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Crucial information concerning conformational changes that occur during the mechanochemical cycle of actin-myosin complexes is lacking due to the difficulties encountered in obtaining their three-dimensional structures. To obtain such information, we employed a solution-based approach through the reaction of Ni(II).tripeptide chelates which are able to induce protein cleavage and cross-linking reactions. Three different myosin motor domain isoforms in the presence of actin and nucleotides were treated with a library of Ni(II).tripeptide chelates and two reactivities were observed: (1) muscle motor domains were cross-linked to actin, as also observed for the skeletal muscle isoform, while (2) the Dictyostelium discoideum motor domain was cleaved at a single locus. All Ni(II).tripeptide chelates tested generated identical reaction products, with Ni(II).Gly-Gly-His, containing a C-terminal carboxylate, exhibiting the highest reactivity. Mass spectrometric analysis showed that protein cleavage occurred within segment 242-265 of the Dictyostelium discoideum myosin heavy chain sequence, while the skeletal myosin cross-linking site was as localized previously within segment 506-561. Using a fusion protein consisting of the yellow and cyan variants of green fluorescent protein linked by Dictyostelium discoideum myosin segment 242-265, we demonstrated that the primary sequence of this segment alone is not a sufficient substrate for Ni(II).Gly-Gly-His-induced cleavage. Importantly, the cross-linking and cleavage reactions both exhibited specific structural sensitivities to the nature of the nucleotide bound to the active site, validating the conformational changes suggested from crystallographic data of the actin-free myosin motor domain.

BASP1 (also known as CAP-23 and NAP-22) is a novel myristoylated calmodulin-binding protein, abundant in nerve terminals. It is considered as a signal protein participating in neurite outgrowth and synaptic plasticity. BASP1 is also present in significant amounts in kidney, testis, and lymphoid tissues. In this study, we show that BASP1 is accompanied by at least six BASP1 immunologically related proteins (BIRPs), which are present in all animal species studied (rat, bovine, human, chicken). BIRPs have lower molecular masses than that of BASP1. Similarly to BASP1, they are myristoylated. Peptide mapping and partial sequencing have shown that BIRPs represent a set of BASP1 N-terminal fragments devoid of C-terminal parts of different length. In a definite species, the same set of BASP1 fragments is present in both brain and other tissues. The sum amount of the fragments is about 50% of the BASP1 amount in a tissue. Obligatory accompanying of BASP1 by a set of specific fragments indicates that these fragments are of physiological significance.

We designed a screen to identify starfish oocyte proteins able to bind monomeric cyclin B by affinity chromatography on a cyclin B splice variant displaying low affinity for cdc2. We identified a 15 kDa protein previously described as a cdk-binding protein [Biochim. Biophys. Acta Mol. Cell Res. 1589 (2002) 219–231]. Cybip is encoded by a single polymorphic gene and the native protein is matured by cleaving a signal peptide. We firmly establish the fact that it is a true cyclin B-binding protein, since the recombinant protein binds recombinant cyclin B in absence of any cdk. Finally, we show that the microinjection of GST-cybip, and of anti-cybip antibody, in maturing starfish oocytes, inhibits H1 kinase and MPF inactivation, and first polar body emission.

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.

Beta 2 microglobulin isoforms in healthy individuals and in amyloid deposits. Beta 2 microglobulin (β2m) is classically known to have isoforms with isoelectric points (pI) 5.7 and 5.3. New isoforms of β2m with lower pI, probably due to modifications with advanced glycation end products, were found in the amyloid deposits of dialysis related amyloidosis (DRA), and they were proposed as the amyloidogenic forms of β2m. The other modifications in β2m from amyloid deposits are partial proteolysis and single amino acid replacement (Asn by Asp at position 17). However, there are no data on the sequence of the different isoforms of β2m from amyloid deposits. Amyloid deposits surgically obtained from the carpal tunnel from 13 dialysis treated patients and urine from 10 healthy volunteers and 5 living-related kidney donors were analyzed for β2m content. Two-dimensional gel electrophoresis (2D-PAGE) of β2m from amyloid deposits showed the presence of four or more isoforms with pis < 5.7. All the spots migrating at 12 kDa Mr region and between 4 and 6 pH reacted with rabbit anti-human β2m antibody by Western blotting, confirming that they were β2m isoforms. β2m isoforms from the amyloid deposits were then separately purified with an IEF column (PB94, Pharmacia™) for analysis. Enough quantities of three pure β2m isoforms could be obtained in two cases. The sequence analysis showed an intact N-terminus in all the isoforms. There was Asn in the 17th residue in all the isoforms sequenced. 2D-PAGE of urine from 8 out of the 10 healthy volunteers showed the presence of β2m. In two of them β2m also displayed four different isoforms. At least four isoforms were observed in urine of all the kidney donors. The present study shows that the elution peaks of three different β2m isoforms in gel isoelectrofocusing contain β2m with intact N-terminus. None of them have deamidated their 17th residue. More importantly, the β2m isoforms with lower pI are not specific for amyloidosis as they were found in urine from kidney donors and in normal volunteers. These results bring into question the hypothesis that dialysis related amyloidosis is due to the known modifications on β2m. They suggest that the precipitation of β2m into amyloid fibrils should result from the interaction of β2m with other factors with amyloid enhancing activity.

