Biomedical subjects

R Kassab

Publications and source records attributed to R Kassab.

At least 19 recordsLinked to original sources

PubMed2006

[Sensitization of tilt-table testing for syncope of unknown etiology: which drug to use?].

OBJECTIVE: The sensitivity of tilt-table testing in the diagnosis of vasovagal syncope is between 30% and 50% only. The most common method currently used to improve the sensitivity of the test is the administration of isoproterenol i.v. However, this method is difficult to perform and time consuming. The objective of our study was to compare sublingual trinitrin administration to i.v. isoproterenol during tilt-table testing. METHODS: We analyzed the results of 257 consecutive patients referred for tilt testing. Patients who had a negative test received either a ten minutes infusion of i.v. isoproterenol at the dose of 4 mcg/kg/min, or 0.4 mg of trinitrin given sublingually. RESULTS: Two hundred (and) fifty-seven patients underwent tilt-table testing. In the first group (isoproterenol group), 42 patients (39%) had a spontaneous positive tilt test, compared to 45 patients (31%) in the trinitrin group (P = NS). After sensitization, 24 additional patients (22%) had a positive test in the isoproterenol group vs 55 patients (37%) in the trinitrin group (P = NS). The total number of positive tests was 66 (61%) in the isoproterenol group compared to 100 (68%) in the trinitrin group (P = NS). CONCLUSION: Sublingual trinitrin is at least as good as IV isoproterenol during tilt-table testing. Because trinitrin is simpler to use and because its administration is much faster than isoproterenol, it should be recommended as the drug of choice to improve the sensitivity of tilt-table testing.

Administration, Sublingual↗

PubMed2006

[Beneficial effect of sildenafil following surgery for mitral stenosis complicated by pre-capillary pulmonary hypertension: report of two cases].

Pulmonary hypertension is a serious disorder, difficult to treat especially in the severe forms. The treatment consists mainly of calcium channel blockers, anti-coagulation, intravenous epoprostenol, inhaled nitric oxide and recent agents as bosentan and sildenafil. Sildenafil, a phosphodiesterase 5 specific inhibitor, has been largely evaluated in primary pulmonary hypertension, and in some cases of secondary pulmonary hypertension including parenchymal and thromboembolic diseases; it has not yet been evaluated in severe pulmonary hypertension with elevated pre-capillary resistance in operated mitral stenosis. We report the cases of two patients operated from mitral valve replacement for severe mitral stenosis with elevated pre-capillary resistance, where oral sildenafil, introduced empirically immediately after the surgical procedure at the dose of 50 mg/d, permitted a significant decrease in pulmonary pressures and resistances, allowing a rapid withdrawal of nitric oxide and reducing therefore hospitalization time in the intensive care unit. We think that this simple treatment, with or without association to nitric oxide, should be generalized to persistent pulmonary hypertension following cardiac surgery.

Capillaries↗

PubMed2005

[Chaotic atrial tachycardia-induced cardiomyopathy: report of an isolated case].

Chaotic atrial tachycardia is a rare arrhythmia that has no known etiology and that usually inflicts upon newborn infants. The diagnosis is established using the surface electrocardiogram (ECG) which shows a spectacular polymorphism and irregularity of the atrial electrical activity. Clinical tolerability is variable depending on the ventricular rhythm. Cases that are not well tolerated and cases who do not recover spontaneously require medical treatment which relies mainly on amiodarone and other class IC anti-arrhythmic drugs. There is usually complete recovery during the first few months of life. The authors present the case of a female patient who was diagnosed with chaotic atrial tachycardia with induced cardiomyopathy following birth. She was successfully treated with amiodarone but had several relapses of the arrhythmia upon discontinuation of the drug. Although this observation is classic in its presentation, we consider that it is useful to remember this rare and frequently forgotten syndrome and to report the unique and particular aspects of our case and its evolution.

Amiodarone↗

PubMed2000

Fluorescence characterization of structural transitions at the strong actin binding motif in skeletal myosin affinity labeled at cysteine 540 with novel spectroscopic cysteaminyl mixed disulfides.

