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At least 1,333 records · Page 74Linked to original sources
PubMed1997

Effect of walking distance on the change in ankle-brachial pressure index in patients with intermittent claudication.

OBJECTIVES: The ankle-brachial pressure index (ABPI) responses to different exercise intensities on a treadmill were evaluated to clarify the relationship between intermittent claudication and the haemodynamics in the leg. PATIENTS AND METHODS: Thirty patients with intermittent claudication (32 symptomatic legs) due to peripheral arterial occlusive disease were exercised on a treadmill to determine their pain-free walking distance, maximum walking distance (MWD) and recovery time. Each subject was exercised at 25% and 50% of the MWD to determine the effect of work intensity on the drop in ABPI, and the recovery time. RESULTS: In the claudicating legs, 25% of the MWD resulted in almost the same reduction in ABPI as 50% of the MWD, and the MWD. In contrast, the ABPI in the asymptomatic legs (13) was significantly decreased in proportion to the walking distance. The recovery time increased linearly in both the groups, as the walking distance increased. CONCLUSION: The recovery time of the ABPI correlated well with the intensity of workload, while the drop in ABPI did not.

Aged↗
PubMed1996

Distance learning in veterinary medicine: enhancing interactivity in continuing education.

Technological advances have made distance veterinary continuing education a reality. Distance interactive continuing education programs using computer and modern are currently being offered via Veterinary Information Network (VIN) on the America Online (AOL) computer network and the Network of Animal Health (NOAH) on CompuServe. Using over 500 hours of course experience on VIN as an example, this article describes the technology involved in delivering online instruction including electronic mail, asynchronous bulletin board interactions, electronic library files, and realtime interactive discussions. These formats take advantage of the standard features of the network AOL are mixed with links to Worldwide Web sites, print and electronic publications, and even coordinated with "offline" meetings. The paradigm of learning is somewhat different than traditional "offline" or "onsite" meetings in that learning takes place over a period of 2 to 20 weeks, allowing the participating veterinarian to interact with instructors while applying the information in practice. Continual learning is encouraged as participants are allowed to participate in a course offered in sequential years. The successful online instructors tend to take advantage of electronic communications to encourage instructor-participant and participant-participant interactivity. The majority of course participants seek an additional challenge or greater interactivity in their continuing education experience; however, some veterinarians seek distance continuing education for practical reasons, including the ability to pursue additional education without leaving a solo practice, temporary or permanent physical disabilities, family considerations, or military service. Distance learning is a viable educational format, the role for which will evolve as technology and the base of experience with it expands.

Computer Communication Networks↗
PubMed1997

Ultrasonically enhanced corrosion of 304L stainless steel II: the effect of frequency, acoustic power and horn to specimen distance.

The effects of frequency in the range 20 to 60 kHz, acoustic power and horn to specimen distance on the corrosion of 304L stainless steel in an ultrasonic field were investigated. At 40 and 60 kHz, the corrosion rate increased to a maximum and thereafter decreased with increasing transmitted power. At 20 kHz, the corrosion rate increased continuously with increasing power over the range investigated. At 18 W transmitted power, the corrosion rate increased continuously with frequency. However, at 40 W transmitted power a pronounced maximum in the corrosion rate occurred at 40 kHz. A significant effect of horn to specimen distance was found, the corrosion rate increasing with decreasing distance. High corrosion rates in excess of 800 mm yr(-1) were observed when the specimen was situated 0.1 mm from the radiating face of the ultrasonic probe. In addition, the area affected by sonication was found to increase with increasing distance.

Journal Article↗
PubMed1996

Homology modelling by distance geometry.

