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D E Wolf

Publications and source records attributed to D E Wolf.

15 recordsLinked to original sources
PubMed1992

Determination of the transbilayer distribution of fluorescent lipid analogues by nonradiative fluorescence resonance energy transfer.

We measured the nonradiative fluorescence resonance energy transfer between 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) labeled lipids (amine labeled phosphatidylethanolamine or acyl chain labeled phosphatidylcholine) and rhodamine labeled lipids in large unilamellar dioleoylphosphatidylcholine vesicles. Two new rhodamine labeled lipid analogues, one a derivative of monolauroylphosphatidylethanolamine and the other of sphingosylphosphorylcholine, were found to exchange through the aqueous phase between vesicle populations but not to be capable of rapid transbilayer movement between leaflets. Energy transfer from NBD to rhodamine was measured using liposomes with symmetric or asymmetric distributions of these new rhodamine labeled lipid analogues to determine the relative contributions of energy transfer between donor and acceptor fluorophores in the same (cis) and opposite (trans) leaflets. Since the characteristic R0 values for energy transfer ranged from 47 to 73 A in all cases, significant contributions from both cis and trans energy transfer were observed. Therefore, neither of these probes acts strictly as a half-bilayer quencher of NBD lipid fluorescence. The dependence of transfer efficiency on acceptor density was fitted to a theoretical treatment of energy transfer to determine the distances of closest approach for cis and trans transfer. These parameters set limits on the positions of the fluorescent groups relative to the bilayer center, 20-31 A for NBD and 31-55 A for rhodamine, and provide a basis for future use of these analogues in measurements of transbilayer distribution and transport.

4-Chloro-7-nitrobenzofurazan↗
PubMed1992

Protein dynamics in sperm membranes: implications for sperm function during gamete interaction.

A number of mammalian sperm plasma membrane antigens have been implicated as playing a functional role in sperm-egg interaction, by virtue of the fact that antibodies against these antigens interfere with fertilization. Two such mouse sperm plasma membrane antigens are M42, a 200/220 kD glycoprotein doublet, and M5, a 150-160 kD glycoprotein. We show that both of these antigens are concentrated on the posterior region of caudal epididymal and capacitated mouse sperm heads and are relatively diffusible, as determined by fluorescence recovery after photobleaching measurements (D = 3-8 x 10(-9) cm2/s with approximately 23% diffusing). Crosslinking of these antigens with bivalent antibodies causes them to redistribute into the anterior region (acrosomal crescent) of the sperm head. In contrast, we describe a third antigen, P220, which is also localized to the posterior region of the sperm head on caudal epididymal sperm but which exhibits very little diffusion and does not redistribute upon crosslinking. Bivalent anti-M42 blocks the ZP3-induced acrosome reaction. We have found that monovalent Fab fragments of anti-M42 do not block the ZP3-induced acrosome reaction, but that inhibition is restored by addition of a second antibody which crosslinks the Fabs. Thus, crosslinking is required for both inhibition of the acrosome reaction and redistribution. This suggests that redistribution of antigen away from the posterior region of the head may be part of the mechanism of inhibition of the ZP3-induced acrosome reaction.

Acrosome↗
PubMed1992

Theory of fluorescence recovery after photobleaching measurements on cylindrical surfaces.

The theory of fluorescence recovery after photobleaching measurements of isotropic diffusion on a cylindrical surface is developed for Gaussian beam illumination centered perpendicular to an infinitely-long cylinder. A general analytical solution is obtained which is a function of the ratio of the cylindrical radius (r) to the beam exp [-2] radius omega. Numerical analysis of this solution demonstrates that significant deviations from one dimensional recovery are observed for omega less than 3r and from two-dimensional recovery for omega greater than or equal to 0.5r. Numerical data and an algorithm for analysis of recovery data where 0.5r less than or equal to omega less than or equal to 3r is presented.

Biophysical Phenomena↗
PubMed1991

Nerve growth factor receptors are preaggregated and immobile on responsive cells.

It has been hypothesized that signal transduction occurs by ligand-induced receptor clustering and immobilization. For many peptide receptors, cross-linking by anti-receptor antibodies is sufficient for receptor activation. This is not, however, the case for nerve growth factor receptor (NGFR). Using fluorescence microscopy and fluorescence recovery after photobleaching (FRAP), we have analyzed the distribution and diffusibility of NGFR on a series of cell lines. We have found the following: (1) Cells expressing high-affinity responsive NGFR's display clustered NGFR's even in the absence of ligand. In contrast, NGFR's in nonresponsive cell lines are diffusely distributed. (2) Receptors on responsive cell lines are largely nondiffusing while most receptors on nonresponsive cell lines are relatively free to diffuse. (3) NGF does not greatly alter the distribution or diffusion properties of the NGFR on either nonresponsive or responsive cell lines. Thus, NGFR is preclustered and immobile on responsive cells, which suggests that immobilization of NGFR prior to ligand binding is required for signal transduction.

