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Biomedical subjects

D M Jameson

Publications and source records attributed to D M Jameson.

At least 19 recordsLinked to original sources
PubMed1995

Polymerization of an Escherichia coli elongation factor Tu.

Elongation factor Tu (EF-Tu) from Escherichia coli is shown here to polymerize under conditions of low ionic strength and slightly acidic pH. Several factors, such as decreasing pH, decreasing ionic strength, increasing temperature, and increasing protein concentration (up to 2.5 microM) enhance the rate of polymerization. Both EF-Tu.GTP and EF-Tu.GDP polymerized equally well under the conditions studied. A lag time was observed between the lowering of the pH and the onset of measurable polymerization, which was not overcome by addition of preformed polymer "seeds." Finally, addition of unpolymerized EF-Tu.GDP to a solution of polymerized EF-Tu.GDP appears to lead to formation of new polymers instead of addition to preexisting ones, which may suggest a size limit for polymers of EF-Tu.GDP.

Escherichia coli↗
PubMed1995

Macromolecular arrangement in the aminoacyl-tRNA.elongation factor Tu.GTP ternary complex. A fluorescence energy transfer study.

The distance between the corner of the L-shaped transfer RNA and the GTP bound to elongation factor Tu (EF-Tu) in the aminoacyl-tRNA.EF-Tu.GTP ternary complex was measured using fluorescence energy transfer. The donor dye, fluorescein (Fl), was attached covalently to the 4-thiouridine base at position 8 of tRNAPhe, and aminoacylation yielded Phe-tRNAPhe-Fl8. The ribose of GTP was covalently modified at the 2'(3') position with the acceptor dye rhodamine (Rh) to form GTP-Rh. Formation of the Phe-tRNAPhe-Fl8.EF-Tu.GTP-Rh ternary complex was verified both by EF-Tu protection of the aminoacyl bond from chemical hydrolysis and by an EF-Tu.GTP-dependent increase in fluorescein intensity. Spectral analyses revealed that both the emission intensity and lifetime of fluorescein were greater in the Phe-tRNAPhe-Fl8.EF-Tu.GTP ternary complex than in the Phe-tRNAPhe-Fl8.EF-Tu.GTP-Rh ternary complex. These spectral differences disappeared when excess GTP was added to replace GTP-Rh in the latter ternary complex, thereby showing that excited-state energy was transferred from fluorescein to rhodamine in the ternary complex. The efficiency of singlet-singlet energy transfer was low (10-12%), corresponding to a distance between the donor and acceptor dyes in the ternary complex of 70 +/- 7 A, where the indicated uncertainty reflects the uncertainty in dye orientation. After correction for the lengths of the probe attachment tethers, the 2'(3')-oxygen of the GTP ribose and the sulfur in the s4U are separated by a minimum of 49 A. This large distance limits the possible arrangements of the EF-Tu and the tRNA in the ternary complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Energy Transfer↗
PubMed1995

Molecular dynamics of the anti-fluorescein 4-4-20 antigen-binding fragment. 2. Time-resolved fluorescence spectroscopy.

Time-resolved fluorescence experiments were performed to investigate the dynamic aspects of the antigen-binding fragment (Fab) of a high-affinity monoclonal antibody (4-4-20) which binds the fluorescent hapten fluorescein. Both the unliganded Fab and a complex of the Fab with a nonfluorescent analog of fluorescein (fluoresceinamine, FLM) were examined. A fluorescence polarization probe [5-[[2-[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid, AEDANS] was covalently attached to the C-terminus of the Fab. Experiments were performed at three different temperatures (10, 25, and 35 degrees C), and phase-modulation data sets were collected for five different molar ratios of FLM to Fab at each temperature. Global analyses were then used to extract values for fluorescence lifetime and rotational correlation time from these data. In the lifetime analysis the best fit was obtained when the emission of AEDANS was described by a Lorentzian distribution of lifetimes (tau = 15.6 ns, distribution width = 3.4 ns, both at 25 degrees C), which suggested that the probe experienced a heterogeneous environment. Anisotropy analyses suggested that two different rotational components were present. The first was attributed to the global motion of the Fab and exhibited a rotational correlation time (theta 1) of ca. 33 ns at 25 degrees C. This component was relatively unaffected by antigen binding. The second rotational component was attributed to the local or segmental motion within the Fab and exhibited a rotational correlation time (theta 2) of 1.1 ns at 25 degrees C. This value increased by more than 50% upon antigen binding, a result which was consistent with molecular dynamics simulations of the same Fab--fluorescein system [Lim & Herron (1995) Biochemistry 34, 6962-6974]. Furthermore, statistical analysis showed that this increase was significant at the 95% confidence level.

