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Biomedical subjects

D M Jameson

Publications and source records attributed to D M Jameson.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1989

Role of BBM lipid composition and fluidity in impaired renal Pi transport in aged rat.

Renal tubular phosphate (Pi) transport is impaired in the aged rat. The decrement in Pi transport is manifested as a decrease in the Vmax of the luminal brush-border membrane (BBM) sodium-dependent Pi transport. In the present study we determined the potential role of alterations in cortical BBM lipid composition and fluidity in the age-related phosphaturia observed in rats. In the aged rat there are significant increases in the BBM cholesterol (Chol, 504 vs. 422 nmol/mg protein in adult, P less than 0.01) and sphingomyelin (Sph, 41.8 vs. 37.5 mol% in adult, P less than 0.01) and a decrease in the BBM fluidity [increase in fluorescence anisotropy of diphenylhexatriene (DPH), rDPH, 0.221 vs. 0.215 in adult, P less than 0.01]. The BBM lipid compositional and fluidity alterations may also play an important role in the impaired renal adaptation to a low-Pi diet in the aged rat. In the adult rat the renal adaptation to a low-Pi diet is associated with a decrease in BBM Chol (370 to 307 nmol/mg protein, P less than 0.01) and an increase in fluidity (decrease in rDPH 0.207 to 0.201, P less than 0.05). In the aged rat the renal adaptation to a low-Pi diet is incomplete and is associated with impaired ability to lower BBM Chol (441 to 429 nmol/mg protein, P = NS) and to increase fluidity (rDPH 0.211 to 0.211, P = NS). The results of this study therefore suggest that in the aged rat the increase in BBM Chol and Sph content, and the decrease in BBM fluidity play an important role in the impaired renal tubular Pi transport and adaptation to a low-Pi diet.

Aging↗
PubMed1989

In vitro evidence that urine composition affects the fraction of active furosemide in the nephrotic syndrome.

Diuretic resistance to furosemide in the nephrotic syndrome (NS) may result from binding of drug to filtered albumin within the renal tubule. In buffer solutions intended to partially mimic the luminal environment of the distal nephron during the NS, we examined several chemical properties to determine their effect on furosemide-albumin binding equilibria. Dissociation constants were obtained by measuring furosemide's quenching of human serum albumin's intrinsic tryptophan fluorescence over ranges of pH, ionic strength (IS) and osmolarity. Neither pH nor osmolarity significantly affected binding; however, incremental increases in IS between 0.0 and 1.0 produced increases in Kd from 0.65 +/- 0.05 to 34.38 +/- 1.72 microM, resulting in a 5- and 28-fold increase in the unbound furosemide fraction when the furosemide-albumin concentrations were 3.0:5.0 and 10.0:45.0 microM, respectively. Our results indicate that human serum albumin contains one high affinity binding site for furosemide that is sensitive to IS. Because of changes in the concentrations of reactants as well as IS that can occur in nephron segments distal to furosemide's site of action, we conclude that the amount of unbound (i.e., pharmacologically active) drug in voided urine will not necessarily correspond to the amount at the active site. To clinically assess the pharmacodynamic consequence of protein binding in the NS, changes in the concentration of the reactants and IS in the distal nephron must be minimized so that the unbound furosemide measured in voided urine will accurately reflect the amount at the drug's active site.

Furosemide↗
PubMed1988

C-reactive protein, leukocytes, and fever after central IL 1 and alpha-MSH in aged rabbits.

Aged and young unanesthetized rabbits with intracerebroventricular cannulas were tested in experiments designed to determine whether increases in plasma C-reactive protein (CRP) level and leukocytosis can be rapidly induced by central administration of crude buffy-coat supernatant commonly called endogenous pyrogen or interleukin 1 (IL 1). The results indicate that both acute-phase responses occur during fever caused by central administration of this supernatant and that they are generally detectable within 2 h. Although the febrile response was smaller in aged female rabbits, there was no decline in CRP or leukocyte responses, an observation that was not predicted. The antipyretic neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH) reduced fever caused by central IL 1 more effectively in the aged rabbits. alpha-MSH likewise inhibited the CRP and leukocyte responses to central IL 1. The results confirm that CRP and leukocyte responses can be driven by a central IL 1 signal and further indicate that the response can occur rapidly, consistent with direct central nervous system control of the acute-phase responses. The findings indicate that the acute-phase responses depend in part on the age of the host and that the responses can be modulated by an endogenous central nervous system peptide with known antipyretic and immune modulatory properties.

Acute-Phase Reaction↗
PubMed1987

Intrinsic fluorescence of elongation factor Tu in its complexes with GDP and elongation factor Ts.

The intrinsic fluorescence properties of elongation factor Tu (EF-Tu) in its complexes with GDP and elongation factor Ts (EF-Ts) have been investigated. The emission spectra for both complexes are dominated by the tyrosine contribution upon excitation at 280 nm whereas excitation at 300 nm leads to exclusive emission from the single tryptophan residue (Trp-184) of EF-Tu. The fluorescence lifetime of this tryptophan residue in both complexes was investigated by using a multifrequency phase fluorometer which achieves a broad range of modulation frequencies utilizing the harmonic content of a mode-locked laser. These results indicated a heterogeneous emission with major components near 4.8 ns for both complexes. Quenching experiments on both complexes indicated limited accessibility of the tryptophan residue to acrylamide and virtually no accessibility to iodide ion. The quenching patterns exhibited by EF-Tu-GDP and EF-Tu X EF-Ts were, however, different; both quenchers were more efficient at quenching the emission from the EF-Tu x EF-Ts complex. Steady-state and dynamic polarization measurements revealed limited local mobility for the tryptophan in the EF-Tu x GDP complex whereas formation of the EF-Tu x EF-Ts complex led to a dramatic increase in this local mobility.

