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Biomedical subjects

L Brand

Publications and source records attributed to L Brand.

At least 91 records · Page 5Linked to original sources
PubMed1983

Sugar transport by the bacterial phosphotransferase system. Preparation of a fluorescein derivative of the glucose-specific phosphocarrier protein IIIGlc and its binding to the phosphocarrier protein HPr.

In diauxic growth, the bacterial phosphoenolpyruvate: glycose phosphotransferase system (PTS) regulates the utilization of certain compounds which are not PTS substrates. It has recently been shown that this PTS regulation is mediated via one of the PTS phosphocarrier proteins, IIIGlc. In the present studies, IIIGlc was derivatized with the fluorescent reagent fluorescein-5-isothiocyanate. One mol of label was incorporated per mol of protein and the label was located at the NH2-terminal amine, as shown by tryptic peptide mapping and one-step Edman-type degradation. The fluorescent moiety was found to be stable and resistant to photodecomposition. The fluorescent IIIGlc was purified and shown to be fully active in its ability to accept phosphate from phospho-HPr (the histidine-containing phosphocarrier protein of the phosphotransferase system), but only 20% active in catalyzing the transfer of the phosphate to methyl alpha-glucoside via the membrane-bound II-BGlc protein. The decay of the fluorescence intensity was dominated by a single component (90%) with a lifetime of 4 ns. The decay of the fluorescence emission anisotropy was determined for excitation in both a negative and positive transition of fluorescein and was best described in terms of a biexponential function, indicating internal motion of the fluorophore and possible anisotropic rotation of the protein as a whole. The formation of a complex between IIIGlc and HPr was demonstrated by using the techniques of time-resolved and steady state fluorescence emission measurements, resonance energy transfer, and equilibrium gel filtration.

Amino Acid Sequence↗
PubMed1982

Sugar transport by the bacterial phosphotransferase system. Nanosecond fluorescence studies of the phosphocarrier protein (HPr) labeled at the NH2-terminal methionine.

HPr is a low molecular weight, phosphocarrier protein of the Salmonella typhimurium phosphoenolpyruvate:glycose phosphotransferase system (PTS). This protein was alkylated with the fluorescent reagent (N-iodoacetylaminoethyl)-5-naphthylamino-1-sulfonate under conditions which favor alkylation of the thioether linkage in methionine residues (Link, T. P. and Stark, G. R. (1968) J. Biol. Chem. 243, 1082-1088) to give the corresponding sulfonium derivatives. The isolated fluorescent protein (95-100% pure) was as active as native HPr both as a phosphoryl acceptor protein (phosphoenolpyruvate and Enzyme I of the PTS), and as a phosphocarrier protein in the phosphorylation of methyl alpha-glucoside by the complete PTS. The fluorescent label was shown to be predominantly, possibly exclusively, at the NH2-terminal methionine residue. The decay of the fluorescence intensity could be described in terms of a biexponential function with the time constants tau 1 approximately 7 ns, tau 2 approximately 15 ns, and a ratio of alpha 2/alpha 1 approximately 3 for the pre-exponential factors. The decay of the fluorescence emission anisotropy was found to be consistent with some internal motion of the probe, in addition to the rotation of the protein conjugate as a whole.

Bacterial Proteins↗
PubMed1982

Decay-associated fluorescence spectra and the heterogeneous emission of alcohol dehydrogenase.

A procedure is described for using nanosecond time resolved fluorescence decay data to obtain decay-associated fluorescence spectra. It is demonstrated that the individual fluorescence spectra of two or more components in a mixture can be extracted without prior knowledge of their spectral shapes or degree of overlap. The procedure is also of value for eliminating scattered light artifacts in the fluorescence spectra of turbid samples. The method was used to separate the overlapping emission spectra of the two tryptophan residues in horse liver alcohol dehydrogenase. Formation of a ternary complex between the enzyme, NAD+, and pyrazole leads to a decrease in the total tryptophan fluorescence. It is shown that the emission of both tryptophan residues decreases. The buried tryptophan (residue 314) undergoes dynamic quenching with no change in the spectral distribution. Under the same conditions, the fluorescence intensity of tryptophan (residue 15) decreases without a change in decay time but with a red shift of the emission spectrum. There is also a decrease in tryptophan fluorescence intensity when the free enzyme is acid denatured (succinate buffer, pH 4.1). The denatured enzyme retains sufficient structure to provide different microenvironments for different tryptophan residues as reflected by biexponential decay and spectrally shifted emission spectra (revealed by decay association). The value of this technique for studies of microheterogeneity in biological macromolecules is discussed.

Alcohol Dehydrogenase↗
PubMed1981

The relationship of amniotic fluid fluorescence polarization to neonatal lung function.

