Identification of gelsolin, a Ca2+-dependent regulatory protein of actin gel-sol transformation, and its intracellular distribution in a variety of cells and tissues.

Antiserum prepared against gelsolin, a major Ca2+-dependent regulatory protein of actin gel-sol transformation in rabbit lung macrophages, was used to detect the presence of proteins immunologically related to gelsolin in a variety of cells and tissues. Cell extracts were electrophoresed on polyacrylamide gels, and replicas of the gels on cellulose nitrate paper were stained by an indirect immunohistochemical technique. A single band of crossreactive material which comigrates with macrophage gelsolin is found in at least nine different kinds of cells and tissues derived from rabbits and humans and in four lines of cultured cells from humans and rats. Gelsolin was also identified in human serum and plasma, raising the possibility that it may contribute to the clearance of actin from the circulatory system. Using this antiserum, we demonstrated, by indirect immunofluorescent staining of acetone-fixed macrophages and polymorphonuclear leukocytes, that gelsolin resides in the cortical cytoplasm and that during phagocytosis it is concentrated in pseudopodia engulfing particles to be ingested, an area of the cytoplasm actively engaged in movement. In longitudinal cryostat sections of contracted rabbit skeletal muscle, antigelsolin staining was associated with the I-band of the myofibril, suggesting that it may be involved, by an as yet undefined mechanism, in skeletal muscle function. In rabbit intestinal epithelial cells, gelsolin was associated with the cytoplasm and the terminal web region of the brush border, a localization distinct from that previously reported for villin, a structurally and functionally similar protein isolated from the brush borders of chicken intestinal epithelial cells. In conclusion, our findings support the idea that gelsolin is involved in the regulation of movement and suggest that gelsolin-mediated Ca2+-regulation of actin cytoskeletal structure, first characterized in macrophages, may be of general importance.

The tyrosyl residues in creatine kinase. Modification by iodine.

The effect of the iodination of tyrosyl residues in creatine kinase from rabbit muscle has been investigated at alkaline pH after reversible masking of the reactive thiol groups. The conversion of 4-5 tyrosyl residues to monoiodotyrosines as measured by spectrotitration and by radioactive iodine labelling resulted in almost total loss of enzymic activity. The modified enzyme was unable to bind its nucleotide substrates but no significant conformational change was revealed by optical rotatory dispersion or Stokes radius measurements. However, change in the reactivity of some non-essential thiol groups, presumably those located near the active thiol groups, was observed.

Yeast 3-phosphoglycerate kinase. Essential arginyl residues at the 3-phosphoglycerate binding site.

Yeast 3-phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phospho-transferase, EC 2.7.2.3) is inactivated by phenylglyoxal. Loss of activity correlates with the modification of two arginyl residues, both of which are protected by all of the substrates. The modification is not accompanied by any significant conformational change as determined by optical rotatory dispersion. Ultraviolet difference spectrophotometry indicates that the inactivated enzyme retains its capacity for binding the nucleotide substrates whereas the spectral perturbation characteristic of 3-phosphoglycerate binding is abolished in the modified enzyme. The data suggest that at least one of the two essential arginyl residues is located at or near the 3-phosphoglycerate binding site. A likely role of this residue could be its interaction with the negatively charged phosphate or carboxylate groups of 3-phosphoglycerate.

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.

Muscle gelsolin: isolation from heart tissue and characterization as an integral myofibrillar protein.

A 92-kDa polypeptide present in rabbit and dog cardiac muscle was purified to homogeneity and some of its properties were investigated using biochemical and cytochemical approaches. The protein was found to be similar, if not identical to macrophage gelsolin; it cross-reacts immunologically with anti-rabbit macrophage gelsolin antibody, has a Ca2+-sensitive shortening effect on the actin filaments as judged by the high shear viscometry and sedimentation experiments, and has a similar amino acid composition. In addition, immunoblot and SDS polyacrylamide gel analysis of cardiac muscle extracts obtained at high and low ionic strength showed that this protein is tightly bound to myofibrils, both in the absence and presence of Ca2+, in ventricular as well as in atrial muscle cells. Indirect immunofluorescence microscopy revealed a striated gelsolin staining pattern analogous to that previously observed for the skeletal muscle gelsolin, suggesting that in the muscle cell this protein is sharing the same localisation as actin. Because of its severing and nucleating properties the gelsolin may play a major role in the organization, assembly and turnover of the thin filaments within the muscle cells.