BACKGROUND: Unknown protein structures can be predicted from known structures (the scaffolds) with sequences sufficiently homologous to that of the target, based on the observation that similar sequences usually adopt the same fold. When structural equivalences between residues in the scaffold and target proteins are expressed in terms of conserved interatomic distances, the resulting 'distance geometry' representation provides an elegant mechanism for simultaneous restraint satisfaction and bias-free conformation space exploration. RESULTS: We present a homology modelling algorithm based on distance geometry that relies on the gradual projection of simple model chain coordinates into Euclidean spaces with decreasing dimensionality. The similarity between the unknown target structure and the scaffold proteins with known structures was described by mapping secondary structure assignments and specific distance restraints between C alpha atoms onto the model through a multiple alignment. This information was complemented by additional restraints derived from stereochemical considerations and other general aspects of protein structure such as hydrophobic core formation or the absence of tangled mainchains. CONCLUSIONS: The method was capable of quickly locating the correct fold even from an alignment with modest average conservation indicating that it could serve as a fast tool for obtaining correct low-resolution starting conformations for detailed refinement.

Amino Acid Sequence↗
PubMed1997

Distance geometry based comparative modelling.

A distance geometry based protein modelling algorithm is presented which relies on the projection of simple model chain coordinates into Euclidean spaces with gradually decreasing dimensionality. Fast embedding was achieved by performing separate distance matrix projections on subsets of the model points. Structural equivalences between the unknown target and related proteins with known structures were deduced either from a mixed sequence/structure multiple alignment or from the output of various fold recognition (threading) approaches. These equivalences were mapped onto the model as structure-specific conserved C alpha atom distances and secondary structure assignments. Additional nonspecific distance restraints derived from general stereochemical properties of folded protein chains were used to guide the modelling process. The method quickly constructed a large number of low-resolution models which could then serve as starting conformations for full-atom refinement. Structure predictions for some targets in the 'Asilomar Challenge' (CASP2) are presented to illustrate potential applications of the approach.

Algorithms↗
PubMed1996

Ethological analysis of crew member behavior: distances, orientations, and postures.

The purpose of this study was to investigate the spatial behavior towards each other of a group of three males and one female isolated and confined for a period of 60 days. Video recordings of the individual crew members in the habitat, laboratory, and transfer modules for a 4-hr period once weekly during the isolation period provided the material for analysis. The observed parameters were: inter-individual distance, orientation (number of other subjects laced), and posture. Three distinct periods are noted. The initial period was characterized by a rather constant spatial behavior of the crew with few changes in inter-individual distances. The middle period of isolation (particularly weeks 5 and 6) showed a tendency towards closer grouping with decreased inter-individual distances (personal and social space). The final period (weeks 7-9) was marked by a dispersion of the crew members with increased inter-individual distances (public space) and more frequent isolated positions. Individual differences in behavioral strategy were noted. The Commander (subject B) kept the largest number of other crew members in his visual field. The orientation of the other subjects showed more variation. Dominant posture were: seated, standing, lying. Here also there were individual differences, for example, the female subject H frequently assumed a very grouped posture. During the second half of the isolation period stretched body positions became more frequent, suggesting reduced tension and stress. The observed spatial behavior indicates a weak cohesion of the crew with little tendency towards formation of a true social group in the course of isolation. The crew remained a group of individuals constituted for the purpose of the experiment. From her closer positions to the other subjects, it appears that the female crew member had a positive effect on group cohesiveness through an active social role. However, it seems that a longer period of confinement would be needed to turn the four crew members into a cohesive crew. The findings from these simulation studies and in earlier spaceflight observations suggest a possible use of ethological observation during space missions for the purpose of optimizing performance, physical and psychological fitness of the individual astronauts and group dynamics of the entire crew. It might be possible to establish a set of individual standards allowing detection of slight behavioral disturbances in an astronaut during the mission.

Astronauts↗
PubMed2005

Relative infection levels and taxonomic distances among the host species used by a parasite: insights into parasite specialization.

