A spectroscopic technique for measuring slow rotational diffusion of macromolecules. 1: Preparation and properties of a triplet probe

Cherry, Richard J.; Cogoli, Augusto; Oppliger, M.; Schneider, Gyula; Semenza, Giorgio

doi:10.1021/bi00662a001

Cited by 97 publications

(37 citation statements)

References 13 publications

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“…Briefly, washed erythrocytes from fresh human blood were mixed with eosin 5-isothiocyanate (Molecular Probes, Plano, TX) in phosphatebuffered saline, pH 7.4, for 3 hr at 21'C, lysed, and washed extensively in 5 mM NaPO4/30 nM phenylmethylsulfonyl fluoride (PhMeSO2F), pH 7.4, and stored up to 7 days at 4VC in 5 mM NaPO4/10 mM NaN3/3O nM PhMeSO2F, pH 7.4. The amount of bound eosin was determined spectrophotometrically (13). Protein was determined by using the method of Lowry et al (14).…”

Section: Methodsmentioning

confidence: 99%

Lateral mobility of band 3 in the human erythrocyte membrane studied by fluorescence photobleaching recovery: Evidence for control by cytoskeletal interactions

Golan

Veatch

1980

Proc. Natl. Acad. Sci. U.S.A.

206

116

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Band 3,. the major intrinsic protein of the human erythrocyte membrane, was specifically labeled with the covalent fluorescent probe eosin isothiocyanate. The lateral mobility of labeled bans 3 in the plane of the membrane under various conditions of ionic strength and temperature was examined by using the fluorescence photobleaching recovery technique. Low temperature (210C) and high ionic strength (46 mM NaPO4) favored immobilization of band 3 (10% mobile) as well as slow diffusion of the mobile fraction (diffusion coefficient D = 4 X 101 cm2 sec'1). Increasing temperature (370C) and decreasing ionic strength (13 mM NaPO4) led to an increase in the fraction of mobile band 3 (90% mobile) and a reversible increase in the diffusion rate of the mobile fraction (D = 200 X 10-11 cm2 sec'). The increase in the fraction of mobile band 3 was markedly dissociated, however, from the increase in the diffusion rate of the mobile fraction. Thus, the fraction of mobile band 3 always increased at higher ionic strength and lower temperature than the ionic strength and temperature at which the diffusion rate increased. This dissociation was manifested kinetically on prolonged incubation of ghosts at constant ionic strength and temperatures the diffusion rate of the mobile fraction increased slowly at first and much more rapidly after the initial lag period, whereas the fraction of mobile band 3 increased almost immediately to 90% and remained maximal for the duration of the experiment. Further, changes in diffusion rate with temperature were promptly and totally reversible, whereas increases in the mobile fraction were only slowly and partially reversible. These effects were shown not to be due to complete dissociation of spectrin, the major protein of the erythrocyte cytoskeleton, from the membrane. This evidence suggests control of band 3 lateral mobility by at least two separate processes. The process that determines the diffusion coefficient of the mobile band 3 is completely reversible, and it probably involves a metastable state of cytoskeleton structure intermediate between tight binding to the membrane and complete dissociation from it.Extensions of the fluid-mosaic model for membrane structure (1) have emphasized the importance of intramembranous (especially transmembranous) forces in the modulation of lateral mobility of integral membrane proteins (2, 3). Immunologic phenomena such as antibody-induced patching and capping and anchorage modulation (reviewed in ref.3) demonstrate a functional role for such forces in mammalian cell membranes. With reference to the human erythrocyte membrane, qualitative evidence for cytoskeletal control of integral membrane protein distribution and mobility has emerged from freeze-fracture electron microscopic studies (4-7). Direct examination of the lateral mobility of fluorescently labeled transmembrane proteins (chiefly band 3 and glycophorin) by the cell fusion (8) and photobleaching recovery (9) techniques has also suggested restricted mobility for these proteins.A definitiv...

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Section: Methodsmentioning

confidence: 99%

Lateral mobility of band 3 in the human erythrocyte membrane studied by fluorescence photobleaching recovery: Evidence for control by cytoskeletal interactions

Golan

Veatch

1980

Proc. Natl. Acad. Sci. U.S.A.