[Isolation of peptide containing essential tyrosine from lobster arginine kinase]. / Isolement du peptide a tyrosine essentielle de l'arginine kinase de homard

The essential tyrosine residue of Lobster muscle arginine kinase, which is part of an antigenic determinant, has been modified by tetranitromethane. Cleavage of the S-carboxymethylated nitrated enzyme with cyanogen bromide gives rise to eight peptides, one of which containing the labelled essential tyrosyl group. Ion exchange chromatography on sulfoethyl-Sephadex C-25 in urea medium has been used with success for isolation and purification of the nitrated peptide. From its amino acid composition and end groups structure this peptide is the N-terminal fragment of the protein.

Spatial proximity of a tyrosyl and a lysyl residue in the active site region of yeast 3-phosphoglycerate kinase.

The effect of 7-chloro-4-nitrobenzofurazan on yeast 3-phosphoglycerate kinase causes a modification of one tyrosyl residue concomitantly with a total loss of activity of the enzyme. The modification is not accompanied by any significant conformational change. A total protection against inactivation is observed with the substrates : furthermore, AMP, tripolyphosphate and pyrophosphate afford an effective protection. At pH 9, a shift in the absorbance spectrum of the tyrosine O-nitrobenzofurazan derivative of 3-phosphoglycerate kinase is observed. It can be related to the transfer of the reagent from tyrosine to lysine. The N-nitrobenzofurazan derivative is also completely inactive. It is concluded that a lysine residue is located close to the essential tyrosyl residue.

Subcellular distribution of calponin and caldesmon in rat hippocampus.

Caldesmon and calponin are two actin- and calmodulin-binding proteins involved in the 'actin-linked' regulation of smooth muscle and non-muscle Mg(2+)-actin-activated myosin II ATPase activity. In the present report we show that caldesmon and calponin are present in the post-synaptic side of symmetric synapses and accumulate in the post-synaptic densities of asymmetric synapses. Caldesmon- and calponin-immunoreactivities are also observed at the plasma membrane of the hippocampal neurones. Finally, while caldesmon seems strictly distributed to neurones, acidic calponin is present in both neurones and astrocytes.

Interaction of yeast 3-phosphoglycerate kinase with negatively charged carriers.

The aim of this study was to investigate the possibility of an interaction of yeast 3-phosphoglycerate kinase with negatively charged carriers such as polyanionic agents or a polarized electrode. Various polyanions were found to promote enzyme aggregation as judged by ultracentrifugation measurements and chemical modification. The data obtained suggest that these interactions are mediated through the N-terminal domain of the protein. However, the most striking property of 3-phosphoglycerate kinase described here is concerned with its significant dipolar moment as evidenced by electrocapillary measurements, which allows an orientation of the macromolecule in an electric field. Further, the enzyme could be absorbed by a negatively charged surface, first by hydrophobic links and then oriented perpendicularly to the surface. Therefore, the intrinsic properties of yeast 3-phosphoglycerate kinase agree with the formation of an enzyme-membrane complex and afford the ability for a specific orientation of the molecule at the lipid bilayer surface or in the cytoplasm.

Cross-linking of smooth muscle caldesmon to the NH2-terminal region of skeletal F-actin.

The cross-linking of the F-actin-caldesmon complex with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide in the presence of N-hydroxysuccinimide generated four major adducts which were identified on polyacrylamide gels. By cross-linking 3H-actin to 14C-caldesmon, these were found to represent 1:1 cross-linked complexes of actin and caldesmon displaying different electrophoretic mobilities. Tropomyosin did not noticeably affect the cross-linking process. The same four fluorescent species resulting from the cross-linking of caldesmon to F-actin labeled with N-[7-(dimethylamino)-4-methyl-3-coumarinyl]maleimide were subjected separately to partial cleavages with hydroxylamine or cyanogen bromide. These treatments yielded fluorescent 41- and 37-kDa fragments, respectively, from each cross-linked entity indicating unambiguously that caldesmon was cross-linked only to the NH2-terminal actin stretch of residues 1-12. This region is also known to serve for the carbodiimide-mediated cross-linking of the myosin subfragment-1 heavy chain (Sutoh, K. (1982) Biochemistry 21, 3654-3661). A covalent caldesmon-F-actin conjugate containing a protein molar ratio close to 1:19 was isolated following dissociation of uncross-linked caldesmon. It showed a low level of activation of the ATPase activity of skeletal myosin subfragment-1, and the binding of Ca2(+)-calmodulin to the derivative did not cause the reversal of the ATPase inhibition. In contrast, the reversible binding of caldesmon to F-actin cross-linked to myosin subfragment-1 did not inhibit the accelerated ATPase of the complex. The overall data point to the dual involvement of the actin's NH2 terminus in the inhibitory binding of caldesmon and in actomyosin interactions in the presence of ATP.