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)

p40MO15, a cdc2-related protein, is the catalytic subunit of the kinase (CAK, cdk-activating kinase) responsible for Thr161/Thr160 phosphorylation and activation of cdk1/cdk2. We have found that strong overexpression of p40MO15 only moderately increases CAK activity in Xenopus oocytes, indicating that a regulatory CAK subunit (possibly a cyclin-like protein) limits the ability to generate CAK activity in p40MO15 overexpressing oocytes. This 36 kDa subunit was microsequenced after extensive purification of CAK activity. Production of Xenopus CAK activity was strongly reduced in enucleated oocytes overexpressing p40MO15 and p40MO15 shown to contain a nuclear localization signal required for nuclear translocation and generation of CAK activity. p40MO15 was found to be phosphorylated on Ser170 and Thr176 by proteolytic degradation, radiosequencing of tryptic peptides and mutagenesis. Thr176 phosphorylation is required and Ser170 phosphorylation is dispensable for p40MO15 to generate CAK activity upon association with the 36 kDa regulatory subunit. Finally, Thr176 and Ser170 phosphorylations are not intramolecular autophosphorylation reactions. Taken together, the above results identify protein-protein interactions, nuclear translocation and phosphorylation (by an unidentified kinase) as features of p40MO15 that are required for the generation of active CAK.

International audience; Human cdc25C protein, a specific tyrosine phosphatase that activates the p34cdc2 protein kinase at mitosis, is itself a phosphoprotein that shows increased phosphorylation during the G2-M transition. In vitro, cdc25C protein is substantially phosphorylated by purified p34cdc2-cyclin B protein kinase. Of seven putative phosphorylation sites for p34cdc2 protein kinase present in human cdc25C, five are phosphorylated by p34cdc2 protein kinase in vitro, as assessed by tryptic phosphopeptide mapping and peptide sequencing. These same sites are also phosphorylated in vivo during the G2-M transition in normal mammalian fibroblasts and have been precisely mapped. The cdc25C phosphorylated in vitro by p34cdc2 protein kinase exhibits a 2-3-fold higher activity than the nonphosphorylated cdc25C, as assayed by activation of inactive cdc2 prokinase. Microinjection of purified cdc25C proteins into living fibroblasts reveals that only the phosphorylated form of cdc25 is highly effective in activating G2 cells into premature prophase in a manner similar to microinjection of purified active p34cdc2 protein kinase. Together these data show that multisite phosphorylation of cdc25C by p34cdc2-cyclin B protein kinase occurs at the G2-M transition and is sufficient to induce the autoamplification of cdc2/M-phase promoting factor necessary to drive somatic mammalian cells into mitosis.

We have isolated an NH2-terminal fragment of filamin (M(r) = 70,000) after digestion with Staphylococus aureus V8 protease. This fragment was shown to interact with filamentous actin in cosedimentation assays. Using cross-reactive anti-peptides antibodies directed against the strongly conserved 27-mer sequence of alpha-actinin, already implicated as an actin binding site (Kuhlman, P. A., Hemmings, L., and Critchley, D. R. (1992) FEBS Lett. 304, 201-206), we obtained evidence suggesting that the homologous sequence of filamin (121-147 sequence) is the major element in the interaction with actin. In particular, we used enzyme-linked immunosorbent assay experiments, in conjunction with a synthetic peptide approach, and found that the hydrophobic part of the 27-mer peptide (141-147 sequence) is largely involved in actin binding. Thus, the filamin sequence 121-147 (or the alpha-actinin sequence 108-134) and the actin counterpart composed of residues 112-125 and 360-372 (we have already implicated) could constitute the main interface between actin and these cytoskeletal proteins. However, the divergent behavior of filamin and alpha-actinin toward conformational changes of actin argues in favor of distinctive interfaces. Finally, the ionic strength dependence of the filamin-actin interaction, in contrast to that with alpha-actinin, strongly suggests that, besides hydrophobic interactions conferred by the 27-mer sequence, more hydrophilic region(s) of filamin participate(s) in the binding.

