Expression of phenotypic markers of mast cells, macrophages and dendritic cells in gallbladder mucosa with calculous cholecystitis.

The study aimed at quantitative analysis of expression involving markers of mast cells (tryptase), monocytes/macrophages (CD68 molecule) and dendritic cells (S100 protein) in gallbladder mucosa with acute and chronic calculous cholecystitis. Routinely prepared tissue material from the patients with acute (ACC) (n = 16) and chronic calculous cholecystitis (CCC) (n = 55) was evaluated. Three cellular markers were localized by immunocytochemistry. Their expression was quantified using spatial visualization technique. The expression of tryptase was similar in acute and chronic cholecystitis. CD68 expression in ACC was significantly higher than in the CCC group. Expression of S100 protein was significantly higher in CCC as compared to the ACC group. No significant correlations were disclosed between expression of studied markers and grading in the gallbladder wall. A weak negative correlation was noted between expression of CD68 and number of gallstones in the CCC group. The spatial visualization technique allowed for a credible quantitative evaluation of expression involving markers of mast cells (MCs), monocytes/macrophages (Mo/Ma) and dendritic cells (DCs) in gallbladder mucosa with ACC and CCC. For the first time mucosal expression of S100 protein-positive DCs was evaluated in calculous cholecystitis. The results point to distinct functions of studied cell types in the non-specific immune response in calculous cholecystitis.

Effect of nucleotides and actin on the orientation of the light chain-binding domain in myosin subfragment 1.

The X-ray structure of myosin head (S1) reveals the presence of a long alpha-helical structure that supports both the essential and the regulatory light chains. It has been proposed that small structural changes in the catalytic domain of S1 are amplified by swinging the long alpha-helix (the "lever arm") to produce approximately 11 nm steps. To probe the spatial position of the putative lever in various S1 states, we have measured, by fluorescence resonance energy transfer (FRET), the effect of nucleotides and actin on the distances between Cys-177 of the essential light chain A1 (which is attached to the alpha-helix) and three loci in the catalytic domain. Cys-177 (donor) was labeled with 1,5-IAEDANS. The trinitrophenylated ADP analog (TNP-ADP, acceptor) was used to measure the distance to the active site. Lys-553 at the actin-binding site, labeled with a fluorescein derivative, and Lys-83 modified with trinitrobenzenesulfonic acid served as two other acceptors. FRET measurements were performed for S1 alone, for its complexes with MgADP and MgATP, for the analogs of the transition state of the ATPase reaction, S1.ADP.BeFx, S1.ADP.AlF4, and S1.ADP.VO4, and for acto-S1 in the absence and in the presence of ADP. When the transition state and acto-S1 complexes were formed, the change in the Cys-177 --> Lys-83 distance was <1.1 A, for the distance Cys-177 --> Lys-553, the change was +/-2.5 A. These distance changes correspond to rotations by <10 degrees and approximately 25 degrees, respectively. For the Cys-177 --> TNP-ADP the interprobe separation decreased by approximately 6 A in the presence of BeFx and AlF4- but only 1.9 A in the presence of vanadate; we do not interpret the 6 A change as resulting from the lever rotation. Using the coordinates of the acto-S1 complex, we have computed the expected changes in these distances resulting from rotation of the lever. These changes were much greater than the ones observed. The above results are inconsistent with models of force generation by S1 in which the head assumes two distinct conformations characterized by large differences in the angle between the motor and the light chain-binding domain. Several alternative mechanisms are proposed.

Myosin subfragment 1 activates ATP hydrolysis on Mg(2+)-G-actin.

