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Iba2 is a homolog of ionized calcium-binding adapter molecule 1 (Iba1), a 17-kDa protein that binds and cross-links filamentous actin (F-actin) and localizes to membrane ruffles and phagocytic cups. Here, we present the crystal structure of human Iba2 and its homodimerization properties, F-actin cross-linking activity, cellular localization and recruitment upon bacterial invasion in comparison with Iba1. The Iba2 structure comprises two central EF-hand motifs lacking bound Ca2+. Iba2 crystallized as a homodimer stabilized by a disulfide bridge and zinc ions. Analytical ultracentrifugation revealed a different mode of dimerization under reducing conditions that was independent of Ca2+. Furthermore, no binding of Ca2+ up to 0.1 mm was detected by equilibrium dialysis. Correspondingly, Iba EF-hand motifs lack residues essential for strong Ca2+ coordination. Sedimentation experiments and microscopy detected pronounced, indistinguishable F-actin binding and cross-linking activity of Iba1 and Iba2 with induction of F-actin bundles. Fluorescent Iba fusion proteins were expressed in HeLa cells and co-localized with F-actin. Iba1 was recruited into cellular projections to a larger extent than Iba2. Additionally, we studied Iba recruitment in a Shigella invasion model that induces cytoskeletal rearrangements. Both proteins were recruited into the bacterial invasion zone and Iba1 was again concentrated slightly higher in the cellular extensions. [ABSTRACT FROM AUTHOR]

In this study we investigated whether the expression of N-terminal myosin light chain-1 (MLC-1) peptides could improve the intrinsic contractility of the whole heart. We generated transgenic rats (TGR) that over expressed minigenes encoding the N-terminal 15 amino acids of human atrial MLC-1 (TGR/hALC-1/1-15, lines 7475 and 3966) or human ventricular MLC-1 (TGR/hVLC-1/1-15, lines 6113 and 6114) isoforms in cardiomyocytes. Synthetic N-terminal peptides revealed specific actin binding, with a significantly (P<0.01) lower dissociation constant (KD) for the hVLC-1/1-15-actln complex compared with the KD value of the hALC-1/1-15-actin complex. Using synthetic hVLC-1/ 1-15 as a TAT fusion peptide labeled with the fluorochrome TAMRA, we observed specific accumulation of the N-terminal MLC-1 peptide at the sarcomere predominantly within the actin-containing I-band, but also within the actin-myosin overlap zone (A-band) in intact adult cardiomyocytes. For the first time we show that the expression of N-terminal human MLC-1 peptides in TGR (range: 3-6 µM) correlated positively with significant (P<0.001) improvements of the intrinsic contracfile state of the isolated perfused heart (Langendorff mode): systolic force generation, as well as the rates of both force generation and relaxation, rose in TGR lines that expressed the transgenic human MLC-1 peptide, but not in a TGR line with undetectable transgene expression levels. The positive inotropic effect of MLC-1 peptides occurred in the absence of a hypertrophic response. Thus, expression of N-terminal domains of MLC-1 represent a valuable tool for the treatment of the failing heart. [ABSTRACT FROM AUTHOR]

Defective L-type Ca2+ channel (ICaL) regulation is one major cause for contractile dysfunction in the heart. The ICaL is enhanced by sympathetic nervous stimulation: via the activation of β-adrenergic receptors, PKA phosphorylates the α1C(CaV1.2)- and β2-channel subunits and ahnak, an associated 5643-amino acid (aa) protein. In this study, we examined the role of a naturally occurring, genetic variant Ile5236Thr-ahnak on ICaL. Binding experiments with ahnak fragments (wild-type, Ile5236Thr mutated) and patch clamp recordings revealed that Ile5236Thr-ahnak critically affected both β2 subunit interaction and ICaL regulation. Binding affinity between ahnak-C1 (aa 4646-5288) and β2 subunit decreased by ≈50% after PKA phosphorylation or in the presence of Ile5236Thr-ahnak peptide. On native cardiomyocytes, intracellular application of this mutated ahnak peptide mimicked the PKA-effects on ICaL increasing the amplitude by ≈60% and slowing its inactivation together with a leftward shift of its voltage dependency. Both mutated Ile5236Thr-peptide and Ile5236Thr-fragment (aa 5215-5288) prevented specifically the further up-regulation of ICaL by isoprenaline. Hence, we suggest the ahnak-C1 domain serves as physiological brake on ICaL. Relief from this inhibition is proposed as common pathway used by sympathetic signaling and Ile5236Thr-ahnak fragments to increase ICaL. This genetic ahnak variant might cause individual differences in ICaL regulation upon physiological challenges or therapeutic interventions. [ABSTRACT FROM AUTHOR]

Examines the interaction of small heat shock proteins with actin cytoskeleton. Inhibition of the polymerization of actin in vitro; Sequence of murine HSP25; Use of fluorescence spectroscopy and electron microscopy.