We describe, for the first time, the F-actin-promoted changes in the spatial relationship of strands in the NH2-terminal 25-kDa and COOH-terminal 20-kDa heavy chain fragments of the skeletal myosin subfragment 1 (S-1), detected by their exclusive chemical cross-linking in the rigor F-actin-S-1 complex with m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS). Quantitative electrophoretic analysis of the reaction products showed extensive conversion of the 95-kDa heavy chain of the actin-bound S-1 into a new species with an apparent mass of 135 kDa (yield = 50-60%), whereas the heavy chain mobility remained unaffected when actin was omitted. The 135-kDa entity retained the fluorescence of AEDANS-S-1 but not of AEDANS-actin, indicating that it was not a cross-linked acto-heavy chain adduct. Its extent of production depended markedly on the S-1: actin molar ratio and was maximum near a ratio of 1:4. The MBS treatment of acto-S-1 led also to some covalent actin-actin oligomers which could be suppressed by using trypsin-truncated F-actin lacking Cys-374, without altering the generation of the 135-kDa heavy chain derivative.(ABSTRACT TRUNCATED AT 250 WORDS)

Stable microtubules (as defined by resistance to Ca2+, drug or cold temperature induced disassembly) form in abundance during tubulin assembly in brain crude extracts. We have previously shown that, in rat brain crude extracts, all microtubule stabilizing activity could be ascribed to a single Ca(2+)-calmodulin binding and Ca(2+)-calmodulin regulated protein, called "stable tubule only polypeptide", STOP145 [Pirollet, F., Rauch, C. T., Job, D., & Margolis, R. L. (1989) Biochemistry 28, 835-842]. We have now performed an exhaustive study of STOP-like effectors in bovine brain high-speed supernatants. All activity binds to cation exchangers and to Ca(2+)-calmodulin affinity columns. The activity can be resolved into two peaks on sizing columns. The first eluted peak contains a prominent 220-kDa protein. The second peak contains an apparently homogeneous 20-kDa polypeptide. A monoclonal antibody specific to rat brain STOP145 recognizes the 220-kDa protein, but not the 20-kDa species. The 220-kDa protein can be purified on a STOP antibody column and accounts for the bulk of stabilizing activity in the first peak. The 20-kDa protein does not bind to STOP antibody affinity columns. Sequence analysis of oligopeptide fragments of the 20-kDa protein shows 100% homology with bovine myelin basic protein (MBP). Anti-MBP antibodies recognize the 20-kDa, but not the 220-kDa species. We conclude that the 220-kDa protein is the bovine equivalent to rat brain STOP145 and that the 20-kDa species is MBP. Microtubule stabilization by MBP and STOP220 is abolished in the presence of Ca(2+)-calmodulin, and inhibition curves are similar for both proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-related changes in progesterone hepatic metabolism were measured in Lacaune ewes in the foetal, neonatal (1 and 4 weeks), growing (7 months), pregnant (11 months) and adult (6 years) stages. 6 beta-Hydroxylation and 20 alpha-reduction were found to be the most efficient metabolic process in ovine microsomes. These activities were detected in 3-month-old foetuses and they increased rapidly during the first month of life, in a similar manner to the developmental expression of the cytochrome P4503A subfamily. 16 alpha- and 21-hydroxylation of progesterone were characterized by low, constant turn over in sheep liver microsomes during development. The hepatic ovine P4502B isozyme was purified to electrophoretic homogeneity by means of successive DEAE cellulose, hydroxylapatite and CM cellulose chromatographic separations. This hemoprotein had an apparent molecular weight of 51 kDa and was characterized by spectral data, NH2-terminal amino-acid sequence, immunological and catalytic properties. The relative contribution of this form and of the previously purified ovine P4503A subfamily was investigated in liver progesterone metabolism by immunoinhibition studies using polyclonal antibodies raised in rabbits and from the existence of induction and of significant correlations between microsomal activity and specific P450 content. In sheep liver microsomes, it would appear that cytochrome P4502B is involved in progesterone 21-hydroxylation whereas P4503A participates in the 6 beta- and 16 alpha-hydroxylation and possibly in the reductive conversion of progesterone in its 20 alpha-hydroxy derivative.

Tryptic digestion of brain spectrin generates a number of fragments from alpha and beta subunits; when these fragments are incubated with F-actin or neurofilament light subunit, four of them with molecular masses below 30 kDa sediment with the cytoskeleton structures. A selective purification of these fragments by ammonium sulfate fractionation and butyl-Sepharose chromatography has been achieved. Two fragments with molecular masses of 28 and 23 kDa bind to F-actin. Native brain spectrin causes half-maximal inhibition of the association at a concentration of 3 microM. Protein sequencing indicates that the actin-binding domain is contained in the beta subunit, in a stretch of amino acids at the N terminus from Ala43 (28-kDa fragment) or from Met104 (23-kDa fragment) and terminate probably at the C-terminal Lys288 or Lys284. Amino acids are numbered by reference to the sequence of the Drosophila beta subunit. The 28-kDa fragment also binds to the low-molecular-mass subunit of neurofilaments; brain spectrin heterodimer disrupts this binding. Hence, spectrin binds to F-actin and to neurofilaments via a common binding domain.