Steady-state fluorescence polarization (FP) of 1,6-diphenyl-1,3,5-hexatriene (DPH) in monkey and human amniotic fluid was studied over a wide range of gestational ages. In both of these systems, the fluorescence polarization decreased with advancing gestational age. In the monkey, these measurements were correlated with both biochemical and physiologic parameters of lung function, including maximal lung volume, alveolar stability, percentage of disaturated phosphatidylcholine in lung homogenate, and lecithin/sphingomyelin ratio of amniotic fluid. Fluorescence polarization values correlated well with the lung disaturated phosphatidylcholine content expressed as a percentage of phosphatidylcholine, thus suggesting that the fluorescent probe interacts with a fraction of the amniotic fluid which is closely related to development of the pulmonary surfactant. system. Comparison of monkey and human amniotic fluid fluorescence polarizations showed a greater anisotropy of DPH in the monkey fluid at all stages of gestation, thereby indicating a greater microviscosity in monkey pulmonary surfactant.

Amniotic Fluid↗
PubMed1981

Time-resolved fluorescence and anisotropy decay of the tryptophan in adrenocorticotropin-(1-24).

The direct time-resolved fluorescence anisotropy of the single tryptophan residue in the polypeptide hormone adrenocorticotropin-(1-24) (ACTH) and the fluorescence decay kinetics of this residue (Trp-9) are reported. Two rotational correlation times are observed. One, occurring on the subnanosecond time scale, reflects the rotation of the indole ring, and the other, which extends into the nanosecond range, is dominated by the complex motions of the polypeptide chain. The fluorescence lifetimes of the single tryptophan in glucagon (Trp-25) and the 23-26 glucagon peptide were also measured. In all cases the fluorescence kinetics were satisfied by a double-exponential decay law. The fluorescence lifetimes of several tryptophan and indole derivatives and two tryptophan dipeptides were examined in order to interpret the kinetics. In close agreement with the findings of Szabo and Rayner [Szabo, A. G., & Rayner, D. M. (1980) J. Am. Chem. Soc. 102, 554-563], the tryptophan zwitterion exhibits emission wavelength dependent double-exponential decay kinetics. At 320 nm tau 1 = 3.2 ns and tau 2 = 0.8 ns, with alpha 1 = 0.7 and alpha 2 = 0.3. Above 380 nm only the 3.2-ns component is observed. By contrast the neutral derivative N-acetyltryptophanamide has a single exponential decay of 3.0 ns. The multiexponential decay kinetics of the polypeptides are discussed in terms of flexibility of the polypeptide chain and neighboring side-chain interactions.

Adrenocorticotropic Hormone↗
PubMed1981

Time-resolved fluorescence of the two tryptophans in horse liver alcohol dehydrogenase.

The tryptophan fluorescence decay of horse liver alcohol dehydrogenase, at 10 degrees C in 0.1 M pH 7.4 sodium phosphate buffer, with excitation at 295 nm, is a double exponential with time constants of 3.8 and 7.2 ns. Within experimental error, the two lifetimes remain constant across the emission spectrum. Only the 3.8-ns lifetime is quenched in the NAD+-pyrazole ternary complex, and only the 7.2-ns lifetime is quenched by 0-0.05 M KI. On the basis of these results, we assign the 3.8-ns lifetime to the buried tryptophan, Trp-314, and the 7.2-ns lifetime to the exposed tryptophan, Trp-15. The steady-state lifetime-resolved emission spectrum of Trp-15 has a maximum at approximately 340 nm and that of Trp-15 is at approximately 325 nm. The total time-resolved emission, after 40 ns of decay, has a maximum between 338 and 340 nm and is primarily due to the Trp-15 emission. As a consequence of the wavelength dependence of the preexponential weighting factors, there is an increase in the average lifetime from the blue to the red edge of the emission. This increase reflects the change in the spectral contributions of Trp-15 and Trp-314. Consideration of the spectral overlap between the emission spectra of the two tryptophans and the absorption due to formation of the ternary complex, as well as the distances between the two residues and the bound NAD+, shows that the selective fluorescence quenching in the ternary complex can be accounted for entirely by singlet-single energy transfer. The decay of the fluorescence anisotropy was measured as a function of temperature from 10 to 40 degrees C and is well described by a monoexponential decay law. Over this temperature range the calculated hydrodynamic radius increases from 33.5 to 35.1 A. Evidently, the indole groups of Trp-15 and Trp-314 rotate with the protein as a whole, and there is some expansion of the protein matrix as the ambient temperature is increased.

Alcohol Dehydrogenase↗
PubMed1981

Investigation of the nature of enzyme--coenzyme interactions in binary and ternary complexes of liver alcohol dehydrogenase with coenzymes, coenzyme analogues, and substrate analogues by ultraviolet absorption and phosphorescence spectroscopy.