Amino Acid Sequence↗
PubMed1995

Oxygen penetration and diffusion into myoglobin revealed by quenching of zincprotoporphyrin IX fluorescence.

Oxygen quenching experiments were carried out on zincprotoporphyrin IX reconstituted myoglobin (MbFe-->Zn) at different temperatures and two solvent viscosities. The data were fit to a dynamic model for quenching of fluorophores in protein interiors previously presented (Biophysical J., 45 (1984) 789-794). The parameters associated with the oxygen entry rate (k+), exit rate (k-), and migration rate (chi) in the protein were obtained at six temperatures and two viscosities (1 and 8 cp), along with the activation enthalpies associated with the above rates (k+ and k-). The partition coefficient (alpha) was calculated at each temperature along with the free energy, delta G0, associated with this partition. The rate parameters (k+, k-, chi) and the partition coefficient (alpha) have also been determined for the sample in 40% sucrose (8 cp), to evaluate the effect of bulk solvent viscosities on these values. The steady-state Stern-Volmer quenching plot was calculated using the rate parameters obtained from the analysis (of the dynamic model). Comparison of the Stern-Volmer points obtained using the dynamic model and those obtained experimentally showed excellent agreement.

Chemical Phenomena↗
PubMed1994

Reversible unfolding of fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase.

Reversible unfolding of rat testis fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase in guanidine hydrochloride was monitored by following enzyme activities as well as by fluorescence methodologies (intensity, emission maximum, polarization, and quenching), using both intrinsic (tryptophan) and extrinsic (5((2-(iodoacetyl)amino) ethyl)naphthalene-1-sulfonic acid) probes. The unfolding reaction is described minimally as a 4-state transition from folded dimer-->partially unfolded dimer-->monomer-->unfolded monomer. The partially unfolded dimer had a high phosphatase/kinase ratio due to preferential unfolding of the kinase domain. The renaturation reaction proceeded by very rapid conversion (less than 1 s) of unfolded monomer to dimer, devoid of any enzyme activity, followed by slow (over 60 min) formation of the active enzyme. The recovery rates of the kinase and the phosphatase were similar. Thus, the refolding appeared to be a reversal of the unfolding pathway involving different forms of the transient dimeric intermediates. Fluorescence quenching studies using iodide and acrylamide showed that the tryptophans, including Trp-15 in the N-terminal peptide, were only slightly accessible to iodide but were much more accessible to acrylamide. Fructose 6-phosphate, but not ATP or fructose 2,6-bisphosphate, diminished the iodide quenching, but all these ligands inhibited the acrylamide quenching by 25%. These results suggested that the N-terminal peptide (containing a tryptophan) was not exposed on the protein surface and may play an important role in shielding other tryptophans from solvent.

Acrylamides↗
PubMed1994

Hydrodynamics of horseradish peroxidase revealed by global analysis of multiple fluorescence probes.

Previous fluorescence studies of horseradish peroxidase conjugated with protoporphyrin IX suggested that the protein behaved hydrodynamically as a prolate ellipsoid of axial ratio 3 to 1. The present study, designed to further investigate the hydrodynamics of this protein, exploits a series of probes, noncovalently bound to the heme binding site of apo-horseradish peroxidase, having different orientations of the excitation and emission transition dipoles with respect to the protein's rotational axes. The probes utilized included protoporphyrin IX and the naphthalene probes 1-anilino-8-naphthalene sulfonate, 2-p-toluidinyl-6-naphthalene sulfonate, and 4,4'-bis(1-anilino-8-naphthalene sulfonate). Time-resolved data were obtained using multifrequency phase fluorometry. The global analysis approach to the determination of molecular shape using multiple probes was evaluated by utilizing all data sets while maintaining a constant molecular shape for the protein. The results indicated that, in such analyses, probes exhibiting a single exponential decay and limited local motion have the major weight in the evaluation of the axial ratio. Probes that show complex decay patterns and local motions, such as the naphthalene derivatives, give rise to significant uncertainties in such global treatments. By explicitly accounting for the effect of such local motion, however, the shape of the protein can be reliably recovered.

Binding Sites↗
PubMed1994

Safety and efficacy of omega-3 fatty acids in the nutrition of very low birth weight infants: soy oil and marine oil supplementation of formula.

