Search

Research has been conducted on the modification of the rabbit muscle myosin S1 at SH1 alone or at both SH1 and SH2. The use of alkylthiolating reagents of increasing size is examined.

Research was conducted to determine whether the hydroxy group of the amino acid serine or its D-configuration is responsible for the conformation-stablizing function of the monocyclic viroisin. Four viroisin analogues were synthesized and submitted to conformational analysis by NMR and to an actin binding assay. Results reveal that the D-configuration of serine is the structural element that maintains the phalloidin-like structure while the hydroxy group does not contribute to conformational stability.

A technique was developed to study the polymeric form of actin. The technique involves the creation of a cross-link between cysteine-374 and cysteine-10 via the disulfide-containing moiety c-A to prevent polymerization even when phalloidin is present. The cross-linked actin was shown to be an effective model for studies of monomeric actin and actin-binding proteins under conditions that would otherwise cause actin polymerization.

A series of thiol-specific cross-linking reagents were prepared for investigating the kinetics of cross-linking between SH1 (Cys707) and SH2 (Cys697) in rabbit skeletal muscle myosin subfragment 1. Cross-linking rates in the presence and absence of MgADP indicated similar dependence on the length of the reagents, while the cross-linking rates for n = 3 or n = 6 were close to those for n = 0, those for n = 7 and 8 were substantially increased. Thus, the distance between SH1 and SH2 seems to be equal in both states and can be estimated as > or = 15 angstroms.

Thymosin β4 (Tβ4), a peptide of 43 amino acids, binds to actin monomers and inhibits filament formation. In preparations of Tβ4 from bovine lung tissue, the peptide is accompanied by a derivative in which the methionine residue in position 6 is replaced by its sulfoxide. Tβ4 sulfoxide inhibits actin polymerization to an extent approximately 20-times less than Tβ4. While an equimolar amount of Tβ4 prevented actin polymerization almost completely, polymerization with the corresponding amount of the sulfoxide proceeded in a manner similar to that of pure actin, except for a slight retardation. We showed that the decrease in the inhibitory activity is reflected by a 20-times lower affinity to actin. Interestingly, under non-polymerizing conditions, the affinity of Tβ4 sulfoxide for actin is as high as that of Tβ4 (approximately 1 μM). In accordance with this, no differences were found between Tβ4 and the sulfoxide in cross-linking experiments with the monomer, where both forms of the peptide, yielded similar amounts of a 47-kDa band representing conjugates of actin and β-thymosin, as proved by Western-blotting analysis. Likewise, both Tβ4 and the sulfoxide retarded the exchange of G-actin-bound nucleotide to similar extents. Although the sulfoxide is presumably a product of autoxidation, it is attractive to speculate that oxidation of the methionine residue in Tβ4 may represent a regulatory switch for starting filament formation in non-muscle cells. [ABSTRACT FROM AUTHOR]

Natural variants and chemically modified derivatives of phalloidin have been found or prepared which show all degrees of toxicity. High toxicity persists if only three of the side chains are modified. Acetylation of two of the four OH groups of phalloidin leads to less toxic products, the triacetyl derivative is nontoxic. On tosylation toxicity disappeared after reaction of only two OH groups. Alkylation of the indole nitrogen leads to equally or less toxic (N-methyl, -ethyl, carbamoylmethyl, -tert.butyl) or nontoxic (N-propyl, -pentyl) products. Toxicity is also completely absent if only one of the two rings of the molecule is intact. In optical rotatory dispersion spectra all of the toxic compounds show in aqueous solution two cotton effects centered around 300 nm and 248 nm, whereas in nearly all of the nontoxic derivatives at least one of the effects is missing. Triacetylphalloidin and tribromophalloidin, both nontoxic, also exhibit “toxic” curves in water. Their conformation, however, changes on going to organic solvents like methanol or acetonitril as observed by optical rotatory dispersion studies. Thus, stability of the “aqueous” conformation in a lipophflic environment is apparently the second prerequisite for a toxic action of phallopeptides. [ABSTRACT FROM AUTHOR]