Animals↗
PubMed1991

Cross-linking a maturation-dependent ram sperm plasma membrane antigen induces the acrosome reaction.

ESA152 is a highly hydrophobic 18 kDa sialoglycoprotein, which becomes expressed on ram sperm in the proximal cauda epididymis. ESA 152 is expressed on all regions of the sperm surface, most strongly on the posterior region of the head, most weakly on the anterior region of the head. In this paper, we show that induction of the acrosome reaction with Ca2+ ionophore causes ESA152 to be redistributed from the posterior to the anterior region of the head plasma membrane. Cross-linking ESA152 with bivalent antibody causes similar redistribution and induces the acrosome reaction. Induction of the acrosome reaction with ESA152 antibody requires Ca2+ but is insensitive to (10 ng/ml) pertussis toxin.

Acrosome↗
PubMed1990

Synthesis, purification, and characterization of 2,4,6-trinitrophenyl-UDP-galactose: a fluorescent substrate for galactosyltransferase.

Glycosyltransferases enzymatically transfer monosaccharides from sugar-nucleotides to complex oligosaccharide chains and, as cell surface molecules, exhibit receptor-like activity. We have modified the substate UDP-galactose to produce a compound that has useful absorbance and fluorescence properties upon binding to galactosyltransferase (GalTase). Using strategies similar to those for preparing fluorescent ATP analogs, we were able to synthesize 2,4,6-trinitrophenyl-5'-UDP-galactose (TUG). In solution, the absorbance properties of TUG are pH dependent, with absorbance maxima at 260, 408, and 453 nm and an isobestic point at 353 nm. In the presence of soluble GalTase extracted from bovine milk, TUG exhibited an excitation maximum at 368 nm with emission maxima at 436 and 533 nm; in the absence of GalTase only the 533-nm peak was present. Under enzymatic conditions, TUG acted as a competitive substrate of UDP-galactose with GalTase. Under noncatalytic conditions, the fluorescence emission of TUG at 436 nm increased monotonically with Gal-Tase concentration, with a half-maximal response at approximately 4 microM. This compound may be useful for quantifying GalTase function as both an enzyme and a cell adhesion molecule.

Binding, Competitive↗
PubMed1990

Lipid domains in the ram sperm plasma membrane demonstrated by differential scanning calorimetry.

Mammalian sperm plasma membranes, in contrast to those of mammalian somatic cells, exhibit a significant fraction of lipid that does not diffuse laterally in the plane of the membrane. This nondiffusing fraction results from lipid-lipid interactions. Similar nondiffusing fractions are found in mixed-lipid model systems that contain coexistent gel and fluid domains. These results suggest that the sperm plasma membrane may also exhibit lateral phase segregations of lipids and may contain significant amounts of gel-phase lipid. In this paper we use differential scanning calorimetry to show that, in contrast to the plasma membranes of mammalian somatic cells, the plasma membrane from the anterior region of the head of ram sperm exhibits at least two major endothermic transitions, one centered at approximately 26 degrees C and one centered at approximately 60 degrees C. The heats of these transitions are consistent with gel-to-fluid transitions in model membranes. These transitions are observed both in plasma membrane vesicles and in rehydrated lipid extracts made from these vesicles. These results demonstrate that at physiological temperatures the lipids of the ram sperm plasma membrane are segregated into coexistent fluid and gel domains. Since sperm encounter a wide range of temperatures during their development, these phase transitions may be important in establishing dynamic domains of lipid requisite for epididymal storage and fertilization.

Animals↗
PubMed1990

Lateral diffusion of nerve growth factor receptor: modulation by ligand-binding and cell-associated factors.