Because formula-fed preterm infants may be at risk of omega 3 essential fatty acid deficiency, we tested experimental formulas supplemented with soy oil to provide alpha-linolenic acid or marine oil to provide preformed omega 3 long-chain polyunsaturated fatty acids at a level comparable to that of human milk. This report addresses the effect of feeding formula supplemented with soy oil or with soy and marine oils on growth, clinical tolerance, coagulation test results, changes in erythrocyte membrane fluidity, and plasma concentrations of vitamins A and E in very low birth weight infants from 30 to 57 weeks of postconceptional age. "Healthy" preterm infants were maternally selected to receive human milk or selected at random to receive commercial ready-to-feed liquid formula, which provided limited omega 3 fatty acid, or experimental formulas supplemented with soy oil or soy and marine oils. Results of this study indicate that formula enriched with soy oil or soy and marine oils containing preformed omega 3 long-chain polyunsaturated fatty acids does not induce abnormalities in growth, clotting function, erythrocyte membrane fluidity, or vitamin A or E levels in healthy very low birth weight preterm infants. Additional studies to evaluate safety in a representative preterm population are required.

Analysis of Variance↗
PubMed1993

Solution dynamics of p21ras proteins bound with fluorescent nucleotides: a time-resolved fluorescence study.

The solution dynamics of normal and transforming p21ras proteins in both the GTP- and GDP-bound forms were examined with time-resolved fluorescence spectroscopy. The fluorescent 2'(3')-O-(N-methylanthraniloyl) derivatives (mant derivatives) of GTP, dGTP, and GDP and the aminocoumarin and fluorescein derivatives of GTP and GDP were synthesized and used as reporter groups. The fluorescence lifetimes at 5 degrees C of the mant nucleotide derivatives increased from approximately 4 ns in solution to approximately 9 ns when bound to p21ras. At 30 degrees C, there was a 7.8% difference in lifetime between normal p21ras.mantGTP and p21ras.mantGDP, but no difference between similar complexes of the [Asp-12]p21ras protein. These data are consistent with steady-state fluorescence intensity differences among p21ras.mantGTP, p21ras.mantGDP, and the free nucleotides. Rotational correlation times for the mantGTP- and mantGDP-bound p21 proteins, N-ras, K-ras, and H-ras, were similar at 26 ns (5 degrees C), which is significantly longer than the 15-ns rotational correlation time predicted for a globular 21,000-Da protein. The p21-bound fluorescein and aminocoumarin nucleotide derivatives reported correlation times of 19 and 29 ns, respectively. Global analysis of the three fluorophore.p21 complexes with linked protein rotational correlation functions were best fit with a common rotational correlation time of 28 ns. Gel permeation chromatography of the GDP and mantGDP complexes of normal p21N-ras also showed greater apparent molecular weights than were expected in both cases, demonstrating that the high rotational correlation times obtained from time-resolved fluorescence measurements were not a result of the introduction of the fluorophore.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical Phenomena↗
PubMed1992

Effect of docosahexaenoic acid on membrane fluidity and function in intact cultured Y-79 retinoblastoma cells.

Considerable metabolic energy is expended in ensuring that membranes possess a characteristic fatty acid composition. The nature of the specific requirement of the retina for high levels of docosahexaenoic acid (DHA) is as yet undefined. Previous work has speculated that DHA is required to maintain the fluid nature and permeability necessary for optimal retinal function. Cultured Y-79 retinoblastoma cells were grown in serum-containing media with and without supplemental DHA. Resultant changes in membrane fluidity were assessed using fluorescent probes. No differences were observed in rotational probe mobility as assessed by fluorescence polarization despite a fourfold increase in cellular DHA content. Lateral probe mobility as assessed by pyrene eximer formation was significantly enhanced in DHA-supplemented cells. Both the DHA content and total fatty acid unsaturation index in retinoblastoma cells were directly correlated with membrane fluidity as reported by eximer formation (Pearson's rho = 0.96 and 0.92, respectively). DHA supplementation also resulted in a significant increase in cellular choline uptake. We speculate that the effect of DHA content on retinal function may be mediated by changes in membrane fluidity and associated enzyme and transport activities.

Cell Line↗
PubMed1991

Oxygen diffusion near the heme binding site of horseradish peroxidase.