The difference spectra of binary and ternary complexes of horse liver alcohol dehydrogenase with oxidized and reduced nicotinamide adenine dinucleotides, nicotinamide 1,N6-ethenoadenine dinucleotide, and adenosine diphosphate ribose along with a number of substrate analogues have been measured. These spectra bear a very close resemblance to those obtained by perturbation of the coenzyme(s) and their analogues by acid, NaCl, dioxane, or tert-butyl alcohol. It is inferred that the coenzymes experience a combination of ionic and nonpolar environments at the adenine binding site of the enzyme. This is borne out by published X-ray crystallographic results. The phosphorescence spectra do not indicate the presence of ionized tyrosine in ternary complexes invovling enzyme, coenzyme, and substrate analogues. The ultraviolet spectra can be explained as arising from the perturbation of the coenzyme chromophores upon binding to the enzyme without having to invoke tyrosine ionization.

Adenosine Diphosphate Ribose↗
PubMed1979

On the kinetics of lead in the human body.

A compartment model is derived for the kinetics of lead in the human body. The parameters are estimated from field data. Numerical solutions of the equations are obtained. They are used to discuss recent theories on lead intake, in particular from the atmosphere.

Air Pollution↗
PubMed1979

Addition of basic amino acids prevents G-1 arrest of nitrogen-starved cultures of Saccharomyces cerevisiae.

Arginase-minus mutants of Saccharomyces cerevisiae were arrested in growth and accumulated at the unbudded G-1 stage of the cell cycle when starved for nitrogen. If, however, arginine was added to the culture medium at the time of starvation, growth ceased but the cells did not collect at the unbudded G-1 stage. We suggest that arginine addition prevented the cells from collecting at the G-1 stage by starving them for histidine and lysine, thereby inhibiting synthesis of proteins needed to complete the cell cycle.

Arginase↗
PubMed1978

The interaction of liver alcohol dehydrogenase with NADH as studied by differential protein denaturation.

Heat denaturation of horse liver alcohol dehydrogenase was followed in the presence of isobutyramide at various degrees of saturation of the binding sites by NADH. A study of the fluorescence enhancement which is observed when an excess of NADH is added to the partially denatured mixtures provides information regarding the relative concentrations of mono- and bioccupied enzyme molecules. This approach is of value in situations when the association constants for coenzyme are so large that the concentration of the free ligand is negligible. The results obtained indicate that the binding of NADH to liver alcohol dehydrogenase follows the statistically predicted distribution. At the same time evidence was obtained for interaction between the two subunits of the enzyme.

Alcohol Oxidoreductases↗
PubMed1978

Fluorescence measurements of environmental relaxation at the lipid-water interface region of bilayer membranes.

Nanosecond time-resolved emission spectroscopy is used to characterize the complex fluorescence behavior of the probe 2-p-toluidinonaphthalene 6-sulfonate (2,6 p-TNS) when adsorbed to several bilayer membrane system. These include egg phosphatidylcholine vesicles with and without added cholesterol as well as erythrocyte ghost membranes. In each case a nanosecond time-dependent shift of the fluorescence emission to lower energy follows pulsed photoexcitation. The properties of the time-resolved surfaces obtained are consistent with a non-exponential decay law which describes a continuous interaction process of 2,6 p-TNS with its local environment in the membrane. This environment consists in part of polar residues (water plus polar head region) undergoing nanosecond motions. The pure phosphatidylcholine bilayer system was studied at four temperatures and electronic and spectral relaxation contributions to the total fluorescence decay were separated. Temperature coefficients for empirical rate parameters derived for the separated processes were obtained. It appears that a treatment of the fluorescence behavior of amphiphilic probes such as 2,6 p-TNS adsorbed to bilayer membranes at temperatures near ambient in which a single lifetime and radiative decay channel have been assumed is inappropriate.

Cholesterol↗
PubMed1977

Rotational relaxation of the "microviscosity" probe diphenylhexatriene in paraffin oil and egg lecithin vesicles.

The rotational relaxation of the widely used "microviscosity" probe, 1,6-diphenyl-1,3,5-hexatriene, was examined by the technique of nanosecond time-resolved fluorescence depolarization. The decays of the emission anisotropy were determined at five temperatures in the range 3-31 degrees both in a reference paraffin oil and in sonicated egg lecithin vesicles. These decays were complex in both media. Marked qualitative as well as quantitative differences were observed in the rotational behavior of the probe in the complex bilayer medium as opposed to the homogeneous reference solvent. The results are discussed in relation to the structure of the hydrophobic bilayer membrane interior and the concept of its "microviscosity".

Mathematics↗
PubMed1977

Nanosecond time-dependent fluorescence depolarization of diphenylhexatriene in dimyristoyllecithin vesicles and the determination of "microviscosity".

The nanosecond time dependence of the fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene in L-alpha-dimyristoyllecithin vesicles was determined at temperatures above and below the midpoint of the gel-liquid crystalline transition. In neither case could the decay of the total fluorescent emission or the decay of the emission anisotropy be described adequately in terms of single exponential decay laws. At the lower temperature, the emission anisotropy did not approach zero in the time window available for measurement, a finding which may indicate that the range over which rotation of the probe can freely occur is restricted. The results are discussed in relation to the concept of microviscosity of bilayer membranes.

Benzene Derivatives↗