Examines the presence of the tetrameric ε-crystallin from the eye of the duck eye lens. Characterization of common vertebrate heart and muscle lactate dehydrogenases; Observation of guanidine-induced and temperature induced denaturation transitions; Structure of lactate dehydrogenases.

Examines the role of mouse Hsp25, a small heat shock protein, C-terminal extension in oligomerization and chaperone action. Stabilization of unfolding proteins; Prevention of proteins' precipitation from solution; Use of CD spectroscopy.

Examines the effect of the ionic environment on the molecular structure of bacteriophage SPP1 portal protein. Flexibility to allow polypeptide chain movements for protein functionality; Importance of cyclic oligomers with rotational symmetry in central biological processes; Discussion on the gene encoding bacteriophase SPP1 portal protein.

Ultracentrifuge studies of intact protein L7/L12, of its fragments 27–120, 1–74 and 74–120 and of protein L7/L12 with oxidized methionine residues, indicate that the N-terminal sequence of the protein L7/L12 is responsible for its dimerization. The symmetry model of the dimer is discussed. [ABSTRACT FROM AUTHOR]

A method for reducing the degree of heterogeneity in the electrophoretic enzyme activity pattern of hypoxanthine phosphoribosyltransferase preparations by incubation with a (magnesium) phosphoribosyl diphosphate substrate is described. Hypoxanthine phosphoribosyltransferase was isolated from human erythrocytes and Chinese hamster livers. A subunit molecular weight of 26000–27000 as reported by other authors was obtained for both enzymes by gel electrophoresis in the presence of dodecylsulfate. Gradient gel electrophoresis revealed that the native enzymes mainly have a molecular weight of 105000–110000 and are thus apparently tetrameric, when held in the active state by the presence of phosphoribosyl diphosphate. The dimeric enzyme with a molecular weight of 52000–55000, was also found under other conditions. The trimer occurred only in the absence of phosphoribosyl diphosphate, for instance by glycerol gradient centrifugation. The enzyme from human erythrocytes was partly degraded during purification in the absence of a protease inhibitor. The purified enzyme has a very low protease contamination level. Proteolysis is an additional cause of heterogeneity and might therefore explain earlier conflicting results. Since the heterogeneous nature of hypoxanthine phosphoribosyltransferase is caused only by the secondary processes of dissociation/association and, in the case of the human erythrocyte enzyme, degradation, we suggest that the use of the term ‘isozyme’ to describe the different forms should be avoided. [ABSTRACT FROM AUTHOR]

On the basis of hydrodynamic, electron microscopic and biochemical investigations a new model of the structure of initiation factor eIF-3 is proposed. From sedimentation and diffusion coefficients of 16.35 S and 2.13 × 1007 cm²/s, respectively, as well as from sedimentation equilibrium measurements, a molecular mass of about 650 kDa was determined for isolated eIF-3. This is in agreement with molecular mass estimations by sodium dodecyl sulphate gel electrophoresis. A partial specific volume of 0.723 cm³/g was determined by means of the amino acid composition and the specific volume increments of the amino acids. From this value and from the molecular mass, a volume of 780 nm³ was calculated for eIF-3. In electron micrographs of isolated eIF-3, images with triangular profiles and side lengths of 14 nm, 16 nm, and 17 nm have been observed. Taking into account the calculated volume and considering the triangular image as one face of the particle, it is suggested that eIF-3 has the shape of a flat triangular prism with a height of about 7 nm and the above-mentioned side-lengths. This model is in agreement with results of electron microscopic investigations of eIF-3 in native small ribosomal subunits [Lutsch, G., Benndorf, R., Westermann, P., Bommer, U.-A. & Bielka, H. (1986) Eur. J. Cell Biol. 40/2, in press]. The high frictional ratio of about 1.7 also supports elF-3 to be rather of a flat than of a globular shape. [ABSTRACT FROM AUTHOR]