The quenching by molecular oxygen of the fluorescence of several probes complexed to apohorseradish peroxidase has been studied by intensity and time-resolved fluorescence methods. The probes utilized include 1-anilino-8-naphthalene sulfonic acid, 4,4'-bis (1-anilino-8-naphthalene sulfonic acid), and 2-p-toluidinylnaphthalene-6-sulfonic acid. These results are contrasted to those obtained using apohorseradish peroxidase complexed with protoporphyrin IX. The resistance of these complexes to denaturation by guanidine hydrochloride was determined. The results demonstrate a dramatic increase in oxygen accessibility to the naphthalene probes compared to protoporphyrin IX, which can be correlated to the increased stability of the protein-protoporphyrin IX complex.

Apoenzymes↗
PubMed1991

Examination of elongation factor Tu for aluminum fluoride binding sites using fluorescence and 19F-NMR methodologies.

This article reports on a comparison of the interaction of Al3+ and F- with two GTP-binding proteins, elongation factor Tu (EF-Tu) and the hormone sensitive regulatory protein (G protein) G0 alpha. The methodologies chosen to elucidate possible interactions between protein and aluminum fluoride were fluorescence spectroscopy and nuclear magnetic resonance (19F-NMR). Both proteins have tryptophan residues near their nucleotide binding sites, the purported site of aluminum fluoride interaction. It has been assumed for G proteins (including G0 alpha) that aluminum fluoride, in the presence of Mg2+ mimics the magnesium coordinated gamma-phosphate group for the GDP-form of the protein and shifts the protein's conformation toward the active GTP-form. Indeed, changes in intrinsic fluorescence of G0 alpha effected by aluminum fluoride are observed. The presence of aluminum fluoride did not affect the intrinsic fluorescence, spectra or lifetimes, of EF-Tu.GDP 19F-NMR was then used to directly test for bound F-. Fluoride alone or in the presence of either protein gave a single 19F-NMR peak at -10 ppm, characteristic of free F-. With the addition of aluminum to the protein and F- samples a second peak, shifted upfield from the first to -29 ppm, was observed for G0 alpha.GDP. This second peak, which has been assigned to protein-bound F-, was not observed for EF-Tu.GDP. These observations show that the interaction of Al3+ and F-, in the presence of Mg2+, may be quite different between the hormone-sensitive G proteins, which bind aluminum fluoride, and the GTP-binding proteins as a whole, which include EF-Tu. Care must therefore be exercised when structural data on the elongation factor, specifically on the nucleotide site, are used to interpret data or compose models intended to describe the hormone-sensitive regulatory G proteins.

Aluminum↗
PubMed1990

Fluorescence studies of nucleotide interactions with bovine adrenal chromogranin A.

The binding of the fluorescent probe bis-ANS to chromogranin A, the major protein of adrenal chromaffin vesicles, caused a marked enhancement and blue shift in the fluorescence emission spectrum. The emission maximum shifted from 515 nm to 480 nm and the yield increased approx. 75-fold upon addition of 10 microM chromogranin A to 1 microM bis-ANS. Adenine nucleotides had clear effects on the bis-ANS fluorescence signal, while other nucleotides such as GTP, UTP and CTP had no discernible effect. Specifically, ATP caused a decrease in the fluorescence, whereas ADP and AMP caused a fluorescence increase. These results indicate adenine nucleotide binding to chromogranin A. Substitution of ATP with epsilon-ATP, an ATP derivative with a modification on the six-membered ring of the adenine base, failed to reduce the fluorescence intensity. Therefore, it was concluded that adenine bases play an important role in the chromogranin A-adenine nucleotide interaction.

Adenosine Triphosphate↗
PubMed1990

Excitation wavelength dependent fluorescence anisotropy of eosin-myosin adducts. Evidence for anisotropic rotations.

Steady-state and time-resolved fluorescence anisotropy measurements of eosin in solution and eosin-5-maleimide bound to purified myosin were made to study localized motions of the "head region" of this protein. The lifetime and apparent Debye rotational relaxation times of eosin in aqueous solution are essentially invariant with changes in excitation wavelength. In more viscous solvents, such as propylene glycol/water mixtures, the apparent Debye rotational relaxation times of eosin differ upon excitation in the regions of positive and negative anisotropy. Using eosin attached to the SH-1 thiol of the myosin head differing rotational modes of the bound probe were detected, dependent upon excitation wavelength. The main features of the anisotropy data for eosin-myosin are consistent with the existence of a 'crevice' or 'pocket' in the myosin head. A model is presented which allows estimation of the ratio of distinct rotational diffusion terms (selected by different excitation wavelengths) that produce both the observed steady-state anisotropy and differential phase results.

Animals↗
PubMed1990

Oxygen diffusion through horseradish peroxidase.

