PubMed · 3724557
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L C Smith, D M Benson, A M Gotto, J Bryan. 1986. Digital imaging fluorescence microscopy.. https://doi.org/10.1016/0076-6879(86)29109-0
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Previous work has shown that bovine prothrombin fragment 1 binds to supported planar membranes composed of phosphatidylcholine and phosphatidylserine in a Ca(2+)-specific manner (Tendian et al. (1991) Biochemistry 30, 10991; Pearce et al. (1992) Biochemistry 31, 5983-5995). In the present work, fluorescence pattern photobleaching recovery has been used to examine the effect of membrane-bound fragment 1 on the translational diffusion coefficients of two fluorescent phospholipids in fluid-like phosphatidylserine/phosphatidylcholine Langmuir-Blodgett monolayers. The results show that saturating concentrations of fragment 1, in the presence of Ca2+, reduce the diffusion coefficient of nitrobenzoxadiazolyl-conjugated phosphatidylserine (NBD-PS) and nitrobenzoxadiazolyl-conjugated phosphatidylcholine (NBD-PC) by factors of approximately four and two, respectively. Ca2+ or fragment 1 alone do not have a statistically significant effect on NBD-PS or NBD-PC diffusion. In addition, a nonspecific protein (ovalbumin) does not change the diffusion coefficients of the fluorescent phospholipids either in the absence or presence of Ca2+. The fractions of the fluorescent phospholipids that are laterally mobile are approximately 0.9 for all samples. These results are interpreted with several models for possible mechanisms by which extrinsically bound proteins might retard phospholipid diffusion in membranes.
The molecular characterization of transport proteins is often limited by transient functional expression or the need for a simple method to select functional cDNA clones. We used a mammalian expression system to obtain long-term expression of GLUT-2, an isoform of glucose permease. Rat GLUT-2 cDNA was ligated into an EBV vector (pLPP) and transfected into B lymphocytes which lack GLUT-2. Northern and Western analyses confirmed expression of GLUT-2 protein in membranes of transfected cells. Two functional assays using flow cytometry were developed to distinguish GLUT-2 transfectants from control/pLPP transfectants. Uptake of NBD-glucosamine, a fluorescent analogue of glucose, was increased in GLUT-2 transfectants. In addition, when exposed to hypertonic glucose medium, GLUT-2 transfectants and control/pLPP transfectants exhibited a difference in forward-angle light scatter (FALS), an index of cell volume, indicating a difference in glucose permeability. Independent measurements of glucose uptake (isotopic) and cell volume (video microscopy) confirmed the flow cytometry observations. This expression system used in combination with flow cytometry is useful for studying the functional properties of glucose and other solute transporters.
The nature of the kinetic intermediates involved in S1-induced polymerization of G-actin into F-acto-S1-decorated filaments has been investigated using as probes light scattering, the fluorescence of pyrenyl- or NBD-labeled actin, and the anisotropy of fluorescence of N-iodoacetyl-N'-(5-sulfo-1-napthyl)ethylene diamine (AEDANS)-labeled actin. With AEDANS-G-actin, the initial formation of a ternary G2S complex between two G-actin and one S1 molecules (Valentin-Ranc, C., Combeau, C., Carlier, M. F., and Pantaloni, D. (1991) J. Biol. Chem. 266, 17871-17879) has been confirmed. Data obtained with all probes are consistent with the subsequent rapid formation of G-actin-S1 oligomers in which the actin/S1 molar ratio is 2:1. Oligomers form above 0.6 microM G-actin with S1(A1) and above 3.5 microM G-actin with S1(A2), at 20 degrees C. Oligomerization is endothermic with a delta H of 14 kcal/mol. A tentative model is proposed to comprehensively account for the data and the structural features of the F-actin-S1 filament. Within this model, longitudinal actin-actin interactions take place in G2S, and lateral, hydrophobic actin-actin interactions appear upon formation of (G2S)n oligomers.