Glutathionyl(cysteine-374) actin forms filaments of low mechanical stability.

Rabbit muscle actin reacts with 2,4-dinitrophenylglutathionyldisulfide, forming a mixed disulfide in position 374. the product S-(cysteine-374)glutathionyl actin forms filaments which are easily disrupted under shearing stress. Even weak mechanical strain, as exerted, for example, during capillary viscometry or heating the solution to 37 degrees C, leads to considerable breakage of these filaments. Because of spontaneous repair which consumes ATP, the mechanically broken filaments exhibit an approx. 6-fold enhanced steady-state ATPase activity as compared to normal F-actin. Monomers of glutathionyl actin have a reduced affinity for their bound nucleotide and a slightly increased critical concentration. Disruption of the filaments and enhanced ATPase activity are reversed by the addition of KCl or the mushroom toxin phalloidin. By the large stabilizing effects of KCl and phalloidin on glutathionyl actin filaments we propose glutathionyl actin as a tool for detecting filament-stabilizing agents and for studying the different mechanisms of filament stabilization.

Stereochemical investigation of the phosphoketolase reaction. The formation of chiral [2H1,3H]acetyl phosphate.

1. D-(1R)-(1-2H1)[1-3H]Fructose 6-phosphate was prepared by the action of glucosephosphate isomerase on D-(1-2H)glucose 6-phosphate in tritiated water. Using the same enzyme but interchanging the labelling pattern of the substrate D-(1S)-(1-2H)[1-3H]fructose 6-phosphate was also obtained. 2. The doubly labelled samples of D-fructose 6-phosphate were converted into chiral acetylphosphate on phosphoketolase from Bifidium globosum (ATCC 25864), and these were reacted in situ with phosphotransacetylase and malate synthase in the presence of coenzyme A and glyoxylate. 3. The steric distribution of the tritium in the samples of malate, as shown by the action of fumarase, revealed that D-(1R)- and D-(1S)-(1-2H1)[1-3H]fructose 6-phosphate was converted into (R)- and (S)-(2H1)[3H]acetyl-phosphate, respectively. 4. The results are discussed in terms of the mechanism of action of phosphoketolase.

Changes in OD at 235 nm do not correspond to the polymerization step of actin.

Discrepancies were observed when the polymerization of rabbit muscle actin was monitored by delta OD235 and viscometry (eta). For example, in the presence of (beta,gamma)-methyleno ATP, the delta OD signal was as large as with ATP although polymerization was very poor (eta 1.1, compared with eta = 1.7 in the presence of ATP). Furthermore, when monomeric actin, kept for 1 h in the presence of a stoichiometric equivalent of ADP, was exposed to conditions favoring polymerization (addition of MgCl2), a considerable delta OD235 signal appeared, although the actin had completely lost its polymerizability (eta = 1.0). We conclude that the observed changes in OD235 cannot reflect polymerization itself, but must be caused by another reaction preceding the assembly. Under normal conditions, this reaction is supposed to be the slowest step of filament formation and so to determine the velocity of the whole process. In conclusion, monitoring of actin polymerization by delta OD235 is a valid method only when polymerization has been assessed by another, independent method.

Thiol group reactivity and polymerization of actin in the presence of ATP analogs.

We have investigated polymerization and the number of SH-groups of monomeric actin exposed in the presence of (beta, gamma)-substituted ATP-analogs. Actin, when depolymerized in a buffer containing 10 equiv. of APPCP exposes 4 thiol groups. The time course of the SH-titration is similar to that obtained when F-actin is depolymerized in a nucleotide free buffer. When actin is depolymerized in a buffer containing 10 equiv. of APPNP it also exposes 4 thiols. However, thiol-titration follows different kinetics. While one SH group reacts quickly the reaction of 3 others is retarded. We conclude that APPNP exhibits a shielding effect on part of the thiols for a period of time, while APPCP does not. In agreement with this, in the presence of APPNP yield of polymerization as well as stability against denaturation are distinctly higher than without added nucleotide or in the presence of APPCP. In line with this a hydrolysis product, most probably APPNH2, was associated with the filaments, as indicated by the replacement of tritiated ADP during polymerization, and from analysis of the attached nucleotide. Under the same conditions APPCP replaced tritiated ADP only to a small extent. The data indicate that APPNP interacts with monomeric actin much less than ATP and still less than ADP, but more so APPCP. APPNP is cleaved by actin ATPase and a hydrolysis product is incorporated into filaments.

Nucleotide in monomeric actin regulates the reactivity of the thiol groups.

A new thiol reagent, 2,4-dinitrophenyl glutathionyl disulfide, allowed the characterization of four thiol groups in monomeric actin by stoichiometric reaction. The number of thiol groups exposed to the reagent was found to depend on the nucleotide bound. In the absence of ATP, G-actin exposed four thiol groups ( G4s ). On the addition of ATP (1 equiv), three of them were shielded. The resulting actin with one thiol group exposed ( G1s ) is the form of monomeric actin normally produced by depolymerization of F-actin in buffers containing ATP. G1s is stable over hours, while G4s , i.e., monomeric actin in ATP-free solution, is not. This must be concluded from the fact that the shielding effect of thiol groups induced by addition of ATP was lost within ca. 30 min probably due to denaturation of G4s to G4s *. Therefore, denaturation of monomeric actin must be understood in terms of loss of thiol shielding, rather than by oxidation of the thiol groups. Addition of equimolar amounts of Ca2+ significantly retarded the denaturation process. ADP (50 equiv) shielded only ca. two of the four thiol groups but, similar to ATP, protected actin from denaturation. Three ATP analogues (10 equiv) were tested but had no shielding effect. In the presence of these analogues actin ( G4s ) rapidly denatured (to G4s *) as in the absence of added nucleotides. It was shown that the thiol-shielding activity and the protective capacity of a nucleotide are interrelated with its binding capability to monomeric actin. G1s was found to be polymerizable as was G approximately 2s on the addition of ATP. No polymerization could be detected for G4s or G4s *.(ABSTRACT TRUNCATED AT 250 WORDS)