Parasites often exploit more than one host species at any stage in their life-cycle, but the extent to which these host species are used varies greatly. Parasites typically achieve their highest prevalence, intensity and/or abundance in one host species (the principal host), whereas infection levels in auxiliary hosts range from relatively high to very low. The present study examines what influences the distribution of parasite individuals among their different host species, using metazoan parasites that use freshwater fish as their definitive or only host. Specifically, I test the hypothesis that differences in relative infection levels by a parasite among its auxiliary hosts are proportional to the taxonomic distance between the respective auxiliary hosts and the parasite's principal host. Taxonomic distance among hosts is a surrogate measure of their similarity in terms of ecology, physiology and immunology. Using data on 29 parasite species and 6 fish communities, for a total of 47 parasite-locality combinations, it was found that taxonomic distance between the auxiliary hosts and the principal host had no real influence on infection levels in auxiliary hosts, measured as either prevalence, intensity or abundance. The analysis revealed differences in the degree of specialization among major groups of parasites: in terms of abundance or intensity, auxiliary hosts were less important for cestodes than for nematodes and copepods. The lack of an effect of taxonomic distance may indicate that ecological similarity among host species, arising from convergence and not from relatedness, is more important than host phylogeny or taxonomy. Although the results are based on a limited number of parasite taxa, they suggest that parasites may be opportunistic in their colonization of new hosts, and not severely constrained by evolutionary baggage.

Animals↗
PubMed1987

Distance geometry and related methods for protein structure determination from NMR data.

Computational tools have been developed in the last few years, to allow a direct determination of protein structures from NMR data. Numerical calculations with simulated and experimental NMR constraints for distances and torsional angles show that data sets available with present NMR techniques carry enough information to determine reliably the global fold of a small protein. The maximum size of a protein for which the direct method can be applied is not limited by the computational tools but rather by the resolution of the two-dimensional spectra. A general estimate of the maximum size would be a molecular weight of about 10,000 (Markley et al. 1984), but parts of larger proteins might be accessible with the method. Effort for improvement of the NMR structures should be concentrated more on the local conformation rather than the global features. The r.m.s. D values for variations of the polypeptide backbone fold are on the order of 1.5-2 A for several of the studied proteins, indicating that the global structure is well determined by the present NMR data and their interpretation. The local structures are sometimes rather poor, with standard deviations for the backbone torsion angles of about 50 degrees. Possible improvements would be stereospecific resonance assignments of individual methylene protons and individual assignments of the methyl groups of the branched side-chains. Accurate estimates of the short-range NOE distance constraints by calibrating the distance constraints, including segmental flexibility effects, and combined use of distance geometry, energy minimization and molecular dynamics calculations, are further tools for improving the structures.

Magnetic Resonance Spectroscopy↗
PubMed1993

Solid-state NMR determination of intra- and intermolecular 31P-13C distances for shikimate 3-phosphate and [1-13C]glyphosate bound to enolpyruvylshikimate-3-phosphate synthase.

Rotational-echo, double-resonance (REDOR) 31P NMR was used to obtain internuclear distances for shikimate 3-phosphate (S3P) and N-(phosphonomethyl)-[1-13C]glycine (glyphosate) bound to 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, a 46-kDa enzyme essential for the synthesis of aromatic amino acids in plants and microorganisms. An intermolecular 31P-13C distance of 7.2 A was measured between the phosphate of S3P and the labeled carbon of glyphosate. This means that S3P and glyphosate are in proximity in the binding site of the enzyme. An intramolecular 31P-13C distance of 5.6 A was measured between the phosphonate 31P and the labeled carbon of glyphosate. This distance can be achieved only if glyphosate is completely extended when bound to EPSP synthase.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2000

Refinement of the geometry of the retinal binding pocket in dark-adapted bacteriorhodopsin by heteronuclear solid-state NMR distance measurements.