Structural studies on yeast 3-phosphoglycerate kinase. Identification by immuno-affinity chromatography of one glutamyl residue essential for yeast 3-phosphoglycerate kinase activity. Its location in the primary structure.

3-Phosphoglycerate kinase is inactivated by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate and nitrotyrosine ethyl ester. The coupling of 1 mol nitrotyrosine/mol enzyme is sufficient to inactivate the protein completely. A weak protection against inactivation is observed with each substrate added separately. In contrast, the complex ATP--3-phosphoglycerate--enzyme or ATP--Mg--3-phosphoglycerate--enzyme affords a considerable protection. The critical residue is identified as a glutamyl residue after isolation by immuno-affinity chromatography of nitrotyrosyl peptide resulting from exhaustive proteolytic digestion of the modified protein. In addition, the determination of the primary sequence of the C-terminal part of the protein leads to the location of the glutamyl residue at position eight from the C-terminus. We conclude that this glutamyl residue is situated in the domain which does not bind the nucleotide substrates [Bryant, T.N., Watson, H.C. and Wendell, P.L. (1974) Nature (Lond.) 247, 14--17]. Its role in the catalysis process is discussed.

Yeast 3-phosphoglycerate kinase: sulfate and substrate binding, their effect on the conformational state of the enzyme.

Anions and particularly sulfate are known to interact with 3-phosphoglycerate kinase and to induce an increase of its catalytic efficiency. The present work affords information on the location of the anionic site and on the conformational change produced by the sulfate binding. We have established that sulfate is able, first, to modify the environment of some critical amino acids (cysteine and arginines) located in the N-terminal half of the protein, second, to induce perturbation of aromatic residues as judged by spectrophotometry, and, third, to slightly decrease the magnitude of the Cotton effect at 233 nm. All these modifications are produced by sulfate concentrations required for the activation of the enzyme. The most striking result consists in a large change in the hydrodynamic properties of the protein upon sulfate interaction as determined by analytical ultracentrifugation studies. Thus, sulfate modifies the shape of the molecular, causing it to become more compact. Furthermore, a study of the binary and ternary complexes between yeast 3-phosphoglycerate kinase and its substrates suggests that such a change of the shape of the molecular only occurs in sulfate-enzyme with or without substrates and in ATP (with or without Mg2+)-3-phosphoglycerate-enzyme complexes.

Characterization of the Ca2+-induced conformational changes in gelsolin and identification of interaction regions between actin and gelsolin.

Serum gelsolin, a Ca2+-dependent protein regulating the length of actin filaments, undergoes conformational changes upon binding Ca2+. These were detected and analyzed by several approaches including ultraviolet difference spectroscopy, circular dichroism studies, analytical ultracentrifugation, thiol group titration, and limited proteolytic digestions. The effect of Ca2+ binding on the UV absorption difference spectrum and the near-UV circular dichroism spectrum was consistent with changes in the environments of tyrosine and phenylalanine residues. In the presence of Ca2+, the S0(20),w value decreased from 5.3 to 4.7. This latter result implies a transformation to a more asymmetric molecular shape. Gelsolin contained only two accessible thiol groups per mole of protein, one of which was titratable in the native protein; it was more accessible to 5,5'-dithiobis(2-nitrobenzoic acid) in the absence than in the presence of Ca2+. The limited digestion of gelsolin from serum and bovine aorta smooth muscle by two different proteases, chymotrypsin and trypsin, proceeded much faster in the presence of Ca2+ than in its absence with the production of three main fragments of about 40K, 32K, and 21K. This fragment mixture was found still able to shorten F-actin in a Ca2+-dependent manner; this severing activity was expressed by the isolated 40K peptide. Gelsolin was cross-linked to F- and G-actin by the zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC), generating a covalent 130K binary complex (actin1-gelsolin1) followed by a covalent 180K ternary complex (actin2-gelsolin1).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the carboxyl-terminal 10-kDa cyanogen bromide fragment of caldesmon as an actin-calmodulin-binding region.

