The excimer fluorescence of N-(1-pyrenyl)iodoacetamide labeled to myosin and its subfragment 1

Myosin and its subfragment 1 were labeled with the fluorescent probe N-(1-pyrenyl)iodoacetamide. Both of the labeled complexes exhibited the excimer band at 480 nm (pH 8.0, 25 degrees C). SH1 and SH2 are labeled with this probe as judged by Ca2+-ATPase of the labeled complex. Excimers arise both from the interaction of PIAAs in the two different heads within a single myosin molecule and also from the interaction of PIAAs in the same head. ATP affects these excimers depending on the concentration of Ca2+.     

Excimer fluorescence of equine platelet tropomyosin labeled with N-(1-pyrenyl)iodoacetamide

Tropomyosin from equine platelets was reacted with N-(1-pyrenyl)iodoacetamide, a sulfhydryl-specific fluorescent reagent, to give an average extent of incorporation of 1.12 pyrene (Py) groups per platelet tropomyosin (P-TM) chain. The predominant site of reaction on P-TM was the penultimate COOH-terminal residue, Cys-246. The high proportion of the total emission that is due to pyrene ecximers and the pretransition observed in thermal denaturation of Py-P-TM point to a rather loose structure for the COOH-terminal amino acid residues of P-TM. The label on Cys-246 also reports on end-to-end overlap interactions that occur between two different tropomyosin molecules. Additions to a Py-P-TM solution at low ionic strength of unlabeled P-TM, rabbit cardiac tropomyosin (C-TM), or a carboxypeptidase A treated, nonpolymerizable derivative of C-TM all reduce the extent of excimer fluorescence from the sample. Addition of salt greatly reduces the effects of the unlabeled TM species on the Py-P-TM emission spectrum. Circular dichroism measurements indicate Py-P-TM still to be greater than 95% helical. However, analysis of excimer fluorescence levels in samples that contained a constant protein concentration but different mole ratios of labeled to unlabeled P-TM suggests that the bulky pyrene group may diminish the tendency of Py-P-TM to polymerize in an end-to-end manner.     

The use of actin labelled with N-(1-pyrenyl)iodoacetamide to study the interaction of actin with myosin subfragments and troponin/tropomyosin.

A pyrene label attached to Cys-374 of actin has been shown to be a useful probe for monitoring the interaction of actin with myosin subfragments [Kouyama & Mihashi (1981) Eur. J. Biochem. 114, 33-38]. We report that the presence of this label decreases the affinity of actin for myosin subfragment 1 by less than a factor of 2. The rate of actin binding is unaffected by the label and the dissociation rate is increased by up to a factor of 2. Both the rate of actin binding to, and the rate of actin dissociation from, heavy meromyosin show two phases when monitored by pyrene fluorescence. Thin filiments reconstituted from pyrene-labelled actin show a 5% increase in pyrene fluorescence on binding Ca2+.     

High performance liquid chromatography analysis of 2-mercaptoethylamine (cysteamine) in biological samples by derivatization with N-(1-pyrenyl) maleimide (NPM) using fluorescence detection

2-Mercaptoethylamine (cysteamine) is an aminothiol compound used as a drug for the treatment of cystinosis, an autosomal recessive lysosomal storage disorder. Because of cysteamine's important role in clinical settings, its analysis by sensitive techniques has become pivotal. Unfortunately, the available methods are either complex or labor intensive. Therefore, we have developed a new rapid, sensitive, and simple method for determining cysteamine in biological samples (brain, kidney, liver, and plasma), using N-(1-pyrenyl) maleimide (NPM) as the derivatizing agent and reversed-phase high performance liquid chromatography (HPLC) with a fluorescence detection method (lambda(ex)=330 nm, lambda(em)=376 nm). The mobile phase was acetonitrile and water (70:30) with acetic acid and o-phosphoric acid (1 mL/L). The calibration curve for cysteamine in serine borate buffer (SBB) was found to be linear over a range of 0-1200 nM (r(2)=0.9993), and in plasma and liver matrix, the r(2) values were 0.9968 and 0.9965, respectively. The coefficients of the variation for the within-run and between-run precisions ranged from 0.68 to 9.90% and 0.63 to 4.17%, respectively. The percentage of relative recovery ranged from 94.1 to 98.6%.     

