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1.
Cyanine dye labeling reagents for sulfhydryl groups   总被引:8,自引:0,他引:8  
Cyanine and merocyanine dyes are introduced as new fluorescent reagents for covalently labeling proteins and other biomolecules. These dyes, which contain iodoacetamide functional groups, have high extinction coefficients and moderate quantum yields. A major advantage of these polymethine dyes is the easy manipulation of their spectral properties during synthesis. Cyanines containing reactive functional groups can be made with absorption maxima ranging from less than 500 nm to greater than 750 nm. This property opens additional regions of the spectrum for experiments involving the simultaneous multicolor analysis of different fluorescent probes. The cyanines, which are relatively insensitive to solvent property changes, are complemented by the merocyanines, which are keen indicators of solvent polarity.  相似文献   

2.
A membrane-bound ATPase from the archaebacterium Halobacterium saccharovorum is inhibited by N-ethylmaleimide in a nucleotide-protectable manner (Stan-Lotter et al., 1991, Arch. Biochem. Biophys. 284, 116-119). When the enzyme was incubated with N-[14C]ethylmaleimide, the bulk of radioactivity was associated with the 87,000-Da subunit (subunit I). ATP, ADP, or AMP reduced incorporation of the inhibitor. No charge shift of subunit I was detected following labeling with N-ethylmaleimide, indicating an electroneutral reaction. The results are consistent with the selective modification of sulfhydryl groups in subunit I at or near the catalytic site and are further evidence of a resemblance between this archaebacterial ATPase and the vacuolar-type ATPases.  相似文献   

3.
Enzyme I is the first protein of the phospho transfer sequence in the bacterial phosphoenolpyruvate:glycose phosphotransferase system. This protein exhibits a temperature-dependent monomer/dimer equilibrium. The nucleotide sequence of Escherichia coli ptsI indicates four -SH residues per subunit (Saffen, D. W., Presper, K. A., Doering, T. L., and Roseman, S. (1987) J. Biol. Chem. 262, 16241-16253). In the present experiments, the sulfhydryl groups of the E. coli enzyme were studied with various -SH-specific reagents. Titration of Enzyme I with 5,5'-dithiobis-2-nitrobenzoic acid also revealed four reacting -SH groups. The kinetics of the 5,5'-dithiobis-2-nitrobenzoic acid reaction with Enzyme I exhibit biphasic character, with pseudo-first order rate constants of 2.3 x 10(-2)/s and 2.3 x 10(-3)/s at pH 7.5, at room temperature. Fractional amplitudes associated with the rate constants were 25 +/- 5% for the fast and 75 +/- 5% for the slow rate. The "slow" rate was influenced by ligands that react with Enzyme I (the protein HPr, Mg2+, Mg2+ plus P-enolpyruvate), and also by temperature (at the temperature range where the monomer/dimer association occurs). The fractional ratio of the two rates remained at 1:3 under these conditions. Thus, under all conditions tested, two classes of -SH groups were detected, one reacting more rapidly than the other three -SH groups. Modification of the "fast" -SH group results in an active enzyme capable of forming dimer, whereas modification of the slow -SH groups results in inactive and monomeric Enzyme I. The enzyme was labeled with pyrene maleimide under conditions where only the more reactive sulfhydryl group was derivatized. Hydrolysis by trypsin followed by reverse-phase high performance liquid chromatography analysis of the peptide mixture resulted in only one fluorescent peak. This peak was not observed when the more reactive sulfhydryl residue was protected prior to pyrene maleimide labeling. Amino acid sequencing of the fluorescent peak indicated that the more reactive residue is the C-terminal amino acid residue, cysteine 575. The results provide a means for selectively labeling Enzyme I with a fluorophore at a single site while retaining full catalytic activity.  相似文献   

4.
Electron paramagnetic resonance was used to characterize the first use of a thiol-specific spin label in membranes. Procedures for use of the spin-label, 1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl (methanethiosulfonate MTS) covalently attached to membrane proteins in human erythrocyte membranes are reported. The major findings are: (1) MTS was found to be thiol-specific in membranes as it is for soluble proteins; (2) MTS labels ghost proteins in as few as 30 min at room temperature, providing a distinct advantage when sensitive or fragile membranes are to be used; (3) the distribution of the spin label suggests that the major cytoskeletal protein, spectrin, and the major transmembrane protein (Band 3) incorporate the highest percentage of spin label. This procedure expands the tools with which the researcher can investigate the physical state of membrane proteins and its alteration upon interaction of membrane perturbants or in pathological conditions.  相似文献   