Fluorescence↗
PubMed1987

Interaction of a fluorescent analogue of GDP with elongation factor Tu: steady-state and time-resolved fluorescence studies.

A fluorescent derivative of GDP was prepared by the reaction of 2'-amino-2'-deoxy-GDP with fluorescamine. This derivative binds tightly (KD approximately 4.5 X 10(-8) M) to elongation factor Tu (EF-Tu) from Escherichia coli. The emission properties, including spectra, polarizations, and lifetimes, for fluorescamine-GDP free in solution and bound to EF-Tu are presented. Emission data on the fluorescamine-ethylamine conjugate are also given. A multifrequency phase and modulation lifetime study (using nine modulation frequencies over the range of 2-80 MHz) indicated that the emissions of these three systems were well characterized by single exponential decays corresponding to 1.45 ns for the fluorescamine-ethylamine derivative in buffer and to 7.74 and 11.03 ns for the fluorescamine-GDP derivative free in buffer and bound to EF-Tu, respectively. Multifrequency differential polarized phase fluorometry results indicated a rotational relaxation time of the protein-probe complex of approximately 88 ns; these data also indicated the lack of significant local motion for the probe. Addition of excess GDP to the EF-Tu-probe complex led to displacement of the fluorescamine-GDP derivative as evidenced by the change in both the steady-state and dynamic polarization values. The observed increase in fluorescence intensity upon displacement allowed us to follow the kinetics of the dissociation reaction; a dissociation rate constant of 5.0 X 10(-3) S-1 was determined. These results demonstrate the utility of this 2'-amino-2'-deoxy-GDP analogue as a probe of guanosine nucleotide dependent systems.

Absorption↗
PubMed1987

Dynamic aspects of the heme-binding site in phylogenetically distant myoglobins.

Dynamic aspects of the heme-binding site of myoglobins derived from two phylogenetically distant species, namely sperm whale and bluefin tuna, have been investigated by studying steady-state and time-resolved emission properties of 2-p-toluidinyl-6-naphthalene sulfonic acid (TNS) apomyoglobin conjugates. Multi-frequency phase and modulation fluorometry data indicate that charge movements occur in the fluorophore environment during the excited state lifetime in the sperm whale myoglobin system. In the case of the bluefin tuna myoglobin TNS adduct these movements were not detected, indicating that the relaxation processes differ in the two types of myoglobins.

Animals↗
PubMed1986

Fluorescence quenching studies of apolipoprotein A-I in solution and in lipid-protein complexes: protein dynamics.

Fluorescence lifetime and intensity quenching studies of human plasma apolipoprotein A-I (apo A-I) in aqueous solution and in recombinant lipoprotein complexes with dimyristoylphosphatidylcholine (DMPC) indicate differences in conformational dynamics. In aqueous solution, the bimolecular quenching constants (k*) for lipid-free apo A-I fluorescence quenching by oxygen and acrylamide are 2.4 X 10(9) and 0.38 X 10(9) M-1 s-1, respectively. These values are independent of the oligomeric form of the protein. There is no correlation between the relatively small k* for apo A-I, which reflects rapid, low-amplitude protein fluctuations, and the labile conformational changes of apo A-I folding reactions, like denaturation, which occur on a slower time scale. In recombinant DMPC/apo A-I complexes (100:1 molar ratio) the protein increases in amphiphilic alpha-helical structure as it blankets the lipid matrix. The apparent k* for oxygen quenching of apo A-I fluorescence in the complex is large and increases in a temperature-dependent manner. We have introduced a two-compartment model, which discriminates the source of quencher molecules as aqueous or lipid, to describe oxygen quenching of DMPC/apo A-I fluorescence. The magnitude and temperature dependence of the apparent k* predominantly reflect the partitioning of oxygen between the two phases rather than being a probe of the lipid physical state. Calculations of the helical hydrophobic moment in apo A-I indicate that tryptophan residues 8 and 72 occur at the lipid-protein interface of amphiphilic alpha-helices, whereas the other two tryptophan residues (50, 108) lie on the nonpolar faces of amphiphilic helices.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1986

Thermodynamic properties of ligand binding by monoclonal anti-fluorescyl antibodies.

The effects of temperature on the binding of fluorescein by three monoclonal anti-fluorescyl antibodies (4-4-20, 20-19-1, and 20-20-3) were assessed by measurements of affinity constants (Ka) over a temperature range of 2-70 degrees C. Values for Ka were determined from the degree of ligand association by using fluorescence methodology. Curvilinear van't Hoff plots (ln Ka vs. T-1) were observed for all three antibodies, indicating that their standard enthalpy changes (delta Ho) were temperature dependent. This phenomenon was further investigated by plotting the changes in unitary free energy (delta Gu), standard enthalpy (delta Ho), and unitary entropy (delta Su) vs. temperature. Strong temperature dependencies were observed for enthalpy and entropy values, while free energy plots were only weakly dependent on temperature. At low temperatures (4 degrees C), entropy played a major role in the binding of fluorescein by all three antibodies, while enthalpy dominated at higher temperatures. This was a consequence of the negative heat capacity changes (delta Cpo approximately equal to -320 cal K-1 mol-1) observed for these antibodies, which produced a negative trend in both enthalpy and entropy values with increasing temperature. The negative heat capacity values also indicated that the hydrophobic effect was instrumental in the binding of fluorescein. Entropy changes were lower than expected for hydrophobic binding alone, suggesting that other forces were acting to mitigate the hydrophobic effect. One possibility was that the binding of fluorescein acted to restrain vibrational fluctuations in the active-site region, producing negative changes in both heat capacity and entropy.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