Antibodies raised against cytochrome P450, which is overexpressed in mouse hepatic tumors, (P450tu) crossreact with two human liver microsomal proteins (49 kDa and 52 kDa). We have quantified these proteins in 60 human liver samples and found great interindividual variability in both of them. The concentration of the 49-kDa protein varies up to 144 fold in the various samples and represents typically 10% of the total mincrosomal P450 content. Its immunologically determined concentration correlates well (R = 0.78) with the microsomal coumarin-7-hydroylase (COH) activity. This activity is strongly and completely inhibited by anti-P450tu antibody (IC50 = 0.13 mg IgG/mg microsomal protein). The crossreacting 49-kDa protein shows an unusually high substrate specificity towards coumarin; it presents all human COH and part of 7-ethoxycoumarin O-deethylase (ECOD). Besides these two activities, we did not find any activity with other typical P450 substrates. In primary cultures of human hepatocytes, it is inducible by phenobarbital and dexamethasone, but not by pyrazole and beta-naphthoflavone. We isolated this protein from human liver microsomes and purified it to homogeneity by a combination of aminooctyl-amino-Sepharose chromatography and immunoaffinity chromatography. The protein was identified as a cytochrome P450 of the IIA subfamily. Its N-terminal amino-acid sequence was identical with the first 20 residues deduced from the nucleotide sequence of P450IIA6.

βIGH3 protein has been recently involved in the pathogenesis of blinding corneal diseases, some of which have characteristic amyloid corneal deposits. The 124 codon of the βig-h3 gene seems to be crucial for the amyloidogenicity of the protein product. We presently report an in vitro system that reproducibly forms amyloid fibrils from βIGH3 (110–131) derived peptides. We also assessed the differences in fibril formation of two 22-amino acid peptides centered on the 124 residue: the native form and the Arg124Cys peptide (mutation linked to lattice corneal amyloid dystrophy type 1). After dialysis of Arg124Cys peptide against PBS 1/15 M pH 7.4 for 72 hours, Congo red staining and electron microscopy demonstrated the presence of abundant material fulfilling the criteria of amyloid. Quantitative analysis with thioflavine T fluorescence studies confirmed the high capacity of Arg124Cys peptide to form amyloid fibrils when compared to the native form.

We have compared the interaction of ncd (non-claret disjunctional), a kinesin related protein, with microtubules and tubulin heterodimer. Ultracentrifugation experiments revealed that the ncd motor domain, residues 335–700 (ncd335), does not induce tubulin polymerization but stabilizes pre-formed microtubules with a maximum effect at a 1:1 ncd335:tubulin ratio. Ncd335 binding to tubulin or microtubules was estimated by following the change in fluorescence polarization of an exogenous dye attached to Cys670 of ncd335. Ncd335 binding to tubulin (containing GTP or GDP-bound) is characterized by a 2:1 stoichiometry, a higher affinity and an increased sensitivity towards salt, ADP, ATP and AMPPNP, as compared with ncd335 binding to microtubules. Maximum ATPases were 0.06–0.08 sec−1 and 1.8–2.0 sec−1 for the ncd335-tubulin and ncd335-microtubules complexes, respectively. Only the polymerized complex is fully functional, suggesting the presence of additional contacts between adjacent protofilaments. Moreover, the data reveal that the oligomeric state of microtubules is a potent regulator for the activity of kinesin related proteins.

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-PNPATP = 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.

Changes in the actin‐myosin interface are thought to play an important role in microfilament-linked cellular movements. In this study, we compared the actin binding properties of the motor domain of Dictyostelium discoideum (M765) and rabbit skeletal muscle myosin subfragment-1 (S1). The Dictyostelium motor domain resembles S1(A2) (S1 carrying the A2 light chain) in its interaction with G-actin. Similar to S1(A2), none of the Dictyostelium motor domain constructs induced G-actin polymerization. The affinity of monomeric actin (G-actin) was 20-fold lower for M765 than for S1(A2) but increasing the number of positive charges in the loop 2 region of the D. discoideum motor domain (residues 613‐623) resulted in equivalent affinities of G-actin for M765 and for S1. Proteolytic cleavage and cross-linking approaches were used to show that M765, like S1, interacts via the loop 2 region with filamentous actin (F-actin). For both types of myosin, F-actin prevents trypsin cleavage in the loop 2 region and F-actin segment 1‐28 can be cross-linked to loop 2 residues by a carbodiimide-induced reaction. In contrast with the S1, loop residues 559‐565 of D. discoideum myosin was not cross-linked to F-actin, probably due to the lower number of positive charges. These results confirm the importance of the loop 2 region of myosin for the interaction with both G-actin and F-actin, regardless of the source of myosin. The differences observed in the way in which M765 and S1 interact with actin may be linked to more general differences in the structure of the actomyosin interface of muscle and nonmuscle myosins.

International audience; Human DNA topoisomerase I not only has DNA relaxing activity, but also splicing factors phosphorylating activity. Topo I shows strong preference for ATP as the phosphate donor. We used photoaffinity labeling with the ATP analogue [α-32 P] 8-azidoadenosine-5′-triphos-phate combined with limited proteolysis to characterize Topo I domains involved in ATP binding. The majority of incorporated analogue was associated with two fragments derived from N-terminal and C-terminal regions of Topo I, respectively. However, mutational analysis showed that deletion of the first 138 N-terminal residues, known to be dispensable for topoisomerase activity, did not change the binding of ATP or the kinase activity. In contrast, deletion of 162 residues from the C-terminal domain was deleterious for ATP binding, kinase and topoisomerase activities. Furthermore, a C-terminal tyrosine 723 mutant lacking topoisomerase activity is still able to bind ATP and to phosphorylate SF2/ASF, suggesting that the two functions of Topo I can be separated. These findings argue in favor of the fact that Topo I is a complex enzyme with a number of potential intra-cellular functions.