5.
D Amir  D P Levy  Y Levin  E Haas 《Biopolymers》1986,25(9):1645-1658
Bovine pancreatic trypsin inhibitor (BPTI) was reductively alkylated with 2-methoxy-1-naphthyl aldehyde and sodium cyanoborohydride (NaCNBH3). All five possible derivatives, each labeled at one of the primary amino groups of BPTI, were obtained. The distribution of yields of the various derivatives can be controlled by changing the reaction conditions. Products were identified by high-performance liquid chromatography (HPLC) tryptic peptide mapping. This procedure was used for the preparation of three pure 2-methoxy-1-naphthyl-methylenyl-BPTI (MNA-BPTI) derivatives. Purification was achieved by means of affinity chromatography and HPLC. The spectral characteristics of the probe, notably monoexponential decay with a lifetime of 6.8 ± 0.1 ns and moderate limiting fluorescence polarization, P = 0.3 ± 0.015, make it a very useful donor in energy-transfer measurements.  相似文献   

6.
Aminoacylase I from porcine kidney (EC 3.5.1.14) contains seven cysteine residues per subunit. Three sulfhydryl groups are accessible to modification by 4-hydroxymercuribenzoate (p-MB). The kinetics of the reaction suggest that only one of these groups affects acylase activity when modified by p-MB. Its reaction rate increases 2-3-fold when the essential metal ion of aminoacylase is removed. Modification of metal-free apoenzyme by N-ethylmaleimide (NEM) abolishes its activity without impairing Zn2+ binding. This indicates that the sulfhydryl group reacting with NEM is not directly coordinated to the metal. DTNB (5,5'-Dithio-bis(2-nitrobenzoate), Ellman's reagent) also modifies three sulfhydryl groups per subunit. In this case, the reactivities of native aminoacylase and apoenzyme are not significantly different. N-Hydroxy-2-aminobutyrate, a strong aminoacylase inhibitor, substantially increases the reactivity of the slowest reacting sulfhydryl in both native enzyme and metal-free aminoacylase. It appears that binding of the inhibitor or removal of the metal ion induces conformational changes of the amino-acylase active site that render a buried sulfhydryl group more accessible to modification.  相似文献   

7.
Reaction of sulfhydryl-containing compounds, RSH, with Ce4+ in the presence of the spin trap phenyl-N-t-butylnitrone results in the appearance of a nitroxide ESR spectrum, which is greatly diminished if the sulfhydryl group is blocked prior to reaction. The spectra have short lifetimes which can be increased two- to fivefold to half-lives of 5-60 min by prior flushing of the solutions with nitrogen. For small molecules, such as cysteine, N-acetylcysteine, glutathione, and 2-mercaptoethanol, the spectrum is that of a freely rotating nitroxide while for the proteins, bovine serum albumin and myosin, the spectrum is characteristic of a strongly immobilized nitroxide spin label rigidly attached to the protein. Since Ce4+ is reported to oxidize the sulfhydryl group via the thiyl radical, RS, the following reactions are proposed to account for the formation of the nitroxide: (formula; see text) These reactions permit the spin labeling of sulfhydryl proteins such that the nitroxide is much closer to the point of attachment than when using conventional spin-labeling methods.  相似文献   

