-domain, modular glycoprotein, consisting of two nearly identical subunits disulfide-bridged close to their C-terminal ends. Three sites that can be differentially labeled with fluorescent probes are present on each fibronectin subunit, the transglutaminase-sensitive Gln3 residue and the two free sulfhydryl residues, Cys1201 and Cys2196. These sites are located, respectively, in the N-terminal heparin/fibrin-binding domain, between the central DNA and cell-binding domains, and just before the C-terminal fibrin-binding domain. To map the relative spatial arrangement of these domains, steady-state and lifetime fluorescence energy transfer techniques were employed. Our results show that the minimal intramolecular distances between the labeled Gln3-Cys1201 and Gln3-Cys2196 pairs are 5.5(+/- 0.6) nm and 5.7(+/- 0.7) nm, respectively, as measured by steady-state methods. Lifetime methods gave somewhat higher distances of 8.1(+/- 0.2) nm and 7.6(+/- 0.2) nm, respectively, between these sites. The binding of heparin or subjection to high ionic strength had only a minor effect, while in the presence of 50% (w/v) glycerol, an increase of about 25% in the intramolecular distances between these sites was observed. A similar effect was induced by binding of fibronectin to the surface of Cytodex beads, an event which was previously shown instead to markedly increase the intersubunit distances between the Gln3-Gln3 and Cys1201-Cys1201 pairs. The solution structure of fibronectin was further investigated by elastic light-scattering and circular dichroism measurements. By elastic light-scattering, the radius of gyration of fibronectin was found to be 15.3(+/- 0.8) nm in the presence of 30% (w/v) glycerol, in contrast to a value of 8.6(+/- 0.3) nm under physiological conditions. Far and near ultraviolet circular dichroism spectra showed that only minor changes in the secondary structure of fibronectin take place on increasing the glycerol content of the solvent up to 34% (w/v). Our results complement previously available information on the solution structure of fibronectin and on its transition from the native compact conformation to a more expanded form on increasing ionic strength or glycerol content. In either situation, fibronectin seems to retain a basic structural core, in which the N-terminal, the central and the C-terminal regions of the two subunits strongly interact with each other. A major role of hydrophobic forces, in stabilizing the fibronectin conformations under these conditions, is therefore postulated. The transition to the extended forms seen in many electron micrographs can instead be explained by disruption of the proposed structural core upon adsorption to surfaces.

Circular Dichroism↗
PubMed1990

Inter-sulfhydryl distances in plasma fibronectin determined by fluorescence energy transfer: effect of environmental factors.

Human plasma fibronectin, a dimeric glycoprotein, contains two cryptic free sulfhydryl groups per chain. Recent observations revealed that upon binding to a gelatin-coated surface the SH1 site, located between the DNA-binding and cell-binding domains, is partially exposed, while the SH2 site, situated within the carboxyl-terminal fibrin-binding domain, remains buried. Utilizing this newly discovered property of plasma fibronectin, we have developed a procedure to introduce maleimide derivatives of fluorescent probes such as N-(1-pyrenyl)maleimide, 7-(diethylamino)-3-(4'-maleimidylphenyl)-4-methylcoumarin, or fluorescein 5-maleimide selectively into either the SH1 or SH2 site of the fibronectin molecule and have measured the inter-sulfhydryl distances in fibronectin by fluorescence energy transfer methods. The results show that the distance between the SH1 site of one subunit and the SH1 site of the other subunit is between 35 and 44 A, indicating the close proximity of the two subunits near the SH1-containing regions. On the other hand, the distance between the SH2 site of one subunit and the SH2 site of the other subunit is found to be greater than 95 A, suggesting that the two SH2-containing regions are well separated. Additionally, the distance between the SH1 and SH2 sites within each subunit is estimated to be 42-53 A, assuming no intersubunit energy transfer between the probes. Heparin or high salt, which drastically affects the hydrodynamic properties of fibronectin, had virtually no effect on the distance between the SH1-SH1 or the SH1-SH2 pair.(ABSTRACT TRUNCATED AT 250 WORDS)

Energy Transfer↗
PubMed1989

The pharmacology of arachidonic acid-induced rat PMN leukocyte infiltration.

Arachidonic Acid (AA) injected into a hindpaw of Lewis rats produces high levels of tissue myeloperoxidase (MPO), a biochemical marker for PMN leukocytes. Treatment with a corticosteroid (prednisolone) or dual 5-LO/CO inhibitors of AA metabolism (phenidone, SKF 86002) produced dose-related inhibition of AA-induced elevations in paw tissue MPO levels. In contrast, administration of high pharmacologic doses of selective cyclooxygenase inhibitors (indomethacin, ibuprofen, naproxen), anti-histamine/serotonin agents (cyproheptadine, chlorpheniramine) or an anti-arthritic gold compound (auranofin) produced only slight or moderate effects. Thus, AA-induced hindpaw inflammation is a useful method for determining pharmacologic effects of 5-LO/CO inhibitors on PMN leukocyte infiltration in vivo.

Animals↗
PubMed1987

The pharmacology of arachidonic acid-induced rat paw edema.

Arachidonic acid (AA) injected into hindpaws of Lewis rats produces a severe edematous response. Treatment with corticosteroids (dexamethasone, prednisolone), dual inhibitors of arachidonate metabolism (phenidone, SK & F 86002), anti-histamine/serotonin agents (chlorpheniramine, cyproheptadine) and a gold compound (auranofin) inhibited AA-induced edema. In contrast, administration of high doses of cyclooxygenase inhibitors (indomethacin, piroxicam, naproxen, ibuprofen, meclofenamic acid and tiflamizole) did not affect AA-induced hind paw edema. The involvement of lipoxygenase products and mast cell mediators in the edematous response to arachidonic acid render this model potentially useful for studying antiinflammatory agents with a mechanism of action different from that of cyclooxygenase inhibitors.

Adrenal Cortex Hormones↗