We have synthesized the luminescent and fluorescent lanthanide chelate S-(2-nitro-5-thiobenzoic acid)cysteaminyldiethylenetriaminepentaacetate-5-[(2-aminoethyl)am ino ]naphthalene-1-sulfonic acid as well as the fluorescent analogue S-(2-nitro-5-thiobenzoic acid)cysteaminyl-5-carboxyfluorescein using the procedure we recently described [Bertrand, R., Capony, J.-P., Derancourt, J., and Kassab, R. (1999) Biochemistry 38, 11914-11925]. Both mixed disulfides react with the skeletal myosin motor domain (S-1) as actin site-directed agents and label exclusively and stoichiometrically Cys 540 in the hydrophobic strong actin binding helix-loop-helix motif, causing only a 1.9-2.4-fold decrease in the V(max) for acto-S-1 ATPase. The covalently attached cysteaminyl probe side chain spans maximally 17 and 8 A, respectively, and the fluorophores have different polarity, volume, and flexibility. Thus, they may provide complementary spectroscopic information on the environmental properties of this critical actin binding region. Here, we have analyzed by extrinsic fluorescence spectroscopy S-1 derivatized with the fluorescein label or with the Tb(3+) or Eu(3+) chelate of the other label to assess the conformational transitions precisely occurring at this site upon interaction with F-actin, nucleotides, or phosphate analogues. For either label, specific spectral changes of significant amplitude were obtained, identifying at least two major structural states. One was mediated by rigor binding of F-actin in the absence or presence of MgADP. It was abolished by MgATP, and it was not produced by the binding of nonpolymerizable G-actin. A modeling of the corresponding changes in the intensity and lambda(max) of the fluorescence emission spectra, achieved using the fluorescent adducts of 2-mercaptoethanol in varying concentrations of dimethylformamide, illustrates the predicted apolar nature of the strong acto-S-1 interface. A second state was promoted by the binding of ATP, AMP-PNP, ADP.AlF4, ADP. BeFx, or PP(i). It should be prevalent in the weak acto-S-1 binding complexes. The accompanying fluorescence intensity reduction, observed with each label, in both the absence and presence of F-actin, would result from a specific modification by these ligands of the probe orientation and/or solvent accessibility as suggested by acrylamide quenching experiments. It could represent the spectral manifestation of the predicted allosteric linkage from the ATPase site to the strong actin binding site of S-1 that modulates the acto-S-1 affinity. Our study offers the basis necessary for further detailed spectroscopic investigations on the conformational dynamics in solution of the stereospecific and hydrophobic actin binding motif during the skeletal cross-bridge cycle.

Actins↗

PubMed2000

X-linked transposition of the great arteries and incomplete penetrance among males with a nonsense mutation in ZIC3.

We report on a Lebanese family in which two maternal cousins suffered and died very early in life from cardiac malformations. Both presented with a transposition of the great arteries associated with one or several other cardiac defects. Various minor midline defects were also observed, but there were no situs abnormalities other than a persistent left superior vena cava in one. A maternal uncle of these two babies was born cyanotic and died on the third post-natal day. Analysis of the ZIC3 gene, revealed the presence of a mutation in the second exon leading to a truncation of the protein. Surprisingly, another maternal uncle of the two affected cousins also had the mutation but was not clinically affected. To our knowledge, this is the first instance of incomplete penetrance in a male for a mutation in a chromosome X gene.

Alternative Splicing↗

PubMed2000

[Autosomal dominant Mendelian midline complex. Secundum atrial septal defect associated with cardiac and facial-thoracic defects. A familial case].

The kindred of 38 individuals reported here have various anomalies: 1. facio-thoracic malformations: hypertelorism, nasal deviation, cleft lip and palate, upper-incisors diastema and pectus excavatum; 2. cardiac anomalies: sinus node bradycardia, atrial fibrillation, nodal rhythm, atrial septal defect. Wolff-Parkinson-White syndrome, low insertion of the septal tricuspid valve corresponding to an Ebstein syndrome, pulmonic "en dôme" valve stenosis, aortic valve stenosis, long QT, and intraventricular conduction blocks. Almost all these defects are septal or para-septal. Mitral stenosis is probably rheumatoid. Such median varied pathology has not been yet reported. All the extra-cardiac anomalies are situated along the vertical upper half-body midline. All cardiac anomalies are in the septal or para-septal region. It is an autosomal dominant trait that implies the early embryonic development of the midline of cardiac and extra-cardiac structures.