The interaction of myosin subfragment 1 isoenzyme A2 (S1A2) with Mg(2+)-G-actin was studied. Polarization titrations of 1,5-IAEDANS-Mg(2+)-G-actin and of epsilon ATP-Mg(2+)-G-actin with S1A2 provided evidence that, similar to Ca(2+)-G-actin, the proteins form a tight binary complex. Significant amounts of oligomeric forms of actin in the presence and absence of S1 were not detected. The effect of S1A2 on the rates of nucleotide and metal dissociation and hydrolysis from Mg(2+)-actin was measured. The hydrolysis rate for [gamma-32P]ATP-actin in the G-acto-S1A2 complex (k- = 0.016 s-1) was faster than the rate of 32P liberation from the complex (k- = 0.004 s-1), obtained by measuring the liberation of [32P]orthophosphate from [alpha-32P]ATP-actin in the presence of a large excess of alkaline phosphatase. This indicates that most of actin's ATP was hydrolyzed before it was released to solution and that the dissociating nucleotide was ADP, for which the dissociation rate is higher than that for ATP. In agreement with this mechanism, S1A2 accelerated the dissociation of epsilon ATP but inhibited the dissociation of epsilon ADP from the complex. The activation of actin's ATPase is specific for Mg(2+)-G-actin and does not occur in Ca(2+)-G-actin. The effect of deoxyribonuclease I on the rates of nucleotide dissociation and hydrolysis was examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Myosin subfragment 1 inhibits dissociation of nucleotide and calcium from G-actin.

The dissociation rates of 1,N6-ethenoadenosine 5'-triphosphate (epsilon ATP) and of Ca2+ from G-actin and its complex with myosin subfragment 1 (S1) were measured by recording a large decrease in the fluorescence intensity of the dissociating nucleotide. Under the experimental conditions employed, the binary G-acto-S1A2 complex does not polymerize (Chaussepied, P., and Kasprzak, A. A. (1989) Nature 342, 950-953). The released nucleotide was hydrolyzed either by alkaline phosphatase or by apyrase; to trap Ca2+, EDTA was used. From the anisotropy of N-iodoacetyl-N'-(5-sulfo-1- naphthyl)ethylenediamine (1,5-IAEDANS)-actin, it was established that during the dissociation of epsilon ATP, the G-acto-S1 complex remained stable and the equilibrium of the system was unaltered. The reactions followed first order kinetics. The dissociation rate constant, kd for epsilon ATP decreased from 5.5 x 10(-4) s-1 for free G-actin to 1 x 10(-4) s-1 for G-acto-S1A2; for Ca2+, kd was also similarly reduced from 2.8 x 10(-2) s-1 to 4 x 10(-3) s-1. Two proteolytically derived actin variants were also examined. For free subtilisin-cleaved actin, kd for epsilon ATP was elevated 2-fold but was almost unchanged for Ca2+. In the complex of the cleaved G-actin with S1A2, kd for both epsilon ATP and for Ca2+ were reduced. The removal of the last 3 amino acids from actin produced a derivative whose behavior in binding to S1, as well as in the kinetics of epsilon ATP and Ca2+ dissociation, was undistinguishable from the unmodified protein.

Isolation and characterization of the G-actin-myosin head complex.

The two main proteins involved in muscular contraction and cell motility, myosin and actin, possess the intrinsic property of being able to form filamentous structures. This property poses a serious impediment to the study of their structures and interactions, and a considerable effort has thus been made to isolate their functional domains. The globular part of myosin, subfragment-1 (S1), which possesses ATPase and actin-binding sites as well as supporting the movement of actin filaments during in vitro assays, has been isolated. But because S1 is efficient in inducing actin polymerization, as is myosin, it has not been possible to prepare and characterize a complex of S1 with monomeric actin (G-actin). We have now used chromatographically purified proteins to show that only the S1 isoenzyme carrying the A1 light-chain subunit promotes actin polymerization. The other isoenzyme, S1 (A2), carrying the A2 light-chain subunit, binds to actin, forming a tight complex of G-actin and S1 in a 1:1 ratio. This new functional difference between myosin isoforms directly implicates the A1 light-chain in myosin-induced actin polymerization. Additionally, this finding should lead to the purification of the stable G-actin-S1 complex needed to resolve the structure and to understand the molecular dynamics of the actin-myosin system.

Change in the actin-myosin subfragment 1 interaction during actin polymerization.