The bacterial proton pump bacteriorhodopsin (BR) is a 26.5 kDa seven-transmembrane helical protein. Several structural models have been published at > or =1.55 A resolution. The initial cis-trans isomerization of the retinal moiety involves structural changes within <1 A. To understand the chromophore-protein interactions that are important for light-driven proton transport, very accurate measurements of the protein geometry are required. To reveal more structural details at the site of the retinal, we have, therefore, selectively labeled the tryptophan side chains of BR with (15)N and metabolically incorporated retinal, (13)C-labeled at position 14 or 15. Using these samples, heteronuclear distances were measured with high accuracy using SFAM REDOR magic angle spinning solid-state NMR spectroscopy in dark-adapted bacteriorhodopsin. This NMR technique is applied for the first time to a high-molecular mass protein. Two retinal conformers are distinguished by their different isotropic 14-(13)C chemical shifts. Whereas the C14 position of 13-cis-15-syn-retinal is 4.2 A from [indole-(15)N]Trp86, this distance is 3.9 A in the all-trans-15-anti conformer. This latter distance allows us to check on the details of the active center of BR in the various published models derived from X-ray and electron diffraction data. The experimental approach and the results reported in this paper enforce the notion that distances between residues of a membrane protein binding pocket and a bound ligand can be determined at subangstrom resolution.

Bacteriorhodopsins↗
PubMed1993

Fluorescence study of conformational flexibility of RNase S-peptide: distance-distribution, end-to-end diffusion, and anisotropy decays.

Frequency-domain fluorescence resonance energy transfer and anisotropy measurements were performed to characterize conformational dynamics of an analog of the RNase S-peptide (residues 1-20). Trp was used as a donor by replacing Phe 8, and a dansyl acceptor group was introduced at position 1 or 18. The distance-distribution parameters, half width of the distribution, end-to-end diffusion coefficient, and to some extent anisotropy decays were sensitive to changes in the S-peptide conformation. The observed mean distance of about 13-14 A between residues 1 and 8 in the presence of 50% TFE and when bound to RNase S-protein is in reasonable accord with the X-ray structure of RNase. The mean distance of 9.3 A between residues 8 and 18 in the presence of 50% TFE is, however, significantly smaller than 15.3 A found for the S-protein complex. The half-width of the distance distribution increased from about 9 to 18 A for residues 1-8 and from about 6 to 14 A for segment 8-18 with the loss of helical structure. The half-widths of 9 A in the case of 1-8 segment when peptide is helical suggests the presence of considerable conformational heterogeneity. Also, the 14 A half-width for segment 8-18 when it is random-coil is smaller than that expected for a random coil 11-residue segment. The donor-to-acceptor diffusion coefficients were less than 1 x 10(-7) cm2/s at 2 degrees C for both segments and increased to 1-2 x 10(-6) cm2/s at 35 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Dansyl Compounds↗
PubMed1992

Using saturation-recovery EPR to measure distances in proteins: applications to photosystem II.

The stable tyrosine radical YD. (tyrosine 160 in the D2 polypeptide) in photosystem II (PSII) exhibits nonexponential electron spin-lattice relaxation transients at low temperature. As previously reported, the tetranuclear Mn complex in PSII significantly enhances the spin-lattice relaxation of YD.. However, in Mn-depleted PSII membranes, the spin-lattice relaxation transients of YD. are also nonexponential, and progressive power saturation (P 1/2) experiments show that it does not behave like an isolated tyrosine radical. A model is developed to treat the interaction of two paramagnets in a rigid lattice at a fixed distance apart but with a random orientation in a magnetic field. This model describes the spin-lattice relaxation of a radical in proximity to another paramagnetic site in terms of three relaxation rate constants: the "intrinsic" relaxation rate, the relaxation rate due to scalar exchange, and the relaxation rate due to dipole-dipole interactions. The intrinsic and the scalar exchange relaxation rates are isotropic and together contribute a single rate constant to the spin-lattice relaxation transients. However, the dipolar relaxation rate is orientation dependent. Each orientation contributes a different dipolar relaxation rate constant to the net spin-lattice relaxation rate constant. The result is a superposition of single-exponential recoveries, each with a different net rate constant, causing the observed saturation-recovery transients to be non-(single)-exponential. Saturation-recovery relaxation transients of YD. are compared with those of a model tyrosine radical, generated by UV photolysis of L-tyrosine in a borate glass. From this comparison, we conclude that scalar exchange does not make a significant contribution to the spin-lattice relaxation of YD. in Mn-depleted PSII. We account for the nonexponential relaxation transients obtained from YD. in Mn-depleted PSII membranes in terms of dipolar-induced relaxation enhancement from the non-heme Fe(II). From simulations of the spin-lattice relaxation transients, we obtain the magnitude of the magnetic dipolar interaction between YD. and the non-heme Fe(II), which can be used to calculate the distance between them. Using data on the non-heme Fe(II) in the reaction center of Rhodobacter sphaeroides to model the non-heme Fe(II) in PSII, we calculate a YD.-Fe(II) distance of greater than or equal to 38 A in PSII. This agrees well with the distance predicted from the structure of the bacterial reaction center.