Pyrene excimer fluorescence in rabbit skeletal alphaalphatropomyosin labeled with N-(1-pyrene)maleimide. A probe of sulfhydryl proximity and local chain separation

Rabbit skeletal alphaalphatropomyosin was specificially labeled at cysteine 190 with the fluorescent reagent, N-(1-pyrene)maleimide. Spectroscopically different products were obtained by labeling at pH 6.0 (PyrI-alphaalphaTm) or pH 7.5 (PyrII-alphaalphaTm). PyrII-alphaalphaTm results from a secondary reaction between the N-(1-pyrene)succinimido moiety at cysteine 190 of PyrI-alphaalphaTm and a lysine group on the same chain, probably lysine 189. Pyrene excimer fluorescence was present in the native state but absent in the unfolded state of both products, thus verifying the proximity of the--SH groups and the chain register model for the structure of tropomyosin. Studies of the guanidinium chloride-dependent unfolding of PyrII-alphaalphaTm showed that loss of excimer fluorescence precedes unfolding, providing evidence for a region of preferential instability in the molecule near cysteine 190. This work suggests that N-(1-pyrene)maleimide could be used to probe both--SH proximity and local conformation in any protein if the presence of two or more proximal--SH groups is suspected.     

Interaction of plasma gelsolin with tropomyosin.

Horse plasma gelsolin labelled with benzophenone-4-isothiocyanate can be photochemically cross-linked to rabbit cardiac tropomyosin. The cross-linking proceeds with greater efficiency in calcium-containing buffers. Further evidence for interaction between these proteins is provided by retention of fluorescently labelled gelsolin on tropomyosin-agarose affinity columns and by the ability of tropomyosin to cause an increase in the fluorescence intensity of gelsolin labelled with fluorescein-5-isothiocyanate. Both of these effects require the presence of calcium ions.     

Comparison of Ellman's reagent with N-(1-pyrenyl)maleimide for the determination of free sulfhydryl groups in reduced cellobiohydrolase I from Trichoderma reesei

The enzyme cellobiohydrolase I (CBH I) from Trichoderma reesei was treated with 5 mM dithiothreitol at different pH values in order to reduce some or all of its 12 disulfide bridges. A discrepancy was found in the number of free sulfhydryl (SH) groups generated upon the reduction of CBH I when they were measured using N-(1-pyrenyl)maleimide (PM) or Ellman's reagent, 5,5′-dithiobis(2-nitrobenzoic acid). For example, the number of SH mol generated/mol CHB I at pH 8.5 was determined to be 16 and < 1 when measured using PM or Ellman's reagent, respectively. The low value obtained with Ellman's reagent may be due to the electrostatic repulsion between the carboxylic acid groups in CBH I and those in Ellman's reagent. The fluorimetric assay used for determining SH molecules in reduced CBH I, based on their reaction with PM, is described.     

Relations between plasma membrane and lysosomal membrane. 1. Fate of covalently labelled plasma membrane protein

To quantify the kinetics of the plasma membrane flow into lysosomes, we covalently labelled at 4 degrees C the pericellular membrane of rat fibroblasts and followed label redistribution to the lysosomal membrane using purified lysosomal preparations. The polypeptides were, either labelled with 125I by the lactoperoxidase procedure, or conjugated to [3H]peroxidase using bisdiazobenzidine as a bifunctional reagent. Both labels were initially bound to plasma membrane, as indicated by their equilibrium density in sucrose or Percoll gradients and their displacement by digitonin, as well as by electron microscopy. Upon cell incubation at 37 degrees C, both covalent labels were lost from cells with diphasic kinetics: a minor component (35% of cell-associated labels) was rapidly released (half-life less than 1 h), and most label (65%) was released slowly (half-life was 20 h for incorporated 125I and 27 h for 3H). Immediately after labelling up to 30 h after incubation at 37 degrees C, the patterns of 125I-polypeptides quantified by autoradiography after SDS-PAGE were indistinguishable, indicating no preferential turnover for the major plasma membrane polypeptides. The redistribution of both labels to lysosomes was next quantified by cell fractionation. At equilibrium (between 6 and 25 h of cell incubation) 2-4% of cell-associated 125I label was recovered with the purified lysosomal membranes. By contrast, when 3H-labelled cells were incubated for 16 h, most of the label codistributed with lysosomes. However, only 6% of cell-associated 3H was bound to lysosomal membrane. These results indicate that in cultured rat fibroblasts, a minor fraction of plasma membrane polypeptides becomes associated with the lysosomal membrane and is constantly equilibrated by membrane traffic.     

The reaction of N-(1-pyrene)maleimide with sarcoplasmic reticulum.