The quenching by molecular oxygen of the fluorescence from a protoporphyrin IX adduct of horseradish peroxidase has been investigated using both intensity and time-resolved techniques. The bimolecular quenching rate constant determined for this process, as evaluated by the conventional Stern-Volmer analysis, was 2 x 10(8) M-1 s-1, among the lowest observed for protein systems. This result suggests that the heme binding site in horseradish peroxidase is relatively inaccessible to oxygen, which may account for the observation of room temperature phosphorescence in aerated solutions from enzymatically created triplet states.

Diffusion↗
PubMed1989

Time-resolved fluorescence studies on protoporphyrin IX-apohorseradish peroxidase.

The hemin moiety of horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) was removed and the apoprotein reconstituted with the fluorescent protoporphyrin IX. Steady-state and time-resolved fluorescence properties of the HRP(desFe) adduct were examined; the multifrequency phase and modulation method was utilized for lifetime and dynamic polarization studies. The emission spectrum of HRP(desFe) had maxima at 633 and 696 nm. The lifetime of this emission was characterized by a single exponential decay of 16.87 ns at 22 degrees C. Debye rotational relaxation times for HRP(desFe) were determined using both static (Perrin plot) and dynamic (differential phase and modulation fluorometry) methods; these two approaches gave values of 96 and 86 ns, respectively. A spherical protein of HRP's molecular weight and partial specific volume would be expected to have a Debye rotational relaxation time, at 22 degrees C, in the range of 50 to 60 ns, depending upon the extent of hydration. Hence our results indicate that HRP(desFe) is asymmetric; the global rotational relaxation times observed are consistent with those of a prolate ellipsoid with an axial ratio of 3:1.

Apoenzymes↗
PubMed1989

Time-resolved fluorescence studies on the ternary complex formed between bacterial elongation factor Tu, guanosine 5'-triphosphate, and phenylalanyl-tRNAPhe.

Time-resolved fluorescence spectroscopy was used to investigate the solution dynamics of Escherichia coli tRNAPhe, Phe-tRNAPhe, and Phe-tRNAPhe associated with GTP and elongation factor Tu (EF-Tu) in a ternary complex. Two fluorescence probes were employed: fluorescein, covalently bound to Phe-tRNAPhe at the s4U8 base (Phe-tRNAPhe-Fl8), and ethidium bromide, noncovalently associated with the tRNA (EB.Phe-tRNAPhe). The lifetimes observed for ethidium bromide were 1.89 ns, free in solution, and 26.3 ns, bound to its tight binding site on tRNA. Fluorescein-labeled tRNA had a lifetime of 4.3 ns, with no significant difference among the values for aminoacylated, unacylated, and EF-Tu-bound Phe-tRNAPhe-Fl8. Differential phase and modulation data for each fluorophore-tRNA system were fit with local and global Debye rotational relaxation times. Local motion of the labeled fluorescein in Phe-tRNAPhe-Fl8, tRNAPhe-Fl8, and Phe-tRNAPhe-Fl8.EF-Tu.GTP was characterized by rotational relaxation times of 2.7 +/- 0.5, 2.4 +/- 0.4, and 2.4 +/- 0.1 ns, respectively. These values are equal, within experimental error, and suggest that the rotational mobility of the s4U8-conjugated dye is unaffected by either tRNAPhe aminoacylation or ternary complex formation. Global rotational relaxation times for Phe-tRNAPhe-Fl8, 97 ns, and EB.Phe-tRNAPhe, 140 ns, were equivalent to those determined for the unacylated species, denoting little change in the overall size or shape of the tRNA molecule upon aminoacylation. These values for (Phe-)tRNA were larger than expected for a hydrated sphere of equivalent volume, 83 ns, and therefore confirm the asymmetric nature of the tRNA structure in solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Escherichia coli↗
PubMed1989

Time-resolved fluorescence studies on NADH bound to mitochondrial malate dehydrogenase.

Time-resolved fluorescence studies on the emission of NADH bound to porcine heart mitochondrial malate dehydrogenase [S)-malate:NAD+ oxidoreductase, EC 1.1.1.37), in the presence and absence of saturating levels of hydroxymalonate, were carried out. The lifetime of NADH bound in the ternary complex was determined to be 9.5 ns compared to 1.74 ns as reported in the literature. Steady-state and dynamic polarization data indicated a Debye rotational relaxation time in the range of 106-109 ns for the dimeric enzyme. This value is significantly larger than that calculated for a spherical protein and is consistent with the asymmetric dimer found by crystallographic studies.

Animals↗