Abnormalities, Multiple↗

PubMed1999

Detection of nucleotide- and F-actin-induced movements in the switch II helix of the skeletal myosin using its differential oxidative cleavage mediated by an iron-EDTA complex disulfide-linked to the strong actin binding site.

We have synthesized the mixed disulfide, S-(2-nitro-5-thiobenzoic acid) cysteaminyl-EDTA, using a rapid procedure and water-soluble chemistry. Its disulfide-thiol exchange reaction with rabbit myosin subfragment-1 (S-1), analyzed by spectrophotometry, ATPase assays, and peptide mapping, led to the incorporation of the cysteaminyl-EDTA group into only Cys 540 on the heavy chain and into the unique cysteine on the alkali light chains. The former thiol, residing in the strong actin binding site, reacted at a much faster rate with a concomitant 3-fold decrease in the V(max) for acto-S-1 ATPase but without change in the essential enzymatic functions of S-1. Upon chelation of Fe(3+) ions to the Cys 540-bound EDTA and incubation of the S-1 derivative-Fe complex with ascorbic acid at pH 7.5, the 95 kDa heavy chain underwent a conformation-dependent, single-cut oxidative fragmentation within 5-15 A of Cys 540. Three pairs of fragments were formed which, after specific fluorescent labeling and SDS-PAGE, could be positioned along the heavy chain sequence as 68 kDa-26 kDa, 62 kDa-32 kDa, and 54 kDa-40 kDa. Densitometric measurements revealed that the yield of the 54 kDa-40 kDa pair of bands, but not that for the two other pairs, was very sensitive to S-1 binding to nucleotides or phosphate analogues as well as to F-actin. In binary complexes, all the former ligands specifically lowered the yield to 40% of S-1 alone, roughly in the following order: ADP = AMP-PNP > ATP = ADP.AlF(4) > ADP.BeF(x)() > PP(i). By contrast, rigor binding to F-actin increased the yield to 130%. In the ternary acto-S-1-ADP complex, the yield was again reduced to 80%, and it fell to 25% in acto-S-1-ADP.AlF(4), the putative transition state analogue complex of the acto-S-1 ATPase. These different quantitative changes reflect distinct ligand-induced conformations of the secondary structure element whose scission generates the 54 kDa-40 kDa species. According to the S-1 crystal structure, this element could be unambiguously assigned to the switch II helix (residues 475-507) whose N-terminus lies 14.2 A from Cys 540 and would include the ligand-responsive cleavage site. This motif is thought to be crucial for the transmission of sub-nanometer structural changes at the ATPase site to both the actin site and the lever arm domain during energy transduction. Our study illustrates this novel, actin site-specific chemical proteolysis of S-1 as a direct probe of the switch II helix conformational transitions in solution most likely associated with the skeletal cross-bridge cycle.

Actins↗

PubMed1999

Autosomal dominant secundum atrial septal defect with various cardiac and noncardiac defects: a new midline disorder.

We report on a Lebanese family in which 12 persons had an atrial septal defect and various cardiac and noncardiac anomalies. Cardiac anomalies are left axis deviation of QRS, right bundle branch block, atrial fibrillation, Wolff-Parkinson-White syndrome, nodal atrioventricular rhythm, aortic stenosis, pulmonic valve stenosis, mitral stenosis (Lutembacher syndrome), and low implantation of the tricuspid valve (Ebstein disease). Noncardiac abnormalities consisted specially of the presence of hypertelorism, cleft lip, and pectus excavatum. This combination appears to constitute a hitherto undescribed autosomal dominant midline disorder of the heart and upper half of the body with almost full penetrance and variable expressivity. The mutation does not map to any known locus involved in atrial septal defect or conduction block.

Abnormalities, Multiple↗

PubMed1999

Fluorescence studies of the carboxyl-terminal domain of smooth muscle calponin effects of F-actin and salts.