To better characterize the conformational differences of G- and F-actin, we have compared the interaction between G- and F-actin with myosin subfragment 1 (S1) which had part of its F-actin binding site (residues 633-642) blocked by a complementary peptide or "antipeptide" (Chaussepied, P., and Morales, M. F. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7471-7475). Light scattering, sedimentation, and electron microscopy measurements showed that, with the antipeptide covalently attached to the S1 heavy chain, S1 was not capable of inducing G-actin polymerization in the absence of salt. Moreover, the antipeptide-carrying S1 did not change the fluorescence polarization of 5-[2-(iodoacetyl)-aminoethyl]aminonaphthalene-1-sulfonic acid (1,5-IAEDANS)-labeled G-actin or of 1,5-IAEDANS-labeled actin dimer, compared to the control S1. This result, interpreted as a lack of interaction between G-actin and antipeptide-carrying S1, was confirmed further by the following experiments: in the presence of G-actin, antipeptide.S1 heavy chain was not protected against trypsin and papain proteolysis, and G-actin could not be cross-linked to antipeptide.S1 by 1-ethyl-3[-3-(dimethylamino)propyl]carbodiimide. In contrast, similar experiments showed that antipeptide.S1 was able to interact with nascent F-actin and with F-actin. Thus, blocking the stretch 633-642 of S1 heavy chain by the antipeptide strongly inhibits G-actin-S1 interaction but only slightly alters F-actin-S1 contact. We, therefore postulate that this stretch of skeletal S1 heavy chain is essential for G-actin-S1 interaction and that the G-F transformation generates new S1 binding site(s) on the actin molecule.

Location of a contact site between actin and myosin in the three-dimensional structure of the acto-S1 complex.

Using fluorescence resonance energy transfer (FRET), we measured distances from chromophores located at or near the actin-binding stretch of amino acids 633-642 of myosin subfragment 1 (S1), to five points in the acto-S1 complex. Specific labeling of this site was achieved by first attaching the desired chromophore to an "antipeptide" that by means of its charge complementarity specifically binds to this segment of S1 [Chaussepied & Morales (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7471] and then cross-linking the fluorescent peptide to the protein. According to this technique, antipeptides containing three different labels, viz., N-dansylaziridine, (iodoacetamido)fluorescein, and monobromobimane, were purified and covalently bound to S1. A second chromophoric group, required for FRET measurements, was selected in such a way as to provide a good spectral overlap with the corresponding peptide chromophore. Cys-707 (SH1) and Cys-697 (SH2) on S1 were modified by using iodoacetamido and maleimido derivatives of rhodamine, 1,N6-ethenoadenosine 5'-diphosphate was trapped at the S1 active site with orthovanadate, Cys-374 on actin was modified with either N-[4-[4-(dimethylamino)phenyl]azo]phenyl]maleimide or N-[(iodoacetyl)-amino]ethyl]-5-naphthylamine-1-sulfonate, and ADP bound to F-actin was exchanged with the fluorescent etheno analogue. By use of excited-state lifetime fluorometry, the following distances from the stretch 633-642 of S1 to other points on S1 or actin have been measured: Cys-707 (S1), 50.3 A; Cys-697 (S1), 49.4 A; active site of S1, greater than or equal to 44 A; nucleotide binding site (actin), 41.1 A; and Cys-374 (actin), approximately 53 A.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactive binding between the substrate and allosteric sites of carbamoyl-phosphate synthetase.