Electron Spin Resonance Spectroscopy↗
PubMed1992

A potential gene target in HIV-1: rationale, selection of a conserved sequence, and determination of NMR distance and torsion angle constraints.

Recently, the capability for determining the high-resolution, sequence-dependent structure of oligonucleotides in solution via careful analysis of multidimensional NMR spectra and structure refinement procedures has been developed. Consequently, the rationale for selection of a genome sequence as a target for drug design based on the detailed three-dimensional structure of the target is presented. The concept is illustrated by the successful search for a highly conserved region of the HIV-1 genome's long terminal repeat which could serve as a molecular target. A compound which could selectively bind the target sequence could inhibit both RNA transcription from the integrated provirus and the reverse transcription process. Of 148 unique HIV-1 sequences examined, 147 exhibit a 21-base conserved sequence (nucleotides 70-90 in HIVHXB2R) in the R region of the long terminal repeat. The only exception, a minor constituent for one individual, has a change in the penultimate base. A 13 base pair duplex sequence, [d(AGCTTGCCTTGAG).d(CTCAAGGCAAGCT)], from this conserved region was selected and synthesized for NMR structure studies. Phase-sensitive proton two-dimensional nuclear Overhauser enhancement (2D NOE) and double-quantum-filtered correlation (2QF-COSY) spectra were obtained at 500 MHz for the DNA duplex. Exchangeable and nonexchangeable proton resonances were assigned. Quantitative assessments of the 2D NOE cross-peak intensities for different mixing times were carried out using conventional Fourier transform NMR and the maximum likelihood method (MLM). Distance constraints, along with upper and lower bounds, were obtained from the 2D NOE intensities using the iterative complete relaxation matrix algorithm MARDIGRAS. Distances entailing both exchangeable and nonexchangeable protons were determined: 7-11 experimental distance constraints per residue including interresidue and interstrand distances. Simulations of the scalar coupling effects manifest in 2QF-COSY cross-peaks by means of the program SPHINX/LINSHA were compared with experimental data to yield torsion angle constraints for the sugar rings. A single conformer was inadequate to describe any of the sugar puckers, but a rapid two-state equilibrium with one conformer strongly dominant (75-95%) provided a good fit of the 2QF-COSY cross-peaks. The sugar pucker of the major conformer exhibited significant variability for the various nucleotides but was roughly 2'-endo. Though derived independently and subject to different time-averaging effects, the 2QF-COSY and 2D NOE results are in accord.

Base Sequence↗
PubMed1994

Hairpin folding of subunit c of F1Fo ATP synthase: 1H distance measurements to nitroxide-derivatized aspartyl-61.