206

116

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“…Eosin isothiocyanate was prepared as described by Cherry et al [34] and dissolved in 50% dimethylsulfoxide in 50 mM Hepes/NaOH pH 8 immediately before use. Its concentration was determined spectrophotometrically using an ~5 2 8 , , , , , of 8.3 x lo4 M-' cm-' at pH 8.…”

Section: Chemicalsmentioning

confidence: 99%

Proximity between fluorescent probes attached to four essential lysyl residues in phosphoenolpyruvate carboxylase

Wagner

Podestá

González

et al. 1988

European Journal of Biochemistry

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Phosphoenolpyruvate carboxylase, purified from maize leaves, is rapidly inactivated by the fluorescence probe dansyl chloride. The loss of activity can be ascribed to the covalent modification of an R-NH2 group, presumably the e-NH2 group of lysine. Analysis of the data by the statistical method of Tsou [Sci. Sin. ZI, 153.5-1.558 (1962)l provides clear evidence that at pH 8 eight R-NH2 groups can be modified in the tetrameric form of the enzyme, four of which are essential for catalytic activity. Essential groups are modified about five times more rapidly than the non-essential ones. The enzyme was completely protected against inactivation by Mg2+ plus phosphoenolpyruvate and consequently binding of the modifier to the essential groups is completely abolished. Hence the four essential groups seemed to be located at or near the active site(s).One of the four essential groups was modified with dansyl chloride and the other three progressively with eosin isot hiocyanate.In the doubly labeled protein non-radiative singlet-singlet energy transfer between dansyl chloride (donor) and eosin isothiocyanate (acceptor) was observed. The low variance (+_ 5%) in the efficiency of energy transfer obtained at a particular acceptor stoichiometry (0.8 -3 .l, 1.9 -2.1, 2.9 -3.1) in triplicate samples provided confidence that the measured transfer efficiency may be interpreted as transfer between specific sites. The distances calculated from the efficiency of resonance energy transfer revealed two acceptor sites, equally separated, 4.8 -5.1 nm from the donor site and the third site being 6.4 nm apart from the donor.Under conditions where the tetrameric enzyme dissociates into the monomers, no transfer of resonance energy between the protein-bound dansyl chloride and eosin isothiocyanate was observed.Most likely the four essential lysyl residues in the tetrameric enzyme are located in different subunits of the enzyme, hence each of the subunits would contain a substrate-binding site with one lysyl residue crucial for activity.Photosynthetic C 0 2 fixation by C4 plants involves a specialized metabolic pathway, the first step of which is the irreversible formation of oxaloacetate from phosphoenolpyruvate (P-pyruvate) and bicarbonate, catalyzed by Ppyruvate carboxylase [l -31. This enzyme, localized in the cytoplasm of leaf mesophyll cells [4], has been described as an oligomeric protein composed of four identical subunits of M , 100000 [5].Cysteine [6-81, histidine [9], arginine [lo] and lysine [ l l , 121 residues have been reported to be essential for catalytic activity.It has been shown that several metabolites and leaf illumination are responsible for the regulation of the carboxylase activity [13 -171. The extent of light activation and kinetic properties such as affinity constants for substrates and effectors are markedly affected by pH [9, 10, 18 -201. Correspondence to C. S. Andreo, Centro de Estudios Fotosintkticos y Bioquimicos, Suipacha 531, RA-2000 Rosario, ArgentinaAbbreviations. P-pyruvate, phosphoenolpyruvate ; Dns-...

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“…Recently, we have developed techniques of labelling proteins covalently using eosin isothiocyanate [59]. In studies with simple model systems we have obtained encouraging evidence that meaningful measurements of slow rotations may be made using this probe [60].…”

Section: Model Systemsmentioning

confidence: 99%

Protein mobility in membranes

Cherry

1975

FEBS Letters

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A spectroscopic technique for measuring slow rotational diffusion of macromolecules. 1: Preparation and properties of a triplet probe

Cited by 97 publications

References 13 publications

Lateral mobility of band 3 in the human erythrocyte membrane studied by fluorescence photobleaching recovery: Evidence for control by cytoskeletal interactions

Lateral mobility of band 3 in the human erythrocyte membrane studied by fluorescence photobleaching recovery: Evidence for control by cytoskeletal interactions

Proximity between fluorescent probes attached to four essential lysyl residues in phosphoenolpyruvate carboxylase

Protein mobility in membranes

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