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Study is conducted to show that the hydrophobic loop is mobile in F- as well as in G-action, fluctuating between the extended and parked conformations. The results show that loop 262-274 fluctuations in G- and F- actin and correlate loop dynamics with actin filament formation and stability.

Site-directed mutagenesis of the actin reveals that the hydrophobic loop comprising the amino acid residues 262-274 plays a crucial role in the polymerization of actin into filaments. Data show that the disulfide cross-linked actin mutant forms amorphous aggregates instead of normal filaments with reduced actin.

Monoclonal antibody complexes formed with only one antibody and complexes formed using two antibodies were investigated using dynamic light scattering and sedimentation velocity techniques. Findings show that the largest complex formed with a single monoclonal antibody was that of an low-density lipoprotein dimer. With two antibodies, a variety of complexes was observed. Monoclonal antibodies MB47 and 4G3, which bound about 55 degrees apart, formed a very stable dimer. Monoclonal antibodies MB47 and 4G3, which bound 136 degrees apart, formed a very stable tetramer.

Actin filaments partially cross-linked with the heterobifunctional photo-cross-linking reagent, N-(4-azido-2-nitrophenyl)-putrescine, between Gln-41 and Cys-374 on adjacent monomers in the long-pitch helix were described. The filaments were depolymerized and fractionated into pools of longitudinal cross-linked dimers, trimers and higher-order oligomers. Experimental results showed the importance of lateral actin-actin interaction for the activation of myosin ATPase and the polymerization of actin by myosin subfragment.

Recombinant apolipoprotein(a) was studied by hydrodynamic and electron microscopy techniques. Recombinant apo(a) forms a tight, noncovalent complex with human low density lipoprotein (LDL). Hydrodynamic modeling suggests that it made contact with the LDL through a few kringles, with the remainder of the molecule extending into solution. This complex dissociates in the presence of 50 mM 6-aminohexanoic acid.

The bifunctional methanothiosulfonate (MTS) reagents were introduced as an interesting alternative tool to examine the effects of the antiparallel dimer (APD) on actin polymerization. The slower decay rate of APD for the filaments elongation revealed the transient incorporation of APD into filaments and confirmed the inhibition of the nucleation and elongation of actin filaments.

Study is presented to show that the myelin protein zero transmembrane (MPZ TM) domain forms stable dimers that are mediated by the glycine zipper motif. Based on this result a new mode is proposed for the MPZ adhesion structure in which dimers of tetramers form a complementary lattice that bonds adjacent layers of the myelin sheath.

The tertiary structure of retrocyclin-2 is determined in a membrane-mimicking environment and a well-defined [beta]-hairpin structure is present in the presence of SDS micelles. The ability to self-associate has contributed to the high-affinity binding of retrocyclins for glycoproteins by increasing the valency and increasing the ability of retrocyclins to cross-link cell surface glycoproteins.

The hydrophobic loop dynamics utilizing four mutants that have cysteine residues introduced at a single location along the yeast actin loop and actin filament stability is analyzed. The data indicate that, in solution, the loop resides predominantly in a parked position within the filament but is able to dynamically populate other conformational states, which stabilize or destabilize the filament.

Metabolismof the hepatotoxicant furan leads to protein adductformation in the target organ. The initial bioactivation step involvescytochrome P450-catalyzed oxidation of furan, generating cis-2-butene-1,4-dial (BDA). BDA reacts with lysine to form pyrrolin-2-oneadducts. Metabolic studies indicate that BDA also reacts with glutathione(GSH) to generate 2-(S-glutathionyl)butanedial (GSH-BDA),which then reacts with lysine to form GSH-BDA-lysine cross-links.To explore the relative reactivity of these two reactive intermediates,cytochrome cwas reacted with BDA in the presenceand absence of GSH. As judged by MALDI-TOF mass spectrometry, BDAreacts extensively with cytochrome cto form adductsthat add 66 Da to the protein, consistent with the formation of pyrrolinoneadducts. Addition of GSH to the reaction mixture reduced the overallextent of adduct formation. The mass of the adducted protein was shiftedby 355 Da as expected for GSH-BDA-protein cross-link formation. LC-MS/MSanalysis of the tryptic digests of the alkylated protein indicatedthat the majority of adducts occurred on lysine residues, with BDAreacting less selectively than GSH-BDA. Both types of adducts maycontribute to the toxic effects of furan. [ABSTRACT FROM AUTHOR]