The fluorescence parameters of the environment-sensitive acrylodan, selectively attached to Cys273 in the C-terminal domain of smooth muscle calponin, were studied in the presence of F-actin and using varying salt concentrations. The formation of the F-actin acrylodan labeled calponin complex at 75 mm NaCl resulted in a 21-nm blue shift of the maximum emission wavelength from 496 nm to 474 nm and a twofold increase of the fluorescent quantum yield at 460 nm. These spectral changes were observed at the low ionic strengths (< 110 mm) where the calponin : F-actin stoichiometry is 1 : 1 as well as at the high ionic strengths (> 110 mm) where the binding stoichiometry is a 1 : 2 ratio of calponin : actin monomers. On the basis of previous three-dimensional reconstruction and chemical crosslinking of the F-actin-calponin complex, the actin effect is shown to derive from the low ionic strength interaction of calponin with the bottom of subdomain-1 of an upper actin monomer in F-actin and not from its further association with the subdomain-1 of the adjacent lower monomer which occurs at the high ionic strength. Remarkably, the F-actin-dependent fluorescence change of acrylodan is qualitatively but not quantitatively similar to that earlier reported for the complexes of calponin and Ca2+-calmodulin or Ca2+-caltropin. As the three calponin ligands bind to the same segment of the protein, encompassing residues 145-182, the acrylodan can be considered as a sensitive probe of the functioning of this critical region. A distance of 29 A was measured by fluorescence resonance energy transfer between Cys273 of calponin and Cys374 of actin in the 1 : 1 F-actin-calponin complex suggesting that the F-actin effect was allosteric reflecting a global conformational change in the C-terminal domain of calponin.

2-Naphthylamine↗

PubMed1998

Interaction of caldesmon with actin subdomain-2.

The polymerization-resistant maleimidobenzoyl-G-actin (MBS-G-actin), which behaves as a functional analogue of native G-actin [Bettache, N., Bertrand, R. & Kassab, R. (1989) Proc. Natl Acad. Sci. USA 86, 6028-6032; Bettache, N., Bertrand, R. & Kassab, R. (1990) Biochemistry 29, 9085-9091) has been employed to probe the solution interaction between monomeric actin and smooth muscle caldesmon, using fluorescence measurements, limited proteolysis and covalent cross-linking reactions. MBS-G-actin associates, without polymerization, to turkey gizzard caldesmon, at about 50 mM ionic strength and 25 degrees C, with a high affinity (Kd approximately 0.04 microM) and with a 1:1 stoichiometry. However, the binding strength of the complex including caldesmon and MBS-G-actin cleaved at the subdomain-2 loop with subtilisin decreased fivefold (Kd approximately 0.20 microM). Conversely, caldesmon strongly protected subdomain-2 of MBS-G-actin from tryptic digestion at the susceptible peptide bond at positions 68-69. Furthermore, caldesmon induced the dissociation of native G-actin from its complex with DNase I, as assessed by cosedimentation assays, and increasing concentrations of the latter protein inhibited the MBS-G-actin-caldesmon interaction, suggesting mutual exclusion binding of caldesmon and DNase I to monomeric actin. MBS-G-actin was specifically coupled, via a maleimidobenzoyl group incorporated into its subdomain-2, to caldesmon, producing in high yield a 205-kDa covalent complex consisting of one actin monomer joined to Cys 580 of caldesmon. A similar conjugation process was observed with the complex of caldesmon and polymerized MBS-F-actin. MBS-G-actin could be also cross-linked to caldesmon by 1-ethyl-3[3-(dimethylamino)propyl]carbodiimide, producing a three-band pattern identical to that of F-actin and caldesmon and previously shown to reflect the covalent union between the NH2-terminal segment of actin and the COOH-terminal actin-binding domain of caldesmon. The overall data point to a direct interaction of the latter region with actin subdomain-2 and suggest that during its binding to monomeric or filamentous actin, the caldesmon functional domain spans the entire length of a single actin and closely contacts the bottom of its subdomain-1 as well as the top portion of its subdomain-2.

Actins↗

PubMed1998

Mycotic aneurysm of the popliteal artery secondary to tuberculosis. A case report and review of the literature.

Mycotic aneurysms of the popliteal artery are rare; 33 cases have been reported in the literature. The treatment of choice is a large excision with extra-anatomic revascularization. In situ revascularization is sometimes possible. To the best of our knowledge, tuberculosis has never been reported as a causal factor of mycotic aneurysms of the popliteal artery. We report a case of a recurrent tuberculous false aneurysm of the popliteal artery. After 2 attempts at in situ revascularization, the femoral artery was ligated with no distal ischemia.