8S Benzo(a)pyrene-binding proteins from liver cytosol of mouse and rabbit have been partially purified by gel permeation chromatography and affinity chromatography on 1-aminopyrene-Sepharose columns. These proteins, which bind polycyclic aromatic hydrocarbons and daunorubicine, have been identified, by microsequencing, as aldehyde dehydrogenases composed of polypeptides of 54 kDa. Using Ah receptor-deficient (AHR-/-) transgenic mice it has been shown that the amount as well as the binding capabilities of 8S protein was strongly altered in these mice, suggesting that its expression was partially under the control of the Ah receptor. The function of these proteins is currently unknown.

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.

The ubiquity of actin, like the functional diversity of many associated proteins, raises a question concerning diversification of motility mechanisms and thus the emergence of an elementary functional system. Our aim was to investigate, in particular, mobiles prokaryotics cells as Synechocystis lacking cilia and flagella, search for actin essential properties and then locate the molecular behaviours. Here we report the presence and purification of a 56-kDa (apparent molecular weight) prokaryotic protein that polymerizes to form filaments, activates myosin Mg ++ -ATPase activity, inhibits DNase-1 activity and affords close antigenic homology to skeletal actin. This protein was found to be associated with thylakoid membranes and extracted in the presence of Triton X-100.

SEVERAL metazoan splicing factors are characterized by ribo-nucleoprotein (RNP) consensus sequences and arginine–serine repeats (RS domain)1–8 which are essential for their function in splicing5,9,10. These include members of the SR-protein family (SC35, SF2/ASF), the U1 small nuclear (sn)RNP protein (U1-70K) and the U2 snRNP auxiliary factor (U2AF). SR proteins are phosphorylated in vivo11 and the phosphorylation state of U1-70K's RS domain influences its splicing activity12. Here we report the purification of a protein kinase that is specific for SR proteins and show that it is DNA topoisomerase I. This enzyme lacks a canonical ATP-binding motif but binds ATP with a dissociation constant of 50 nM. Camptothecin and derivatives, known to be specific inhibitors of DNA topoisomerase I, strongly inhibit the kinase activity in the presence of DNA and affect the phosphorylation state of SR proteins. Thus, DNA topoisomerase I may well be one of the SR protein kinases operating in vivo.

We purified a beta-glucosidase from the mutant strain Candida molischiana 35M5N. Analysis of the kinetic properties of this enzyme did not show any differences between the previously purified wild-type enzyme and that of the mutant. Nevertheless, a study of the stability of the enzyme at different pH levels and temperatures showed the increase resistance of this protein. This enzyme was found to be stable at pH 5 for 145 h and retained 78% of its initial activity after the same time at pH 3.5 (optimal pH) and 30 degrees C. This difference between the wild-type and the mutant enzyme could be explained by differences in the quantity or quality of glycosylation. This glycoprotein showed different forms after deglycosylation. Some peptides from this protein were also sequenced. An homology analysis found similarities between this beta-glucosidase and beta-glucosidases of Candida pelliculosa and Schizophyllum commune.

The precise location of the regulatory F-actin- and calmodulin-binding sites in the COOH-terminal sequence Trp659-Pro756 of gizzard caldesmon was investigated by subjecting the corresponding 10-kDa CNBr fragment, characterized earlier (Bartegi, A., Fattoum, A., Derancourt, J., and Kassab, R. (1990) J. Biol. Chem. 265, 15231-15238), to limited chymotryptic reactions conducted in the absence and presence of F-actin-tropomyosin. As a result, the F-actin-binding and actomyosin ATPase inhibitory activity was separated from the regulatory Ca(2+)-calmodulin-binding site. Seven chymotryptic peptides accounting for the entire primary structure of the CB10 fragment were isolated, and their complete amino acid sequences were established by combining NH2-terminal sequencing, mass spectrometry, and gel electrophoresis. Reversed-phase high performance liquid chromatography analyses of the binding of F-actin to these peptides revealed the 30-residue sequence Leu693-Trp722 as the unique crucial stretch for actin interaction and ATPase inhibition. This segment was also specifically protected by F-actin against proteolytic degradation. We further determined the functional properties of three synthetic peptides which successively cover the sequences Asn675-Lys695, Leu693-Trp722, and Arg711-Lys729. The first peptide segment specifically bound Ca(2+)-calmodulin as assessed by affinity chromatography and spectrofluorometry and should contain a potent novel calmodulin-binding subsite. The second immediately adjacent peptide inhibited the actomyosin ATPase in a tropomyosin-sensitive manner, as expected. In contrast, the third peptide displayed no detectable function. The results indicate that the overall sequence Asn675-Trp722 represents the essential regulatory unit of the COOH-terminal 10-kDa domain of caldesmon.

