No abstract

The neurotoxic gamma-diketone, 2,5-hexanedione (2,5-HD), induces neurofilamentous swellings at prenodal sites in proximal axons as a consequence of pyrrolation of lysine epsilon-amino groups on neurofilament proteins. However, there is disagreement as to whether pyrrole formation and the associated alteration of noncovalent interactions is sufficient to cause neurofilament accumulation, or whether pyrrole autoxidation and subsequent protein-protein cross-linking is an obligatory event. To investigate gamma-diketones that might form pyrroles inert to autoxidative-induced cross-linking, we synthesized 1,1,1-trifluoro-2,5-hexanedione, 3-(trifluoromethyl)-2,5-hexanedione (3-TFMHD), and two 3-(dialkylaminocarbonyl)-2,5-diketones and assessed their rates of pyrrole formation with amines, the oxidation susceptibility of the resulting pyrroles, and the protein cross-linking potential in vitro, relative to those of 3-methyl-2,5-hexanedione. 1,1,1-Trifluoro-2,5-hexanedione does not form pyrroles, but the three 2,5-HD analogues with an electron-withdrawing 3-substituent all rapidly formed pyrroles that were inert to autoxidation. Although 3-TMFHD nonetheless still induced cross-linking of ribonuclease A, by a nonoxidative mechanism independent of the pyrrole, the two 3-(dialkylaminocarbonyl)-2,5-diketones did not exhibit any protein cross-linking. As these two gamma-diketones possess aqueous-organic partitioning properties similar to those of 2,5-HD, they should serve as useful mechanistic probes in further studies.

Methylation of 2-[(2,4-dinitrophenyl)amino]ethyl triphosphate (dNOTP) was found to abolish its ability to support actin sliding in the in vitro motility assay. A comparative study of the interaction of myosin subfragment 1 (S1) and actoS1 with methylated (MdNOTP) and non-methylated dNOTP was undertaken. Both analogues were shown to be substrates for S1 NTPase in the presence of K+/EDTA, Ca2+, or Mg2+, although their rates of hydrolysis in the presence of the divalent cations were significantly greater than that occurring for ATP. However, actin had only a marginal effect on the rate of hydrolysis of MdNOTP, in sharp contrast to its effect on the hydrolysis of dNOTP and ATP which were quite similar. Moreover, while dNODP is able to form stable ternary S1 complexes with orthovanadate (Vi) or berylium fluoride (BeFx), whose formation results in increased thermal stability of S1, the methylated diphosphate analogue was unable to do so. These differences can be related to methylation-induced changes in the conformation of dNOTP indicated by molecular-modeling approaches. These studies suggest that methylation prevents the specific interaction of the aryl ring of dNOTP with S1 in the adenine binding region necessary for the formation of the force-producing intermediate (M. D. P*) during the S1 Mg(2+)-NTPase cycle.

Myosin subfragment 1 hydrophobicity was found to be sensitive to the occupancy and nature of bound nucleotide at its active site, as shown by changes in elution behavior of unmodified and chemically modified S1 during phenyl hydrophobic chromatography. The elution properties of S1 were unaltered by alkylation of SH1 (Cys-707) with N-ethylmaleimide or by covalent bridging between SH1 and SH2 (Cys-697) with p-phenylenedimaleimide with trapping of MgADP. Although addition of MgADP or MgATP to the elution buffers had minimal effect on the elution properties of these modified S1 species, the presence of these nucleotides was found to produce differential effects with unmodified S1. With MgADP, where S1 is in the S1** MgADP state, the elution times were decreased slightly, whereas with MgATP, where S1 is primarily in the S1** MgADP.Pi state, the elution times were significantly lowered, indicating reduced accessibility for the immobilized phenyl ligand. Stable S1 ternary complexes, formed with MgADP and various Pi analogues, showed elution times similar to that for S1 in the buffers containing MgATP. Thus, two main classes of nucleotide-induced S1 conformations can be defined according to their interaction with immobilized phenyl. These nucleotide-induced changes in S1 hydrophobicity correlate well with reported changes in radius of gyration of S1 associated with different states of the bound nucleotide [Wakabayashi, K., Tokunga, M., Kohno, I., Sugimoto, Y.; Hamanaka, T., Takezawa, Y., Wakabayashi, T., & Amemiya, Y. (1992) Science 258, 443-447], suggesting that the observed hydrophobicity interaction may be measuring accessibility of the immobilized phenyl ligand into a hydrophobic crevice, and that this crevice is closed or tightened when S1 is in the S1** MgADP.Pi state.

Evidence is presented that MgADP can be noncovalently trapped in myosin subfragment 1 in the presence of ScFx resulting in the concomitant loss of ATPase function. The rate of inactivation in the presence of MgCl2 at 25 degrees C is 8.7 M-1 s-1 which is too slow for a simple collisional mechanism and suggests that a subsequent slow isomerization step is responsible for formation of a stable tenary complex, S1.MgADP.ScFx in a manner analogous to that proposed for the Vi stabilized complex by Goodno (Goodno, C. C. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2620-2624). It is also found that ADP can be trapped in subfragment 1 in the absence of MgCl2 indicating the formation of an S1.ADP.ScFx complex. The stability of these complexes at 4 degrees C was studied by following the loss of trapped [14C]ADP with a chase with ADP. The rate of nucleotide loss at 4 degrees C was biphasic for both complexes suggesting that the inhibitory complexes exist in two distinct states as previously proposed for the ternary complex stabilized by Vi (Mihashi, K., Ooi, A., and Hiratsuka, T. (1990) J. Biochem. (Tokyo) 107, 464-469). Formation of these complexes resulted in a marked enhancement of the intrinsic tryptophyl fluorescence suggesting that conformationally they may resemble the steady-state intermediate formed with MgATP. The failure to observe photolysis in the presence of excess Vi at sites associated with the ATP consensus sequence suggests that in these complexes ScFx occupies the site responsible for these cleavage reactions and that it is not displaced by the added Vi.