Adult↗

PubMed1997

Interaction of F-actin with synthetic peptides spanning the loop region of human cardiac beta-myosin heavy chain containing Arg403.

The atomic model of the F-actin-myosin subfragment 1 complex (acto-S-1) from skeletal muscle suggests that the transition of the complex from a weakly to a strongly binding state, generating mechanical force during the contractile cycle, may involve the attachment of the upper 50-kDa subdomain of myosin subfragment 1 (S-1) to the interface between subdomains 1 and 3 of actin. For the human cardiac myosin, this putative interaction would take place at the ordered loop including Arg403 of the beta-heavy chain sequence, a residue whose mutation into Gln is known to elicit a severe hypertrophic cardiomyopathy caused by a decrease of the rate of the actomyosin ATPase activity. Moreover, in several nonmuscle myosins the replacement of a Glu residue within the homolog loop by Ser or Thr also results in the reduction of the actomyosin ATPase rate that is alleviated by phosphorylation. As an approach to the characterization of the unknown interaction properties of F-actin with this particular S-1 loop region, we have synthesized four 17-residue peptides corresponding to the sequence Gly398-Gly414 of the human beta-cardiac myosin. Three peptides included Arg403 (GG17) or Gln403 (GG17Q) or Ser409 (GG17S) and the fourth peptide (GG17sc) was a scrambled version of the normal GG17 sequence. Using fluorescence polarization, cosedimentation analyses and photocross-linking, we show that the three former peptides, but not the scrambled sequence, directly associate in solution to F-actin, at a nearly physiological ionic strength, with almost identical affinities (Kd approximately 40 microM). The binding strength of the F-actin-GG17 peptide complex was increased fivefold (Kd = 8 microM) in the presence of subsaturating concentrations of added skeletal S-1 relative to actin, without apparent competition between the peptide and S-1. Each of the three actin-binding peptides inhibited the steady-state actin-activated MgATPase of skeletal S-1 by specifically decreasing about twofold the Vmax of the reaction without changing the actin affinity for the S-1-ATP intermediate. Cosedimentation assays indicated the binding of about 0.65 mol peptide/mol actin under conditions inducing 70% inhibition. Collectively, the data point to a specific and stoichiometric interaction of the peptides with F-actin that uncouples its binding to S-1 from ATP hydrolysis, probably by interfering with the proper attachment of the S-1 loop segment to the interdomain connection of actin.

Actins↗

PubMed1997

Probing the hydrophobic interactions in the skeletal actomyosin subfragment 1 and its nucleotide complexes by zero-length cross-linking with a nickel-peptide chelate.

The complex of Ni(II) and the tripeptide Gly-Gly-His catalyzes, in the presence of monoperoxyphthalic acid, a zero-length protein-protein cross-linking via an oxidative radical pathway involving mainly aromatic amino acids and not at all nucleophilic residues [Brown, K. C., Yang, S.-H., and Kodadek, T. (1995) Biochemistry 34, 4733-4739]. We have taken advantage of this unprecedented cross-linking system to directly and selectively probe the solution structure and functioning of the hydrophobic interface between F-actin and skeletal myosin subfragment 1 (S-1) at the level of its aromatic components, in the absence and in the presence of nucleotides (ATP and ADP) or nucleotide analogs (AMPPNP, PPi, and ADP. AlF4). Following verification of the structure of the Ni(II)-peptide chelate and of its oxidized active form by electrospray mass spectrometry, complexes of F-actin and S-1 or proteolytic S-1 derivatives and complexes of S-1 and proteolytic F-actin derivatives were readily cross-linked under various controlled conditions without apparent alteration of the acto-S-1 recognition. The covalent adducts were identified on electrophoretic gels using specific protein labeling with the oxidation-resistant fluorophor, monobromobimane, combined with immunochemical staining. Two types of actin-heavy chain conjugates were produced. One, with a mass of 180 kDa, was formed in the rigor state or with ADP bound; the other one, with a mass of 200 kDa, was generated from the ternary complexes comprising a gamma-P-containing ligand. They were accumulated with an efficiency of 8 and 6%, respectively. For each reversible complex, the 180 kDa:200 kDa band ratio was essentially as predicted from the nucleotide-dependent A to R equilibrium mechanism of the acto-S-1 interaction in solution [Geeves, A. M., and Conibear, P. B. (1995) Biosphys. J. 68, 194s-201s]. Both covalent species resulted from the cross-linking of an actin monomer to the central 50 kDa segment, and their distinct mobilities reflect gamma-P-mediated structural changes at or near the actin-50 kDa fragment interface. Peptide mapping showed the cross-linking to take place between the 506-561 S-1 segment and the 48-113 actin stretch. The localization of these regions in the atomic F-actin-S-1 model implies that nucleotide-modulated close contacts, involving aromatic residues, are operating between the C-terminal helix of the hydrophobic strong actin-binding motif of S-1 bound to the primary actin monomer and the top portion of the adjacent lower actin subunit. The specificity of the nickel-peptide cross-linking, as assessed with the acto-S-1 complex, makes it a candidate for potential general use in investigations of the hydrophobic interactions within other protein motor-based assemblies.