The interaction between Escherichia coli carbamoyl-phosphate synthetase (CPS) and a fluorescent analogue of an allosteric effector molecule, 1,N6-ethenoadenosine 5'-monophosphate (epsilon-AMP), has been detected by using fluorescence techniques and kinetic measurements. From fluorescence anisotropy titrations, it was found that epsilon-AMP binds to a single site on CPS with Kd = 0.033 mM. The nucleotide had a small activating effect on the rate of synthesis of carbamoyl phosphate but had no effect on the Km for ATP. To test whether epsilon-AMP binds to an allosteric site, allosteric effectors (UMP, IMP, and CMP), known to bind at the UMP/IMP site, were added to solutions containing the epsilon-AMP-CPS complex. With addition of these effector molecules, a progressive decrease of the fluorescence anisotropy was observed, indicating that bound epsilon-AMP was displaced by the allosteric effectors examined. From these titrations, the dissociation constants for UMP, IMP, CMP, ribose 5-phosphate, 2-deoxyribose 5-phosphate, and orthophosphate were determined. When MgATP, a substrate, was employed as a titrant, the observed decrease in anisotropy was consistent with the formation of a ternary complex (epsilon-AMP-CPS-MgATP). The effect of ATP binding, monitored at the allosteric site, was magnesium dependent, and free magnesium in solution was required to obtain a hyperbolic binding isotherm. Solvent accessibility of epsilon-AMP in binary (epsilon-AMP-CPS) and ternary (epsilon-AMP-CPS-MgATP) complexes was determined from acrylamide quenching, showing that the base of epsilon-AMP is well shielded from the solvent even in the presence of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Orientation of actin monomer in the F-actin filament: radial coordinate of glutamine-41 and effect of myosin subfragment 1 binding on the monomer orientation.

We have employed the method of radial distance measurements in order to orient the actin monomer in the F-actin filament. This method utilizes fluorescence resonance energy transfer measurements of the distance between two equivalent chemical points located on two different monomers. The interprobe distance obtained this way is used to compute the radial coordinate of the labeled amino acid [Taylor, D. L., Reidler, J., Spudich, J. A., & Stryer, L. (1981) J. Cell Biol. 89, 362-367]. Theoretical analysis has indicated that if radial coordinates of four points are determined and six intramolecular distances are known, one can, within symmetry limits, position the monomer about the filament axis. The radial distance of Gln-41 that had been enzymatically modified with dansyl, rhodamine, and fluorescein derivatives of cadaverine was found to be approximately 40-42 A. The determination of the radial distance of Cys-374 was accomplished by using monobromobimane and N-[[(iodoacetyl)amino]ethyl]-5- naphthylamine-1-sulfonate as donors and N-[4-[[4-(dimethylamino)phenyl]azo]phenyl]maleimide as acceptor; the results were consistent with a radial coordinate for this residue of 20-25 A. The effect of myosin subfragment 1 (S1) binding on the radial coordinates of (1) Gln-41, (2) Cys-374, and (3) the nucleotide binding site was also examined. S1 had a small effect on the radial coordinate of Gln-41, increasing it to 44-47 A. In the two remaining lases the change in the radial coordinate due to the S1 binding was negligible. This finding excludes certain models of the interaction between actin and S1 in which actin monomer rotates by a large angle when subfragment 1 binds to it.

Measurement of interprotein distances in the acto-subfragment 1 rigor complex.

Using enzymatic labeling, we have conjugated the fluorescence probe dansylcadaverine (DNC) to Gln-41 of rabbit skeletal muscle actin with the intention of utilizing the dansyl chromophore as a donor in fluorescence resonance energy transfer (FRET) distance measurements. The fluorescence decay of DNC-actin was found to consist of two decay constants (8.23 and 21.2 ns) that were associated with two different but partially overlapping spectra of the dye. Three different chemical points on myosin subfragment 1 (S1) were labeled with suitable acceptors: reactive thiol 1 (SH1) and Cys-136 on LC3 were modified with tetramethylrhodamine 5- (and 6-) iodoacetamide (ITMR); Lys-83 (RLR) was derivatized with trinitrobenzenesulfonate. In the rigor complex of the two labeled proteins, fluorescence resonance energy transfer took place, the efficiency of which was 10.9, 9.28, and 3.73% for the transfer from Gln-41 to SH1, Cys-136 (LC3), and RLR, respectively. The limits of the Förster critical distance for each pair were obtained from the analysis of the polarization spectra of the donor and of the acceptors. The kappa 2(2/3) distances from actin Gln-41 to the three points on S1 were 63, 66, and greater than 37 A for SH1, Cys-136 (LC3), and RLR, respectively.