8.
Inhibition of hexose transport by N-ethylmaleimide was studied with regard to alkylation of different types of sulfhydryl group on the hexose carrier of the human erythrocyte. Uptake of 3-O-methylglucose was progressively and irreversibly inhibited by N-ethylmaleimide, with a half-maximal effect at 10-13 mM. A sulfhydryl group known to exist on the exofacial carrier was not involved in transport inhibition by N-ethylmaleimide, since reversible protection of this group by the impermeant sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) had no effect on the ability of N-ethylmaleimide to inhibit transport, or on its ability to decrease the affinity of the exofacial carrier for maltose. Nevertheless, the exofacial sulfhydryl was quite reactive with N-ethylmaleimide, since it was possible using a differential labeling technique to specifically label this group in protein-depleted ghosts with a half-maximal effect at 0.3 mM N-[3H]ethylmaleimide, and to localize it to the Mr 19,000 tryptic carrier fragment. Transport inhibition by N-ethylmaleimide correlated best with labeling of a single cytochalasin B-sensitive internal sulfhydryl group on the glycosylated Mr 23,000-40,000 tryptic fragment of the carrier, which was half-maximally labeled at about 4 mM reagent. Whereas N-ethylmaleimide readily alkylates the exofacial carrier sulfhydryl, it inhibits transport by reacting with at least one internal carrier sulfhydryl located on the glycosylated tryptic carrier fragment.  相似文献   

9.
10.
Glutathione homeostasis and redox-regulation by sulfhydryl groups   总被引:1,自引:0,他引:1  
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11.
A method is described in which the glycoprotein transferrin was double labeled. Its sialic acid residues were labeled with 3H through a consecutive oxidation-reduction technique utilizing tritiated NaBH4. Its protein moiety was labeled with either 125I or 59Fe. Incubation of this double-labeled molecule at 4 degrees C with K562 cells gave overlapping curves, indicating identical patterns of binding for all labels. At 37 degrees C, 3H and 125I demonstrated identical patterns while 59Fe was cummulatively retained. This method can be used to follow the fate of other glycoproteins and their possible desialation in vivo.  相似文献   

12.
13.
14.
Sulfhydryl groups of membrane-bound rhodopsin are studied with the spin label technique by using five maleimide derivative probes of different lengths. Two sulfhydryl groups are titrated per molecule of rhodopsin. These groups are located in protected but probably different environments, less then 12 Å away from the aqueous phase. A distance of about 37 Å is measured between the two groups. These results are consistent with a model in which the two groups would be located close by the external surface of the protein but embedded within the membrane layer, the strong immobilization of the label molecules resulting from phospholipid-protein interactions.  相似文献   

15.
16.
There are two classes of proteins that can be phosphorylated on tyrosine in HeLa cells. One class can be detected by metabolic labeling with [32P]Pi and affinity chromatography using anti-phosphotyrosine antibodies. The other cannot be detected by this technique but can be detected among the proteins which bind to the antibodies by in vitro iodination with 125I. Presumably proteins of the second class contain phosphotyrosine at which the phosphate undergoes very slow turnover. The incubation of cells in phosphate-minus medium caused a marked reduction in the levels of phosphotyrosine-containing proteins, this explaining the failure of detection of the second class proteins even after prolonged labeling with [32P]Pi.  相似文献   

17.
Sulfhydryl groups of membrane-bound rhodopsin are studied with the spin label technique by using five maleimide derivative probes of different lengths. Two sulfhydryl groups are titrated per molecule of rhodopsin, These groups are located in protected but probably different environments, less than 12 A away from the aqueous phase. A distance of about 37 A is measured between the two groups. These results are consistent with a model in which the two groups would be located close by the external surface of the protein but embedded within the membrane layer, the strong immobilization of the label molecules resulting from phosphlipid-protein interactions.  相似文献   

18.
19.
The number of reactive cysteine residues of yeast RNA polymerase I was determined and their function was studied using parachloromercury benzoate (pCMB), dithiobisnitrobenzoate (DTNB) and N-ethyl-maleimide (NEM) as modifying agents. By treatment with DTNB about 45 sulfhydryl groups react in the presence of 8M urea. Under non-denaturing conditions only 20 sulfhydryl groups are reactive with pCMB and DTNB. Both reagents completely inactivate the enzyme and this effect can be reversed by reducing agents. The sedimentation coefficient and the subunit composition are not affected when the enzyme is inactivated. Two of the most reactive sulfhydryl groups are necessary for activity. The modification of these groups is partially protected by substrates and DNA, suggesting that they are involved either in catalysis or in the maintenance of the conformation of the active site. Experiments with 14C-NEM indicate that the most reactive groups are located in subunits of 185,000, 137,000 and 41,000 daltons.  相似文献   

20.
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