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The interaction of dynamin II with giant unilamellar vesicles was studied using two-photon fluorescence microscopy. Dynamin II, labeled with fluorescein, was injected into a microscope chamber containing giant unilamellar vesicles, which were composed of either pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or a mixture of POPC and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Binding of the fluorescent dynamin II to giant unilamellar vesicles, in the presence and absence of PI(4,5)P2, was directly observed using two-photon fluorescence microscopy. This binding was also visualized using the fluorescent N-methylanthraniloyl guanosine 5'-[gamma-thio]triphosphate analogue. The membrane probe 6-dodecanoyl-2-dimethylamine-naphthalene was used to monitor the physical state of the lipid in the giant unilamellar vesicles in the absence and presence of dynamin. A surprising finding was the fact that dynamin II bound to vesicles in the absence of PI(4,5)P2. Activation of the GTPase activity of dynamin II by pure POPC was then shown.

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Cholesterol is known to affect the activity of membrane-bound enzymes, including Na(+)/K(+)-ATPase. To gain insight into the mechanism of cholesterol's effect, we have used various hydrophobic fluorescent probes which insert into different regions of the membrane bilayer and report on the degree of hydration of their environment. Specifially, we have measured the generalized polarization of Laurdan and the lifetime of DPH and derivatives of DPH inserted into membranes from pig kidneys enriched in Na(+)/K(+)-ATPase. Spectral measurements were also carried out on these membranes after modification of their cholesterol content. The generalized polarization of Laurdan increased with increasing cholesterol, showing an abrupt modification at the native cholesterol content. The fluorescence lifetimes of DPH and the DPH derivatives were analyzed using a distribution model. The center value of these lifetime distributions and their widths also changed with increasing cholesterol. One DPH derivative, DPH-PC, showed a minimum value for the lifetime center at the native cholesterol concentration, whereas the other derivatives showed a maximum value for the lifetime center at that cholesterol concentration. DPH-PC is known to sense the protein-lipid interface, whereas the other derivatives sense the bulk lipid phase. These data suggest that hydration at the protein-lipid interface is maximal at the native cholesterol concentration as is the enzymatic activity. Hydration at the protein-lipid interface is therefore proposed to be required for activity. These results are in agreement with current models of membrane dynamics and thermodynamics of protein function.

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The unfolding and refolding of apohorseradish peroxidase, as a function of guanidinium chloride concentration, were monitored by the intrinsic fluorescence intensity, polarization, and lifetime of the single tryptophan residue. The unfolding was reversible and characterized by at least three distinct stages-the intensity and lifetime data, for example, were both characterized by an initial increase followed by a decrease and then a plateau region. The lifetime data, in the absence and presence of guanidinium chloride, were heterogeneous and fit best to a model consisting of a major Gaussian distribution component and a minor, short discrete component. The observed increase in intensity in the initial stage of the unfolding process is attributed to the conversion of this short component into the longer, distributed component as the guanidinium chloride concentration increases. Our results clarify and amplify previous studies on the unfolding of apohorseradish peroxidase by guanidinium chloride.

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The oligomeric state of fluorescein-labeled mitochondrial malate dehydrogenase (L-malate NAD+ oxidoreductase; mMDH; EC 1.1.1.37), as a function of protein concentration, has been examined using steady-state and dynamic polarization methodologies. A "global" rotational relaxation time of 103 +/- 7 ns was found for micromolar concentrations of mMDH-fluorescein, which is consistent with the reported size and shape of mMDH. Dilution of the mMDH-fluorescein conjugates, prepared using a phosphate buffer protocol, to nanomolar concentrations had no significant effect on the rotational relaxation time of the adduct, indicating that the dimer-monomer dissociation constant for mMDH is below 10(-9) M. In contrast to reports in the literature suggesting a pH-dependent dissociation of mMDH, the oligomeric state of this mMDH-fluorescein preparation remained unchanged between pH 5.0 and 8.0. Application of hydrostatic pressure up to 2.5 kilobars was ineffective in dissociating the mMDH dimer. However, the mMDH dimer was completely dissociated in 1.5 M guanidinium hydrochloride. Dilution of a mMDH-fluorescein conjugate, prepared using a Tris buffer protocol, did show dissociation, which can be attributed to aggregates present in these preparations. These results are considered in light of the disparities in the literature concerning the properties of the mMDH dimer-monomer equilibrium.

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The environmentally sensitive fluorescent probes 6-propionyl-2-(N,N-dimethylamino)naphthalene (PRODAN) and 2'-(N,N-dimethylamino)-6-naphthoyl-4-trans-cyclohexanioc acid (DANCA) form complexes with the heme binding site of apohorseradish peroxidase. The dissociation constants of the PRODAN and DANCA complexes were determined from anisotropy titration data to be approximately 8.7 x 10(-5) and 3.3 x 10(-4) M, respectively. From comparison of the steady state fluorescence spectra of PRODAN and DANCA in solvents of varying dielectric constants, and in the apohorseradish peroxidase complex, we conclude that the heme binding site of horseradish peroxidase is relatively polar. The lifetimes of PRODAN and DANCA in organic solvents of varying polarities can be fit to single exponential decays. However, the lifetimes of PRODAN and DANCA associated with apohorseradish peroxidase, determined using a background subtraction method to correct for the non-negligible fluorescence of unbound probe, fit best to a distribution of lifetime values. We attribute these lifetime distributions to microenvironmental heterogeneity which is also consistent with the observed dependence of the emission maxima of PRODAN-apohorseradish peroxidase upon the excitation wavelength. In neither the PRODAN nor the DANCA case was evidence found in the time-resolved data for relaxation of the protein matrix around the excited state probe dipole.

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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.

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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.

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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.

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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.

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