The excimer fluorescence of the adduct of N-(1-pyrene)maleimide (PMI) with the Ca2+-ATPase was proposed as a probe of ATPase-ATPase interactions in sarcoplasmic reticulum (Lüdi and Hasselbach, Eur. J. Biochem., 1983, 130:5-8). We tested this proposition by analyzing the spectral properties and stoichiometry of the adducts of pyrenemaleimide with sarcoplasmic reticulum and with dithiothreitol and by comparing the effects of various detergents on the excimer fluorescence of the two adducts, with their influence on the sedimentation characteristics, ATPase activity, and light scattering of the pyrenemaleimide-labeled sarcoplasmic reticulum. These studies indicate that pyrenemaleimide reacts nearly randomly with several SH groups on the Ca2+-ATPase, and suggest that the observed excimer fluorescence of pyrenemaleimide-labeled sarcoplasmic reticulum may reflect intramolecular phenomena rather than ATPase-ATPase interactions. Further work is required to establish the relative contribution of intra- and intermolecular mechanisms to the excimer fluorescence.     

Characterisation of the alpha 1-protease inhibitor system in Thoroughbred horse plasma by horizontal two-dimensional (ISO-DALT) electrophoresis. 1. Protein staining

The isoelectric points and the molecular weights of the major components of the eight Thoroughbred protease inhibitor (Pi) types have been determined by polyacrylamide gel isoelectric focusing and polyacrylamide gel pore gradient (ISO-DALT) electrophoresis respectively. The major Pi proteins focus in the range pH 3.74-4.43 and have molecular weights ranging from 55 000-72 000 daltons. Using the ISO-DALT method of electrophoresis, protein maps for the eight Thoroughbred Pi types have been presented for the first time. None of the homozygous Pi types are identical except for the types S1 and S2 which show partial identity. The results do not necessarily support Juneja et al.'s (1979) contention of two closely linked alpha 1 Pi systems based on molecular weight differences. It is suggested that the traditional nomenclature originally proposed by Braend (1970) be maintained to describe the eight Pi alleles in Thoroughbred horse plasma. The ISO-DALT method provides a sensitive technique which is superior to existing techniques for the analysis of the horse Pi system.     

Crystallization of the complex of actin with gelsolin segment 1.

Crystals of a 1:1 complex between human gelsolin segment 1 and actin have been grown from solutions containing polyethylene glycol 6000. The crystals are orthorhombic, space group P2(1)2(1)2(1); the axes are a = 57.4 A, b = 70.4 A, c = 184.5 A. They are moderately stable to X-rays and diffract to beyond 2.5 A. There is one molecule of complex in the asymmetric unit.     

Interactions of plasma gelsolin with actin. Isolation and characterization of binary and ternary plasma-gelsolin-actin complexes

We have studied the interactions between plasma gelsolin and actin: firstly the complex formation between both proteins, secondly the effects of gelsolin and its complexes on G-actin polymerization and F-actin fragmentation. Complex formation has been studied by high-performance gel permeation chromatography; plasma gelsolin alone elutes at an Mr of about 77000 and a Stokes radius of 3.7 nm; complex formation occurs in the presence of Ca2+: by chromatography in the presence of EGTA, a binary complex is obtained with an Mr of 134000 and a Stokes radius of 4.7 nm; and by chromatography in the presence of Ca2+, a ternary complex is obtained with an Mr of 173000 and a Stokes radius of 5.2 nm. The binary complex is EGTA-stable. In relation to this stability of the binary complex, the depolymerizing function of gelsolin is not reversed upon chelation of Ca2+. The effects of plasma gelsolin and its complexes on both G-actin polymerization and F-actin fragmentation, and their Ca2+ dependence have been examined by viscometry and electron microscopy. The main conclusions of these studies are the following: the fast processes are the formation of ternary complex, which acts as a heteronucleus for G-actin polymerization, and the severing function of gelsolin, these fast processes are Ca2+-dependent; the slow processes are related to the capping ability of gelsolin or its complexes and are Ca2+-independent.     

Role of the N- and C-terminal actin-binding domains of gelsolin in barbed filament end capping.

Gelsolin is a bivalent Ca(2+)-modulated actin-binding protein that severs, nucleates, and caps filaments. In order to gain a better understanding of the capping mechanism we have studied N- and C-terminal gelsolin fragments, 14NT and 41CT, each of which contains a single functional actin-binding site. The very tight binding measured between gelsolin and the barbed filament end requires gelsolin to greatly decrease the dissociation rate constant of the terminal actin from this end. A mechanism that could account for the observed decrease in dissociation is one in which gelsolin links two actin monomers so that they dissociate more slowly as a dimer. This cannot be the only mechanism, however, since, as shown here, 14NT and 41CT, fragments with single actin-binding sites, decrease the dissociation rate of the capped terminal actin molecule. The observations suggest that these fragments induce a conformational change in the actin monomer that either increases the affinity or alters the kinetics of the terminal actin-actin bond. The available data argue for strengthening of the terminal actin-actin bond.     