F-actin specifically substituted with the photocross-linker, p-azidophenylglyoxal, at Arg95 and Arg28 was isolated and characterized. Upon complexation with myosin subfragment-1 (S1) and photolysis at 365 nm, it was readily cross-linked to the S1 heavy chain with a yield of about 13-25%, generating four major actin-heavy-chain adducts with molecular masses in the range 165-240 kDa. The elevated Mg(2+)-ATPase of the covalent complexes displayed a turnover rate of 33 +/- 8 s-1 which is similar to the values reported earlier for other acto-S1 conjugates. The cross-linking between various proteolytic S1 and actin derivatives, combined with the fluorescent and immunochemical detection of the photocross-linked products, indicated that the arylnitrene group on Arg95 was inserted predominantly in the central 50-kDa region, whereas that attached to Arg28 mediated the selective cross-linking of the COOH-terminal 22-21-kDa fragments of the heavy chain, most probably by reacting at or near the connector segment between the 50-kDa and 20-kDa fragments. The rapid photoactivation and cross-linking to S1 of the substituted F-actin, which can be accomplished on a millisecond time scale, may serve to probe the structural dynamics of the interaction of the S1 heavy chain with subdomain-1 of actin during the ATPase cycle.

We have purified to near homogeneity the M-phase-specific protein kinase from starfish oocytes at first meiotic metaphase, using an improved procedure based on affinity chromatography on the immobilized yeast protein suc1. As already reported, this is identical to MPF, the cytoplasmic factor that controls entry of eukaryotic cells into M-phase. MPF is a complex formed by the stoichiometric association of a 34-kd polypeptide previously identified as cdc2 with a polypeptide that migrates with the same mobility as starfish cyclin in SDS-PAGE (apparent mol. wt 47 kd). A cDNA clone encoding starfish cyclin B has been isolated and its sequence determined. It contains a single open reading frame encoding a predicted 43 729-dalton protein. Partial microsequencing of the 47-kd polypeptide component of MPF allowed its identification as the starfish cyclin. Since the apparent mol. wt of native starfish MPF was found to be less than 100 kd, it is a heterodimer comprising one molecule of cdc2 and one molecule of cyclin B.

Maitotoxin is the most potent of the known marine toxins. The effect of maitotoxin on muscle contraction or hormone release was consistent with its action on the voltage-sensitive channel. Indeed, calcium antagonists such as nifedipine or diltiazem were able to reverse the maitotoxin effects. Using smooth muscle cells, we have analysed the effects of maitotoxin on the inositol phosphate metabolism. Maitotoxin stimulates the inositol phosphate formation (5 ± 1.8-fold in the presence of 10 mM LiCI). Moreover, this effect is not reversed, even partially by calcium antagonists, by α 1 antagonists and is not mimicked by Ca 2+ ionophores such as A23187 or calcium agonists such as Bay-K 8644. The action of maitotoxin is further discussed in this paper.

The existence of the two connector segments linking the tryptic 50 kDA fragment of skeletal S1 heavy chain to the adjacent 27 kDa and 20 kDa peptides was ascertained by digestion of S1 with staphylococcal protease which was found to act specifically at these particular regions. Three new peptides of Mr 28 000, 48 000 and 22 000 were produced and the novel S1 derivative formed had an intact actin-activated ATPase activity. Amino acid sequence analyses indicated that the 48 kDa and 22 kDa peptides overlap the two connector elements.

A 67 kDa actin-binding protein was isolated from bovine aorta. Partial amino acid sequence determination of two large thermolysin peptides were used to compare 67 kDa bovine aorta protein and p36 the substrate of pp60src tyrosine kinase. Sequence analysis shows that 67 kDa bovine aorta protein shares common domains with p36 and possesses the consensus aminoacid sequences of mammalian Ca2+-dependent membrane-binding protein and p36/gelsolin.

We have isolated and chemically characterized several 5-thio-2-nitrobenzoate-subfragment 1 derivatives (TNB-S-1) generated by the reaction of 5,5'-dithiobis(2-nitrobenzoic acid) (DNTB, up to 10-fold molar excess) with native S-1, N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine-S-1 (AEDANS-S-1), and N,N'-p-phenylenedimaleimide-S-1 (pPDM-S-1) at 4 degrees C, pH 8.0. The reaction of the reagent with AEDANS-S-1, which has a blocked -SH1 group, induced the formation of an intramolecular cystine disulfide between two vicinal -SH groups in S-1; in contrast, the treatment of pPDM-S-1 with DTNB resulted in the formation of TNB mixed disulfides only. The incorporation of the TNB groups (up to 3 mol/mol of S-1) into the native or premodified S-1 led to a local conformational change in the 50K heavy chain region that was fully reversed upon disulfide reduction. Exploiting this peculiarity of the DTNB-modified S-1's, we have realized a highly selective proteolysis of the S-1 heavy chain by thrombin and chymotrypsin, which do not act at all on the normal S-1. The 95K heavy chain was cut by thrombin into two fragments with apparent masses of 68K and 30K, whereas the "connector segments" and the light chains were unaffected. The two new fragments were issued from a primary peptide-bound cleavage between Lys-560 and Ser-561 within the amino acid sequence of the 50K region (M. Elzinga, personal communication).(ABSTRACT TRUNCATED AT 250 WORDS)