Adenosine Diphosphate↗

PubMed1997

[Cardiac failure caused by renal arteriovenous fistula fifty years after nephrectomy. A new case and review of the literature].

Arterio-venous fistulae are rare after nephrectomy. They are the cause of congestive cardiac failure. This case report has two particularities: an exceptionally long interval, 50 years after nephrectomy, and a severe hypertension due to stenosis of the contra-lateral renal artery. Surgical treatment of these fistulae is essential and gives excellent results with rapid regression of the signs of cardiac failure. Percutaneous embolization is an alternative in patients with a high surgical risk. All previously reported cases are reviewed (n = 72) with their aetiological, clinical and therapeutic features.

Aortography↗

PubMed1996

Disruption of the actin cytoskeleton in living nonmuscle cells by microinjection of antibodies to domain-3 of caldesmon.

Two classes of affinity-purified polyclonal antibodies directed against the four different domains of gizzard caldesmon were prepared and their specificity was verified by immunochemical assays. One set of antibodies recognized exclusively domain-3 spanning residues 483-578 and the other one included IgG reactive toward the remaining domains-1, -2 and -4 corresponding to residues 1-482 and 581-756. Microinjection into cultured fibroblasts of each antibody preparation was employed to probe the functional importance of the interaction between caldesmon and tropomyosin within living nonmuscle cells. Low concentrations of the antibody to domain-3 caused a rapid, severe and reversible disassembly of the microfilament network. In contrast, the antibodies to domains-1, -2 and -4 were ineffective. The effects of the anti-domain-3 IgG were observed not only with the purified antibody but also when present in the whole caldesmon antiserum serving for its isolation. The microfilament disintegrating activity of this antibody was completely abolished upon preincubation with native caldesmon or a proteolytic caldesmon fragment encompassing amino acids 483-578. In enzyme-linked immunosorbent assay competition experiments, tropomyosin, but not F-actin, significantly decreased the binding of the domain-3 antibody to caldesmon. Consistent with recent mutational studies pinpointing to the contribution of domain-3 in the in vitro binding of cellular caldesmon to tropomyosin, the findings suggest that the microinjected domain-3 antibody selectively disrupts the association of tropomyosin with caldesmon domain-3, thereby destabilizing the protein complex and alleviating its known protective action against F-actin severing in the cell. Thus, our data further highlight the in vivo involvement of this particular domain in the attachment of non-muscle caldesmon to tropomyosin as well as the direct participation of the caldesmon-tropomyosin complex in the cytoskeletal organization of the microfilaments.

Actin Cytoskeleton↗

PubMed1995

Production and properties of skeletal myosin subfragment 1 selectively labeled with fluorescein at lysine-553 proximal to the strong actin-binding site.