A fluorescence stopped-flow study on troponin labeled with N-ethyl maleimide and N-(p-(2-benzimidazolyl)phenyl) maleimide

The kinetics of the conformational change of the troponin-C (TN-C) subunit in N-(p-(2-benzimidazolyl)phenyl) maleimide (BIPM)-N-ethyl maleimide (NEM)-labeled troponin induced by calcium binding or removal were studied with the fluorescence stopped-flow method. The kinetic process of the conformational change was biphasic, the rate constants of the two phases were determined as a function of the free calcium ion concentration of the protein solution. The kinetic behaviour of the conformational change of TN-C in BIPM-NEM-labeled troponin was explained by a simple molecular kinetic mechanism: (Formula: see text) This molecular kinetic mechanism is different from that of the isolated TN-C which we found in the previous work (1). That is, formation of a complex of TN-C with troponin-I (TN-I) and troponin-T (TN-T) modifies the molecular kinetic mechanism of the conformational change of TN-C.     

Formation and movement of Fe(III) in horse spleen, H- and L-recombinant ferritins. A fluorescence study.

Iron oxidation and incorporation into apoferritins of different subunit composition, namely the recombinant H and L homopolymers and the natural horse spleen heteropolymer (10-15% H), have been followed by steady-state and time-resolved fluorescence. After aerobic addition of 100 Fe(II) atoms/polymer, markedly different kinetic profiles are observed. In the rL-homopolymer a slow monotonic fluorescence quenching is observed which reflects binding, slow oxidation at the threefold apoferritin channels, and diffusion into the protein cavity. In the rH-homopolymer a fast fluorescence quenching is followed by a partial, slow recovery. The two processes have been attributed to Fe(II) binding and oxidation at the ferroxidase centers and to Fe(III) released into the cavity, respectively. The fluorescence kinetics of horse spleen apoferritin is dominated by the H chain contribution and resembles that of the H homopolymer. It brings out clearly that the rate of the overall process is limited by the rate at which Fe(III) leaves the ferroxidase centers of the H chains where binding of incoming Fe(II) and its oxidation take place. The data obtained upon stepwise addition of iron and the results of optical absorption measurements confirm this picture. The correspondence between steady-state and time-resolved data is remarkably good; this is manifest when the latter are used to calculate the change in fluorescence intensity as apparent in the steady-state measurements.     

N-(1-pyrene)maleimide: a fluorescent cross-linking reagent.

N-(1-Pyrene)maleimide is nonfluorescent in aqueous solution but forms strongly fluorescent adducts with sulfhydryl groups of organic compounds or proteins. The conjugation reactions of N-(1-pyrene)maleimide are relatively fast and can be monitored by the increase in fluorescence intensity of the pyrene chromophore. In cases where primary amino groups are also present in the system, we have observed a red shift of the emission spectra of the fluorescent adducts subsequent to the initial conjugation, as characterized by the disappearance of three emission peaks at 376, 396, and 416 nm, and the appearance of two new peaks at 386 and 405 nm. Model studies with N-(1-pyrene)maleimide adducts of L-cysteine and cysteamine indicate that the spectral shift is the result of an intramolecular aminolysis of the succinimido ring in the adducts. Evidence from both chemical analysis and nuclear magnetic resonance studies of the addition products supports this reaction scheme. N-(1-Pyrene)maleimide adducts of N-acetyl-L-cysteine and beta-mercaptoethanol, which have no free amino group, do not exhibit a spectral shift. Among several protein conjugates only the N-(1-pyrene)maleimide adduct of bovine serum albumin (PM-BSA) shows the spectral shift resembling that of PM-cysteine. N-(1-Pyrene)maleimide reacts with the sulfhydryl group of the single cysteine residue at position 34 in BSA. The finding that the alpha-amino group of the N-terminus in PM-BSA is blocked after the spectral shift is completed strongly suggests that N-(1-pyrene)maleimide cross-links the N-terminus and the cysteine residue in BSA. The relative proximity of the sulfhydryl and amino groups is very critical in the cross-linking as demonstrated by the observation that the spectral shift observed with PM-BSA can be prevented by addition of denaturing reagents such as 1% sodium dodecyl sulfate immediately after labeling, and by the failure of PM-glutathione to undergo the intramolecular aminolysis. Since the intramolecular rearrangement of PM adducts is associated with characteristic fluorescence changes, N-(1-pyrene)maleimide can serve as a fluorescent cross-linking reagent which provides information about the spatial proximity of sulfhydryl and amino groups in proteins.