The heavy chain fragments generated by restricted proteolysis of the smooth chicken gizzard myosin subfragment-1 (S-1) with trypsin, Staphylococcus aureus V8 protease, and chymotrypsin were isolated and submitted to partial amino acid sequencing. The comparison between the smooth and striated muscle myosin sequences permitted the unambiguous structural characterization of the two protease-vulnerable segments joining the three putative domain-like regions of the smooth head heavy chain. The smooth carboxyl-terminal connector is a serine-rich region located around positions 632-640 of the rabbit skeletal sequence and would represent the "A" site that is conformationally sensitive to the myosin 10 S-6 transition and to its interaction with actin (Ikebe, M., and Hartshorne, D. J. (1986) Biochemistry 25, 6177-6185). A third site which undergoes a nucleotide-dependent chymotryptic cleavage which inactivates the Mg2+-ATPase (Okamoto, Y., and Sekine, T. (1981) J. Biochem. (Tokyo) 90, 833-842, 843-849) was identified at Trp-31/Ser-32. It is vicinal to Lys-34 that is monomethylated in the skeletal heavy chain but not at all in the smooth sequence. However, the two trimethyl lysine residues present in the skeletal sequence are conserved in the same regions of the smooth S-1 and may play a general functional role in myosin. The smooth central 50-kDa segment could be selectively destroyed by a mild tryptic digestion in the absence of any unfolding agent, with a concomitant inhibition of the ATPase activities. This feature is in line with the proposed domain structure of the S-1 heavy chain and also suggests a relationship between the specific biochemical properties of the smooth S-1 and the particular conformation of its 50-kDa region.

Summary A quantitative modification of free carboxyl groups in peptides and proteins can be obtained, under mild conditions, by reacting them with ethylenediamine in the presence of N-ethyl-N′-(3-dimethylaminopropyl)-Carbodiimide. Aminoethylasparagine and aminoethylglutamine side chains are thus generated in place of the corresponding carboxylic ones. The first kind of residue because of its structure closer to that of lysine, is a point of greater potential trypsic cleavage than the second one. The specificity and yields of this enzymatic cleavage reaction and its possible application in sequence studies are discussed.

International audience; Maitotoxin, a potent marine toxin extracted from peredinians, was found to mimic fertilization in Xenopus oocytes and to trigger the breakdown of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2, the precursor of inositol 1,4,5-trisphosphate], an increase of intracellular pCa and the cortical reaction, including the exocytosis of cortical granules and a wave-like propagation of contraction in the animal hemisphere. All these effects of maitotoxin required the presence of external calcium. Moreover, the toxin considerably increased Ca2+ influx in amphibian oocytes arrested at first meiotic prophase, due to the permanent activation of voltage-dependent Ca2+ channels. Nevertheless it is doubtful that maitotoxin acts primarily as a Ca2+ ionophore or at the level of Ca2+ channels. Indeed no stimulation of Ca2+ uptake was observed in metaphase-II-arrested oocytes, although maitotoxin readily triggered the breakdown of PtdIns(4,5)P2 as well as the cortical reaction in such cells. On the other hand, PtdIns(4,5)P2 breakdown was not reduced in oocytes microinjected with EGTA, although the calcium chelator prevented the oocytes from undergoing the cortical reaction. Taken together, these findings support the view that the toxin might act primarily by increasing PtdIns(4,5)P2 phosphodiesterase activity.

A high molecular mass complex isolated from Xenopus laevis oocytes contains three main proteins, respectively p30, p36 and p47. The p47 protein has been reported to be an in vivo substrate of the cell division control protein kinase p34cdc2. From polypeptide sequencing, we now show that the p30 and the p47 correspond to elongation factor EF-1 beta and EF-1 gamma. Furthermore, the p30 and p36 proteins were phosphorylated in vitro by casein kinase II.

We have identified the region of actin involved in the covalent coupling of maleimidobenzoyl-G-actin to the central 50 kDa segment of the myosin-S-1 heavy chain by analyzing the structure of the maleimidobenzoyl-G-actin-S-1 conjugate using selective proteolytic digestions, amino acid sequence determinations and novel cross-linking reactions between S-1 and different maleimidobenzoyl-G-actin derivatives. The cross-linking is shown to occur only on the stretch of residues 48–67 in actin subdomain-2 with Lys-50, residing on the outer part of the DNase-I-binding loop, as the most likely site of cross-linking. Because the maleimidobenzoyl-F-actin-S-1 complex undergoes the same coupling process, the data provide experimental evidence in favor of the recent model of the rigor F-actin-S-1 complex suggesting a close contact between structural elements of the lower domain of the 50 kDa fragment and the top of actin subdomain-2.