A pair of 10-kDa peptides, designated CB-a and CB-b, was isolated by calmodulin-Sepharose chromatography from a total CNBr digest of turkey gizzard caldesmon. CB-a encompasses the COOH-terminal segment of residues 659-756, according to the sequence of adult chicken gizzard caldesmon (Bryan, J., Imai, M., Lee, R., Moore, P., Cook, R.G., and Lin, W.G. (1989) J. Biol. Chem. 264, 13873-13879), whereas CB-b comprises the same structure but was a few amino acids shorter at its COOH terminus. Both peptides cosedimented with F-actin, and their binding was increased by smooth muscle tropomyosin. The Kd values were 1.3 and 0.5 microM, in the absence and presence of tropomyosin, respectively, with a maximum binding capacity of 6.9 actins/mol of peptides. The CB-a/CB-b fragments inhibited, in a tropomyosin-sensitive and Ca2(+)-calmodulin-dependent manner, the skeletal actomyosin subfragment 1 ATPase activity to a level close but not identical to that observed for the parent caldesmon. Ca2(+)-calmodulin was selectively cross-linked to either caldesmon or the CNBr peptides with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide producing 1:1 covalent complexes that were retained neither by phenyl-Sepharose nor by immobilized calmodulin. Moreover, the cross-linked caldesmon bound weakly to F-actin and did not inhibit the actomyosin subfragment 1 ATPase in the absence of Ca2+. The results suggest that the CB-a/CB-b peptide region contains major regulatory determinants of caldesmon.

Mapping of the functional domains in the amino-terminal region of calponin.

Three chymotryptic fragments accounting for almost the entire amino acid sequence of gizzard calponin (Takahashi, K., and Nadal-Ginard, B. (1991) J. Biol. Chem. 266, 13284-13288) were isolated and characterized. They encompass the segments of residues 7-144 (NH2-terminal 13-kDa peptide), 7-182 (NH2-terminal 22-kDa peptide), and 183-292 (COOH-terminal 13-kDa peptide). They arise from the sequential hydrolysis of the peptide bonds at Tyr182-Gly183 and Tyr144-Ala145 which were protected by the binding of F-actin to calponin. Only the NH2-terminal 13- and 22-kDa fragments were retained by immobilized Ca(2+)-calmodulin, but only the larger 22 kDa entity cosedimented with F-actin and inhibited, in the absence of Ca(2+)-calmodulin, the skeletal actomyosin subfragment-1 ATPase activity as the intact calponin. Since the latter peptide differs from the NH2-terminal 13-kDa fragment by a COOH-terminal 38-residue extension, this difference segment appears to contain the actin-binding domain of calponin. Zero-length cross-linked complexes of F-actin and either calponin or its 22-kDa peptide were produced. The total CNBr digest of the F-actin-calponin conjugate was fractionated over immobilized calmodulin. The EGTA-eluted pair of cross-linked actin-calponin peptides was composed of the COOH-terminal actin segment of residues 326-355 joined to the NH2-terminal calponin region of residues 52-168 which seems to contain the major determinants for F-actin and Ca(2+)-calmodulin binding.

Expressing functional domains of mouse calponin: involvement of the region around alanine 145 in the actomyosin ATPase inhibitory activity of calponin.

Previously, we attributed the binding of F-actin to the 38-residue stretch of gizzard calponin encompassing the sequence A145-Y182 and postulated the hexapeptide motif VKYAEK, representing residues 142-147, as a putative actin-binding site [Mezgueldi, M., Fattoum, A., Derancourt, J. & Kassab, R. (1992) J. Biol. Chem. 267, 15943-15951]. Herein, the nature of the ATPase inhibitory amino acids of calponin and their relative position within the actin binding domain was investigated by expressing the following fragments of mouse calponin with or without substitution or deletion of the hexapeptide V142-K147: amino acids 1-228 (CaP1-228), 45-228 (CaP45-228), 131-228 (CaP131-228), and CaP1-228 with substitution of A145 with S (CaP1-228A145S) or deletion of V142-K147 (CaP1-228de1142-147). All the recombinant fragments displayed most of the biochemical properties of the smooth muscle purified calponin including (a) expected electrophoretic mobility, (b) heat stability, (c) binding to actin, tropomyosin and calmodulin, and (d) zero-length cross-linking to actin switched by calmodulin in a calcium-dependent fashion. However, while the wild-type recombinant fragments inhibit the acto-S-1 ATPase activity to the same extent as do the parent calponin, modulation of the hexapeptide by either substitution or deletion strongly affect the inhibitory activity with only slightly decreasing actin binding capacity. The data indicate that the stretch VKYAEK is crucial for ATPase inhibition by calponin but represents only part of the actin-binding domain. These results are discussed in terms of multiple contact sites between actin and calponin.

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.