Characterization of the isolated 20 kDa and 50 kDa fragments of the myosin head.

We have isolated two proteolytic fragments of subfragment 1 (S-1) of myosin from rabbit skeletal muscle. These fragments, identified by their molecular weights of 20 and 50 kDa, may be functional domains that, when isolated, retain their specific function. We have studied several structural and functional features of the 20 and 50 kDa fragments. Considerable secondary structure in both fragments has been observed in CD spectrum studies. Previously CD spectra showed 64% ordered structure for the 20 kDa fragment (Muhlrad and Morales, M.F. (1984) Proc. Natl. Acad. Sci. 81, 1003) and here we show 71% ordered structures for the 50 kDa fragment. Fluorescence lifetime studies of tryptophan residues in the 50 kDa fragment and 1,5-IAEDANS-labeled SH-1 in the 20 kDa fragment are used to investigate the tertiary structure of the fragments. We find the tertiary structure relating to this measurement of both fragments to be intact; however, the reaction of 1,5-IAEDANS with SH-1 on the isolated 20 kDa fragment is less specific than with S-1. Furthermore, the fragments showed a tendency to aggregate. The domain concept of S-1 was supported by the characteristic biochemical function of the isolated fragments. Both of the fragments were effective in competing with S-1 for binding to actin in acto-S-1 ATPase measurements. From these studies and in direct binding measurement the 20 kDa fragment proved to bind with higher affinity to actin than did the 50 kDa fragment.

Localization of the major dehydrogenases in two methylotrophs by radiochemical labeling.

The localization of prominent proteins in intact cells of two methylotrophic bacteria, Hyphomicrobium sp. strain X and bacterium W3A1, was investigated by radiochemical labeling with [14C]isethionyl acetimidate. In bacterium W3A1, trimethylamine dehydrogenase was not labeled by the reagent and is, therefore, an intracellular protein, whereas the periplasmic location of the methylamine and methanol dehydrogenases was evidenced by being readily labeled in intact cells. Similarly, an intracellular location of the trimethylamine and dimethylamine dehydrogenases in Hyphomicrobium sp. strain X was indicated, whereas methanol dehydrogenase was periplasmic.

Identity of the subunits and the stoicheiometry of prosthetic groups in trimethylamine dehydrogenase and dimethylamine dehydrogenase.

Trimethylamine dehydrogenases from bacterium W3A1 and Hyphomicrobium X and the dimethylamine dehydrogenase from Hyphomicrobium X were found to contain only one kind of subunit. The millimolar absorption coefficient of a single [4Fe-4S] cluster in trimethylamine dehydrogenase from bacterium W3A1 was estimated to be 14.8 mM-1 . cm-1 at 443 nm. From this value a 1:1 stoicheiometry of the prosthetic groups, 6-S-cysteinyl-FMN and the [4Fe-4S] cluster, was established. Millimolar absorption coefficients of the three enzymes were in the range 49.4-58.7 mM-1 . cm-1 at approx. 440 nm. This range of values is consistent with the presence of two [4Fe-4S] clusters and two flavin residues, for which the millimolar absorption coefficient had earlier been found to be 12.3 mM-1 . cm-1 at 437 nm. The N-terminal amino acid was alanine in each of the three enzymes. Sequence analysis of the first 15 residues from the N-terminus of dimethylamine dehydrogenase indicated a single unique sequence. Two identical subunits, each containing covalently bound 6-S-cysteinyl-FMN and a [4Fe-4S] cluster, in each of the enzymes are therefore indicated.

Binding of nicotinamide-adenine dinucleotide to rabbit muscle aldolase.

A direct interaction of rabbit muscle fructose-1,6-bisphosphate aldolase with NAD+, NADH, and NAD-agarose was demonstrated. The electrostatic forces are primary involved in this interaction. Two specific binding sites for the dinucleotide were observed. One of them is located at the active site of the enzyme, the second is in a region of weak binding site for ATP and fructose 1,6-bisphosphate.