P67, a new protein binding to a specific RNA probe, was purified from radish seedlings [Echeverria, M. and Lahmy, S. (1995) Nucleic Acids Res. 23, 4963–4970]. Amino acid sequence information obtained from P67 microsequencing allowed the isolation of genes encoding P67 in radish and Arabidopsis thaliana. Immunolocalisation experiments in transfected protoplasts demonstrated that this protein is addressed to the chloroplast. The RNA-binding activity of recombinant P67 was found to be similar to that of the native protein. A significant similarity with the maize protein CRP1 [Fisk, D.G., Walker, M.B. and Barkan, A. (1999) EMBO J. 18, 2621–2630] suggests that P67 belongs to the PPR family and could be involved in chloroplast RNA processing.

A study of plasma and cardiac concentrations of amiodarone during the course of long-term oral therapy was made possible by the improvement of analytical high performance liquid chromatography of plasma and tissue extracts. The plasma level was found to increase linearly with the daily dose of the drug, above a threshold value of c. 1 . 9 mg kg-1 day-1. Similarly, the cardiac content increased linearly with the daily dose, with no threshold, showing that the drug is taken up and accumulated in the cardiac tissue, with no obvious difference between atrial and ventricular samples (P greater than 0 . 05). Both plasma and heart showed no saturation at high drug intake, a justification for increasing the oral intake in severe cases. The linear relationship between tissue and blood concentrations allows a prediction of the cardiac level from a simple and routine blood analysis.

P. Berta , S. Phaneuf , J. Derancourt , J. Casanova , M. Durand-Clement , C. le Peuch , J. Haiech and J.-C. Cavadore . The effects of maitotoxin on phosphoinositides and calcium metabolism in a primary culture of aortic smooth muscle cells. Toxicon 26, 133 – 141, 1988. — Maitotoxin, a potent marine toxin isolated from toxic tropical dinoflagellates and poisonous fishes induces contraction of different smooth muscle preparations. Actions of maitotoxin on phosphoinositides and calcium metabolism were studied using a primary culture of aortic smooth muscle cells. Maitotoxin induced a very large increase of cytosolic calcium concentration as evaluated by fura-2 acetoxymethyl ester fluorescence. This increase was concomitant with stimulation of inositol-phosphate accumulation and loss of viability of aortic smooth muscle cells. These responses to maitotoxin were abolished in Ca 2+ -free medium, and were mimicked by saponin. Calcium ionophores or K + depolarisation did not induce inositol-phosphate formation. These results suggest that maitotoxin acts by altering smooth muscle cells permeability allowing a sustained calcium influx which is able to activate inositol-phosphate formation and which is lethal for the cells.

We have cloned the cDNA coding the beta-tropomyosin of human muscle in an expression vector whose expression depends upon a promotor that can be induced by isopropyl-beta-thiogalactopyranoside. We show that a new protein was synthesized by bacteria containing the engineered plasmid. This protein was heat stable and reacted with antibodies against tropomyosin. We have purified this protein and further identified it by determining its amino acid composition and sequencing the NH2 terminal. Unlike the native muscle tropomyosin, the NH2 terminal is not acetylated and contains a methionine. The circular dichroism spectrum is compatible with 100% alpha-helices. These results show that the protein synthesized in E. coli possesses a native structure.

The automatic sequencing of proteins starting from N-terminus by solid-phase technique should be performed following the same principles as for peptides [l] . In this case the solid-phase sequencing could entirely cover the sequencing field from smaller to larger peptides and proteins and became an alternative rather than a complementary route for automatic sequence analysis if comparated to the homogenous phase technique. However, the serious difficultiwwhich arise when large peptides are to be linked to a solid support for their subsequent sequential analysis constitute the principal limit for routine work. Nevertheless the chemical and physical problems associated with the insolubilization step seems to be eliminated or minimised as shown by the successful sequencing of proteins linked to aminated porous glass [2]. Despite some inconveniencies, essentially a low degree of substitution [3] and the potential lability of the -%-O-C linkage, the aminated porous glass possesses the general properties of an ideal support for large polypeptide sequencing. In other words the best results should be obtained using an inert and rigid support possessing a high concentration of suitable functional groups on its surface. On the basis of these considerations we synthetised N-aminoethyl polyacrylamide starting from a commercial polyacrylic resin

Parvalbumin fragments from carp pI 4.47 parvalbumin corresponding to its residues 1--75 and 76--108 bind Ca2+ with affinities corresponding to Kd 0.9 . 10(-4) M and Kd 3 . 10(-3) M, respectively.

The reaction of endoproteinase Arg-C on the skeletal myosin head heavy chain was investigated through characterization of peptides and amino acid sequence analysis. The protease splits exclusively the 50 kDa-20 kDa junction at the lysine cluster spanning residues 639–641 and does not affect any other protease-sensitive region of the entire myosin heavy chain. The sensitivity of the cleavage to actin and nucleotide binding makes this protease a very specific conformational probe of S-1. The nicked S-1 derivative, containing an intact NH2-terminal 75 kDa fragment, may serve as a tool for gaining further insights into the domain structure and function of the myosin head.