We compared the properties in human melanoma cell line A875 and rat pheochromocytoma cell line PC12 of nerve growth factor receptor (NGFr). We also analyzed NGFr and a truncated NGFR lacking the cytoplasmic domain, which were transiently expressed in COS cells. The full-length NGFR expressed in COS cells bound nerve growth factor (NGF) with positive cooperativity, but A875 NGFr and truncated NGFr in COS cells did not display positive cooperativity. The anti-human NGFr monoclonal antibody NGFR5 was characterized and found not to compete with NGF for binding to NGFr. Fabs were prepared from NGFR5 and 192, an anti-rat NGFR monoclonal antibody that was previously shown not to compete with NGF for binding. Fluorescein-labeled Fabs were used to measure the distribution and lateral diffusion of the NGFr. NGFr expressed on COS and A875 cells are diffusely distributed, but NGFr on the surface of PC12 cells appeared, for some cells, to be patched. In A875 cells, 51% of the NGFr was free to diffuse with diffusion coefficient (D) approximately 7 X 10(-10) cm2/s. In COS cells, 43% diffused with D approximately 5 X 10(-10) cm2/s. There was no significant difference in diffusibility between the full-length NGFr and the truncated NGFr. We compared NGFr diffusion on PC12 cells in suspension or adherent to collagen-coated coverslips. For suspension cells, we obtained 32% recovery with D approximately 2.5 X 10(-9) cm2/s. On adherent cells, we obtained 17% recovery with 6 X 10(-9) cm2/s. Binding of NGF enhanced lateral diffusion of NGFr in A875 cells and in PC12 cells in suspension but did not alter lateral diffusion of NGFr in COS cells or in adherent PC12 cells. NGF had no effect on the diffusing fraction or the distribution of NGFR for any cell line.

Animals↗
PubMed1988

Causes of nondiffusing lipid in the plasma membrane of mammalian spermatozoa.

In the plasma membranes of most mammalian somatic cells, lipid is nearly completely free to diffuse laterally in the plane of the membrane. In mammalian spermatozoa and certain other highly polarized mammalian cells, a significant fraction of the plasma membrane lipid is not free to diffuse laterally. Using the technique of fluorescence recovery after photobleaching, we have demonstrated that a variety of fluorescent lipid analogues exhibit a nondiffusing fraction in the plasma membrane of the anterior region of the ram sperm head. The possible causes of this nondiffusing fraction were investigated. The nondiffusing lipid fraction is not the result of lipid oxidation during handling, and it is not released by extensive enzymatic digestion of the membrane surface proteins or the "bleeding" of the membrane by hypoosmotic shock. When lipid bilayers were prepared from protein-free lipid extracts of the plasma membranes of spermatozoa, most of the nondiffusing fraction was retained. These results suggest that the nondiffusing lipid fraction results from lipid factors such as lateral phase separations, which can cause such a nondiffusing fraction in model systems.

Animals↗
PubMed1988

Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer.

Three approaches were used to study hybridization of complementary oligodeoxynucleotides by nonradiative fluorescence resonance energy transfer. (i) Fluorescein (donor) and rhodamine (acceptor) were covalently attached to the 5' ends of complementary oligodeoxynucleotides of various lengths. Upon hybridization of the complementary oligodeoxynucleotides, energy transfer was detected by both a decrease in fluorescein emission intensity and an enhancement in rhodamine emission intensity. In all cases, fluorescein emission intensity was quenched by about 26% in the presence of unlabeled complement. Transfer efficiency at 5 degrees C decreased from 0.50 to 0.22 to 0.04 as the distance between donor and acceptor fluorophores in the hybrid increased from 8 to 12 to 16 nucleotides. Modeling of these hybrids as double helices showed that transfer efficiency decreased as the reciprocal of the sixth power of the donor-acceptor separation R, as predicted by theory with a corresponding R0 of 49 A. (ii) Fluorescence resonance energy transfer was used to study hybridization of two fluorophore-labeled oligonucleotides to a longer, unlabeled oligodeoxynucleotide. Two 12-mers were prepared that were complementary to two adjacent sequences separated by four bases on a 29-mer. The adjacent 5' and 3' ends of the two 12-mers labeled with fluorescein and rhodamine exhibited a transfer efficiency of approximately 0.60 at 5 degrees C when they both hybridized to the unlabeled 29-mer. (iii) An intercalating dye, acridine orange, was used as the donor fluorophore to a single rhodamine covalently attached to the 5' end of one oligodeoxynucleotide in a 12-base-pair hybrid. Under these conditions, the transfer efficiency was approximately 0.47 at 5 degrees C. These results establish that fluorescence modulation and nonradiative fluorescence resonance energy transfer can detect nucleic acid hybridization in solution. These techniques, with further development, may also prove useful for detecting and quantifying nucleic acid hybridization in living cells.

Chromatography, High Pressure Liquid↗
PubMed1977

Lateral diffusion in planar lipid bilayers.

Direct measurements by fluorescence correlation spectroscopy of lateral diffusion coefficients of fluorescent lipid analogs in lipid bilaryer membranes indicate self-diffusion coefficients D greater than 10(-7) square centimeters per second for various lipid systems above their reported transition temperatures. Cholesterol in egg lecithin at mole ratio of 1 : 2 reduces D by about twofold, while retained hydrocarbon solvent can increase it by two- to threefold.

Cholesterol↗