We describe, for the first time, the reaction of skeletal myosin subfragment 1 (S-1) with the succinimido ester of 6-[fluorescein-5(and 6)-carboxamido]hexanoic acid (FHS), which takes place at pH 7.0, 20 degrees C, within a 15 min period, in the presence of 1.5-1.8-fold molar excess of reagent over protein. As a result, 0.9-1.0 mol of fluorescyl group/mol of S-1 was covalently incorporated exclusively into the 95 kDa heavy chain as monitored by spectroscopic measurements. The central 50 kDa segment included the main site of fluorescence attachment as assessed by gel electrophoresis. The extent of S-1--FHS conjugation is strongly sensitive to F-actin binding but not to the interaction of nucleotides. The formation of the rigor F-actin--S-1 complex decreased the level of S-1 labeling to 20% without any competition between actin and S-1 for FHS binding. The derivatization of S-1 did not alter the K(+)-ATPase activity, but it enhanced the Ca(2+)-ATPase and Mg(2+)-ATPase to 150% and 225%, respectively, whereas it lowered the actin-activated ATPase to only 75% of the original activity. A double-reciprocal plot of the ATPase rate against actin concentration indicated a 2-fold decrease of the Vmax value for modified S-1, while the Km for actin was unchanged. Cosedimentation experiments did not reveal disruption of the rigor acto-S-1 interaction by the bound fluorophore. The labeled S-1 heavy chain was isolated, and its total tryptic digest was fractionated by reverse-phase HPLC.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1995

Flexibility of actin filaments derived from thermal fluctuations. Effect of bound nucleotide, phalloidin, and muscle regulatory proteins.

Single actin filaments undergoing brownian movement in two dimensions were observed at 20 degrees C in fluorescence optical video microscopy. The persistence length (Lp) was derived from the analysis of either the cosine correlation function or the average transverse fluctuations of a series of recorded shapes of filaments assembled from rhodamine-action. Phalloidin-stabilized filaments had a persistence length of 18 +/- 1 micron, in agreement with recent observations. In the absence of phalloidin, rhodamine-labeled filaments could be observed under a variety of solution conditions once diluted in free unlabeled G-actin at the appropriate critical concentration. Such nonstabilized F-ADP-actin filaments had the same Lp of 9 +/- 0.5 microns, whether they had been assembled from ATP-G-actin or from ADP-G-actin, and independently of the tightly bound divalent metal ion. In the presence of BeF3-, which mimics the gamma-phosphate of ATP, F-ADP-BeF3-actin was appreciably more rigid, with Lp = 13.5 microns. Hence, newly formed F-ADP-Pi-actin filaments are more rigid than "old" F-ADP-actin filaments, a fact which has implications in actin-based motility processes. In the presence of skeletal tropomyosin and troponin, filaments were rigid (Lp = 20 +/- 1 micron) in the off state (-Ca2+), and flexible (Lp = 12 microns) in the on state (+Ca2+), consistent with the steric blocking model. In agreement with x-ray diffraction data, no appreciable difference was recorded between the off and on states using smooth muscle tropomyosin and caldesmon (Lp = 20 +/- 1 micron). In conclusion, this method allows accurate measurement of small (< or = 15%) changes in mechanical properties of actin filaments in correlation with their biological functions.

Actin Cytoskeleton↗

PubMed1995

Characterization of the regulatory domain of gizzard calponin. Interactions of the 145-163 region with F-actin, calcium-binding proteins, and tropomyosin.

Earlier, we proposed that the interaction of gizzard calponin with F-actin, promoting the inhibition of the actomyosin ATPase activity, involves the NH2-terminal portion of the calponin segment Ala145-Tyr182 (Mezgueldi, M., Fattoum, A., Derancourt, J., and Kassab, R. (1992) J. Biol. Chem. 267, 15943-15951). In this work, we have directly probed this region for actin binding sites using five peptide analogs covering different stretches of the sequence Thr133-Ile163. Co-sedimentation with F-actin, actomyosin ATPase measurements, and zero-length cross-linking reactions demonstrated that the 19-residue sequence Ala145-Ile163 is essential for actin interaction and ATPase inhibition. Furthermore, each peptide was tested for binding to the Ca(2+)-dependent proteins, caltropin and calmodulin, in both an actomyosin ATPase assay and an affinity chromatographic assay. The results revealed the 11-residue segment Gln153-Ile163, representing the COOH-terminal moiety of the F-actin binding sequence, as a crucial region for the high affinity binding of these regulatory proteins with concomitant removal of the ATPase inhibition. The 153-163 stretch contained also interactive sites for tropomyosin as assessed by affinity chromatography and spectrofluorometry. Collectively, the data support our initial results and highlight the ability of the multifunctional 145-163 region to serve as a potent regulatory domain of the smooth muscle calponin.

Actins↗