Subunit c from the F1Fo ATP synthase of Escherichia coli folds in a hairpinlike structure of two alpha-helices in a solution of chloroform-methanol-H2O, and thus resembles the structure predicted for the folded protein in the membrane. The relevance of the structure in solution to the native structure was demonstrated. Asp61 in the second helical arm was shown to retain its unique reactivity with dicyclohexylcarbodiimide (DCCD) in chloroform-methanol-H2O solution. Further, the protein purified from the Ile28-->Thr DCCD-resistant mutant proved to be less reactive with DCCD in solution. This suggested that the protein folded with Ile28 of the first helical arm close to Asp61 in the second helical arm. Subunit c in wild-type E. coli membranes was specifically labeled with a nitroxide analog of DCCD (NCCD), and the derivative protein was purified. DQF COSY spectra were recorded, and the distances between the paramagnetic nitroxide and resolved protons in the spectra were calculated based upon paramagnetic broadening of the 1H resonances. The paramagnetic contribution to T2 relaxation in the NCCD-labeled sample was calculated by an iterative computer-fitting method, where a control spectrum of a phenylhydrazine-reduced sample was broadened until the line shape of one-dimensional slices through each COSY cross-peak maximally mimicked the line shape of the paramagnetic sample. The distances calculated from paramagnetic broadening indicate that Ala24 and Ala25 in helix-1 lie close (ca. 12 A) to the derivatized Asp61 in helix-2. A model for the interaction of helices in the NCCD-modified protein was generated by restrained molecular mechanics and molecular dynamics using 25 distances of < 10-20 A derived from paramagnetic broadening in combination with 15 long-range nuclear Overhauser enhancement (NOE) restraints (2-5 A) for distances between helices and the 89 intrahelical NOEs that defined helical structure in the DCCD-modified protein.

Aspartic Acid↗
PubMed1991

Proximity relationships between sites on myosin and actin. Resonance energy transfer determination of the following distances, using a hybrid myosin: those between Cys-55 on the Mercenaria regulatory light chain, SH-1 on the Aequipecten myosin heavy chain, and Cys-374 of actin.

Resonance energy transfer measurements have been made on hybrid myosins in order to map distances between sites on the regulatory light chain, heavy chain, and actin as well as to assess potential conformational changes of functional importance. Using scallop (Aequipecten) myosin hybrid molecules possessing clam (Mercenaria) regulatory light chains, we have been able to map the distance between Cys-55 on the regulatory light chain and the fast-reacting thiol on the myosin heavy chain (SH-1). This distance is shown to be approximately 6.4 nm, and it is not altered by the presence or absence of Ca2+, MgATP, or actin. Experiments performed at low ionc strength on heavy meromyosin (HMM) derived from these hybrid myosins gave results similar to those performed on the soluble parent myosin preparations. The distances between Cys-374 on actin and each of the above sites were also measured. Mercenaria regulatory light-chain Cys-55, within the hybrid myosin molecule, was found to be greater than 8.0 nm away from actin Cys-374. Scallop heavy-chain SH-1 is shown to be approximately 4.5 nm away from actin Cys-374, in broad agreement with earlier measurements made by others in nonregulatory myosins. The significance of our results is discussed with respect to putative conformational changes within the region of the heavy chain connecting SH-1 to the N-terminal region of the light chain.

Actins↗
PubMed1983

Förster energy transfer measurements of thiol 1 to thiol 2 distances in myosin subfragment 1.

Förster energy transfer was used to measure the distance between reporter groups on the two reactive thiols of myosin, SH1 and SH2, and to detect changes in this distance upon binding of nucleotide. SH1 was labeled with the fluorophore 5-[[2-[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid (1,5-IAEDANS) and SH2 with the chromophoric acceptor N-[4-(dimethylamino)-3,5-dinitrophenyl]-maleimide (DDPM). Peptide studies verified that [3H]-1,5-IAEDANS reacted specifically with SH1, while [14C]DDPM labeled both SH2 and the alkali light chains. The [14C]-DDPM-modified alkali light chains were replaced with unmodified light chains by the exchange procedure of Wagner and Weeds [Wagner, P.D., & Weeds, A. G. (1977) J. Mol. Biol. 109, 455-473]. Subfragment 1 labeled with 1,5-IAEDANS and then with DDPM exhibited two fluorescence lifetimes, 20.6 (AEDANS-SF1, unquenched) and 9.3 ns (AEDANS-SF1, quenched by DDPM). The latter lifetime decreased to an average of 2.85 ns after the addition of MgAMP-PNP, MgADP, or MgPPi (no change with MgAMP), indicating that the distance between the donor and acceptor decreased. An R0 of 29 A was calculated for the AEDANS/DDPM system assuming random orientation of the donor/acceptor pair. The decrease in the observed lifetimes upon the addition of Mg nucleotide corresponds to a change in the donor-acceptor distance from 28 to 21-22 A. This observation is consistent with the proposal that nucleotide binding juxtaposes SH1 and SH2 to enhance their cross-linking with various bifunctional reagents [Burke, M., & Reisler, E. (1977) Biochemistry 16, 5559-5563; Wells, J. A., & Yount, R.G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4966-4970].