Furan is toxic and carcinogenic in rodents. Because of the large potential for human exposure, furan is classified as a possible human carcinogen. The detailed mechanism by which furan causes toxicity and cancer is not yet known. Since furan toxicity requires cytochrome P4S0-catafyzed oxidation of furan, we have characterized the urinary and hepatocyte metabolites of furan to gain insight into the chemical nature of the reactive intermediate. Previous studies in hepatocytes indicated that furan is oxidized to the reactive α,β-unsaturated dialdehyde, cis-2-butene- 1,4-dial (BDA), which reacts with glutathione (GSH) to form 2-(S-glutathionyl)succinaldehyde (GSH-BDA). This intermediate forms pyrrole cross-links with cellular amines such as lysine and glutamine. In this article, we demonstrate that GSH-BDA also forms cross-links with ornithine, putrescine, and spermidine when furan is incubated with rat hepatocytes. The relative levels of these metabolites are not completely explained by hepatocellular levels of the amines or by their reactivity with GSH-BDA. Mercapruric acid derivatives of the spermidine cross-links were detected in the urine of furan-treated rats, which indicates that this metabolic pathway occurs in vivo. Their detection in furan-treated hepatocytes and in urine from furan-treated rats indicates that polyamines may play an important role in the toxicity of furan. [ABSTRACT FROM AUTHOR]

Provides information on a study which describes the purification and properties of isolated actin oligomers cross-linked by 4-azido-2-nitrophenyl (ANP) between Gln-41 and cys-374 on two adjacent monomers of the same strand in the long-pitch helix in F-actin. Materials and methods; Results; Discussion.

We show by high-resolution atomic force microscopy analysis that drebrin A (a major neuronal actin binding protein) induced F-actin structural and mechanical remodeling involves significant changes in helical twist and filament stiffness (+55% persistence length). These results provide evidence of a unique mechanical role of drebrin in the dendrites, contribute to current molecular-level understanding of the properties of the neuronal cytoskeleton, and reflect the role of biomechanics at the nanoscale, to modulate nanofilament-structure assemblies such as F-actin.

Furan is a liver toxicant and carcinogen in rodents. On the basis of these observations and the large potential for human exposure, furan has been classified as a possible human carcinogen. The mechanism of tumor induction by furan is unknown. However, the toxicity requires cytochrome P450-catalyzed oxidation of furan. The product of this oxidation, cis-2-butene-1,4-dial (BDA), reacts readily with glutathione, amino acids, and DNA and is a bacterial mutagen in Ames assay strain TA104. Characterization of the urinary metabolites of furan is expected to provide information regarding the structure(s) of the reactive metabolite(s). Recently, several urinary metabolites have been identified. We reported the presence of a monoglutathione-BDA reaction product, N-[4-carboxy-4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-l-cysteinylglycine cyclic sulfide. Three additional urinary metabolites of furan were also characterized as follows: R-2-acetylamino-6-(2,5-dihydro-2-oxo-1H-pyrrol-1-yl)-1-hexanoic acid, N-acetyl-S-[1-(5-acetylamino-5-carboxypentyl)-1H-pyrrol-3-yl]-l-cysteine, and its sulfoxide. It was postulated that these three metabolites are derived from degraded protein adducts. However, the possibility that these metabolites result from the reaction of BDA with free lysine and/or cysteine was not ruled out. In this latter case, one might predict that the reaction of thiol-BDA with free lysine would not occur exclusively on the epsilon-amino group. Reaction of BDA with N-acetylcysteine or GSH in the presence of lysine indicated that both the alpha- and the epsilon-amino groups of lysine can be modified by thiol-BDA. The N-acetylcysteine-BDA-N-acetyllysine urinary metabolites were solely linked through the epsilon-amino group of lysine. A GSH-BDA-lysine cross-link was a significant hepatocyte metabolite of furan. In this case, the major product resulted from reaction with the epsilon-amino group of lysine; however, small amounts of the alpha-amino reaction product were also observed. Western analysis of liver and hepatocyte protein extracts using anti-GSH antibody indicated that GSH was covalently linked to proteins in tissues or cells exposed to furan. Our data support the hypothesis that GSH-BDA can react with either free lysine or protein lysine groups. These data suggest that there are multiple pathways by which furan can modify cellular nucleophiles. In one pathway, BDA reacts directly with proteins to form cysteine-lysine reaction products. In another, BDA reacts with GSH to form GSH-BDA conjugates, which then react with cellular nucleophiles like free lysine or lysine moieties in proteins. Both pathways will give rise to N-acetyl-S-[1-(5-acetylamino-5-carboxypentyl)-1H-pyrrol-3-yl]-l-cysteine. Given the abundance of these metabolites in urine of furan-treated rats, these pathways appear to be major pathways of furan biotransformation in vivo.