The location of initiation factor eIF-2 and of its subunits in quaternary initiation complexes (40S-ribosomalsubunit, eIF-2 · GuoPP[CH2]P · Met-tRNAf) was investigated by immunoelectron microscopy. Quaternary complexes were fixed with glutaraldehyde and reacted with affinity-purified polyclonal antibodies against eIF-2α, eIF-2β or eIF-2γ. The dimeric immune complexes obtained by sucrose gradient centrifugation were investigated electron microscopically after negative staining. Antibody-binding sites were observed on the interface side of the 40S ribosomal subunit in the region between the 'head' and the 'body' (neck region) of the 40S ribosomal subunit. Within this region, eIF-2α points to the rear side, whereas eIF-2β and eIF-2γ point to the frontal side of the 40S subunit indicating an elongated shape of eIF-2 about 15 nm long. By analytical ultracentrifugation of isolated eIF-2 the sedimentation and diffusion coefficients were determined to be 6.54 S and 4.74 × 10-7 cm²/s respectively. From these data, a molar mass of 122.4 kg/mol and a dry volume of 147.4 nm³ were calculated. For the shape of eIF-2 a prolate ellipsoid of revolution is assumed with a maximal length of about 15 nm and with an axial ratio of about 1:3.5. This conclusion is further confirmed by a calculated frictional ratio of 1.37 and a Stokes radius of about 4.54 nm. [ABSTRACT FROM AUTHOR]

The amino acid sequence of insulin of carp (Cyprinus carpio) has been determined and correlated with its biological activity in a fat-cell test and its structural properties as measured by circular dichroism and sedimentation analysis. The amino acid sequence of carp insulin displays some unusual features: the B chain is longer at the N terminus by two residues as compared with mammalian insulins and there are substitutions of the charged residues, found in most insulins at positions B21 and B22, by proline and threonine respectively. On the other hand, all amino acid residues essential for biological activity and for the association of insulin monomers are the same in carp insulin. Accordingly, the half-maximal response in a fat-cell test is reached with carp insulin at concentrations which are only three times higher than with porcine insulin and the maximal response is the same. The circular dichroism spectrum of carp insulin resembles greatly that of bovine insulin indicating that it has a similar spatial structure. Despite amino acid substitutions in the dimer-dimer contact region, carp insulin is able to form hexamers. [ABSTRACT FROM AUTHOR]

Structural alterations of albumin, their dependence on concentration and the role of free -SH groups at thermal denaturation, as well as the reversibility of thermally induced structural changes, were studied. Application of various physical methods provides information on a series of structural parameters in a major concentration range. Apart from changes of the helix content, heat treatment gives rise to β structures which are amplified on cooling and which are correlated with the aggregation of albumin. With rising temperature and concentration the proportion of β structures and aggregates increases. At degrees of denaturation of up to 20% complete renaturation is possible in every case. The structure content is concentration-dependent even at room temperature. It may be that intermolecular interactions induce additional α-helix structures which are less stable, however, than the ones stabilized by intramolecular interactions. Unfolding of the pocket containing the free -SH group of cysteine-34 enables disulphide bridges to be formed leading to stable aggregates and irreversible structural alterations. Through binding of N-ethylmaleimide to free -SH groups, which blocks the formation of disulphide bridges, it is possible to prevent aggregation and irreversible conformational changes. At temperatures below 65–70%, oligomers are formed mainly via intermolecular β structures. [ABSTRACT FROM AUTHOR]

The dissociation-association equilibrium of lamprey methemoglobin as a function of protein concentration, pH, and ligandation of the heine Fe3+ has been studied using sedimentation analysis. The sedimentation constant s20, wº of MetHb in the pH range 4.5 < pH < 10 amounts to 1.94 S leading to a calculated molecular weight of 17 000 which corresponds to the Hb protomer. At pH 7,5 the MetHb · N3 complex is monomeric. Below pH 5.7 and at high protein concentration the dimer of 34000, s20, wº = 3.13 S, is formed, the dimerization being accompanied by an uptake of two protons per protomer: (a) 2 MetHb + 2 Na- + 2 H+ ↔ (H+ · MetHb · N3)2. At high concentrations of protein and azide the association of MetHb · N3 becomes a function of pit only: (b) 2 MetHb · N3 + 2 H+ ↔ (H+ · MetHb · N3)2. The pK value of equilibrium (b) is found to be 6.7 pointing to a heme-linked imidazole determining the equilibrium. As compared to the azide complex, other ligands, such as F- > OCN- > CN- > SCN-, favour the dimerization of lamprey MetHb to a lesser degree, Dimerization of the heme polyvpeptide chains in the MetHb-ligand complexes and their association in DeoxyHb supposedly involves different aminoacids in the interface between the protomers. [ABSTRACT FROM AUTHOR]