Adenylyl Imidodiphosphate↗
PubMed1983

Distance measurement between the active site and cysteine-177 of the alkali one light chain of subfragment 1 from rabbit skeletal muscle.

Förster energy-transfer techniques have been applied to labeled myosin subfragment 1 from rabbit skeletal muscle to determine an intramolecular distance and whether this distance changes during magnesium-dependent ATPase activity. The alkali one light chain was labeled at Cys-177 with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS) and then exchanged into subfragment 1. High specificity of labeling was indicated by high-performance liquid chromatography analysis of a tryptic digest of the labeled light chain. 2'(3')-O-(2,4,6-Trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) was bound to the labeled protein at the ATPase active site. The efficiency of energy transfer between the probes was 0.09 when measured by both steady-state and time-resolved fluorescence. Anisotropy measurements of the bound AEDANS indicated considerable freedom of motion of the probe. The probable distance between the probes was 57 A. This distance was unchanged during triphosphatase activity. Two further sites of TNP-ADP interaction with subfragment 1 were found. The effect of these interactions on the energy-transfer measurements was reduced to a minimum by careful choice of reaction conditions.

Adenosine Triphosphatases↗
PubMed1984

Distance relationships between the catalytic site labeled with 4-(iodoacetamido)salicylic acid and regulatory sites of glutamate dehydrogenase.

The distance between the catalytic site on bovine liver glutamate dehydrogenase labeled with 4-(iodoacetamido)salicylic acid (ISA) and the adenosine 5'-diphosphate (ADP) activatory site occupied by the analogue 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)adenosine 5'-diphosphate (TNP-ADP) was evaluated by energy transfer. Native enzyme and enzyme containing about 1 mol of acetamidosalicylate/mol of subunit bind about 0.5 mol of TNP-ADP/mol of subunit, and TNP-ADP competes for binding with ADP to native and modified enzyme, indicating that the analogue is a satisfactory probe of the ADP site. From the quenching of acetamidosalicylate donor fluorescence upon addition of TNP-ADP, an average distance of 33 A was determined between the catalytic and ADP sites. The fluorescent nucleotide analogue 5'-[p-(fluorosulfonyl)benzoyl]-2-aza-1,N6-ethenoadenosine (5'-FSBa epsilon A) reacts covalently with glutamate dehydrogenase to about 1 mol/peptide chain. As compared to native enzyme, the SBa epsilon A-enzyme exhibits decreased sensitivity to GTP inhibition but retains its catalytic activity as well as its ability to be activated by ADP and inhibited by high concentrations of NADH. Complete protection against decreased sensitivity to GTP inhibition is provided by GTP in the presence of NADH. It is concluded that 5'-FSBa epsilon A modifies a GTP site on glutamate dehydrogenase. The distance of 23 A between the catalytic site labeled with ISA and a GTP site labeled with 5'-FSBa epsilon A was measured from the quenching of salicylate donor fluorescence in the presence of the SBa epsilon A acceptor on a doubly labeled enzyme. The average distance between the ADP and GTP sites was previously measured as 18 A [Jacobson, M. A., & Colman, R. F. (1983) Biochemistry 22, 4247-4257], indicating that the regulatory sites of glutamate dehydrogenase are closer to each other than to the catalytic site.

Adenosine↗