Kontraktionszyklus und Interaktion zwischen Aktin und l-Myosin unter der Wirkung spezifischer Interaktions-Inhibitoren

Contraction cycle and inhibitors of actin-L-myosin interaction 1. 1. The term interaction inhibitor describes such substances that never affect L-myosin induced ATP splitting but that, when in the presence of ATP, do inhibit the enzymic interaction between actin and L-myosin, i.e., they convert the Mg-activated actomyosin ATPase reversibly into L-myosin ATPase. 2. 2. Examples of interaction inhibitors are the relaxing factor, discovered by Marsh and Bendall, some polycations, polysulfonates, EDTA, and SCN- in low concentrations. 3. 3. It is shown for polythensulfonate, EDTA, Fuadin, and SCN- and confirmed for the relaxing factor that in the presence of ATP these substances inhibit reversibly the association of actin and L-myosin as well as their chemical interaction. 4. 4. For the fibrils in the water-glycerol extracted fibers this is demonstrated by measuring the resistance to stretching. 5. 5. For gels from natural actomyosin the same conclusion can be drawn, because the actin-component can be almost completely extracted from these gels by an ATP-containing polyethensulfonate solution. 6. 6. Contracted water-glycerol extracted fibers relax as soon as the actin and L-myosin filaments dissociate through the action of ATP and an interaction inhibitor. 7. 7. The dissociation of actin and L-myosin disappears if either the concentration of ATP or that of the interaction inhibitor is sufficiently lowered. 8. 8. In both cases contraction and ATP splitting by Mg-activated actomyosin ATPase occurs anew along with the reappearing association. 9. 9. In the presence of ATP and an interaction inhibitor relaxation even occurs if the Mg-activated actomysin ATPase is converted into the highly effective Ca-activated L-myosin ATPase instead of into the slightly effective Mg-activated L-myosin ATPase. Hence contraction is only produced when the mechanism of ATP splitting is based on the interaction between actin and L-myosin. Contraction is independent of ATP-splitting by L-myosin ATPase

Die erschöpfende Reinigung von Aktin-Präparaten Zahl und Art der phosphathaltigen prosthetischen Gruppen von G- und F-Aktin

Exhaustive purification of actin preparations. Number and kind of phosphate containing prosthetic groups of G- and F-actin 1. 1. Previous investigations on the nucleoside phosphate content of G- and F-actin have all been carried out with unpurified or little purified protein preparations. It has never been tested whether the purification was complete or whether the purification itself inactivated the preparation. 2. 2. In F-actin solutions prepared according to Straub the content of adenine decreases by repeated ultracentrifugal sedimentation or Mg-precipitation according to Bárány to a constant level which is identical with both methods (16 μmoles/g protein). The adenine and phosphate content of the actin remains constant after the second purification procedure. Both procedures remove protein impurities present in the crude extract. 3. 3. The protein impurities are, however, not removed by repeated isoelectric precipitation of the crude unpolymerized Straub-extract. This procedure removes only contaminating phosphate and nucleoside phosphate of the crude extract. The actin polymerizes spontaneously during isoelectric precipitation. 4. 4. Ultracentrifugal sedimentation of F-actin, precipitation by MgCl2 or isolectric precipitation in presence of ATP do not inactivate the actin. The viscosity of F-actin, the ability for activating the ATP-ase of added L-myosin and the ATP-sensitivity of the resulting actomyosin remain constant even after repeating the purification procedure five times. 5. 5. Repeated isoelectric precipitation of actin in absence of ATP leads to an increasing loss of adenine phosphate and also to a stepwise decrease of Zν and ATP-sensitivity. 6. 6. In the nucleoside phosphate of purified F-actin the proportion of adenine to phosphate is 1:2 as in ADP. Paperchromatographic methods reveal in addition traces of AMP and ATP. 7. 7. G-actin and the contaminating proteins in the crude extracts contain also 16 μmoles adenine/g protein. 8. 8. From the content of adenosine phosphates bound to G- or F-actin (16 μmoles/g protein) the minimal molecular weight of the actin monomer is calculated as 62.000. 9. 9. The proportion adenine: phosphate and paperchromatographic methods show, that in the crude unpolymerized extract the protein-bound nucleoside-phosphate consists of 70–75% ATP and 25–30% ADP. Only 70–75% of the protein in the crude extract are able to polymerize. 10. 10. However, G-actin obtained from purified F-actin containing also 70–75% of its nucleoside phosphates as ATP does polymerize entirely. Thus whether or not ADP-G-actin polymerizes seems to depend on the history of the protein preparation. 11. 11. G-actin, whose ability for polymerization has been destroyed by X-rays, nevertheless activates the L-myosin-ATP-ase to a normal extent. The same holds for actin partly denatured by isoelectric precipitation in the absence of ATP. Thus, the ability of actin for polymerisation and its ability to activate the L-myosin-ATP-ase are independent properties. 12. 12. In phosphate or ATP containing solutions, purified F-actin ATP and inorganic phosphate reversibly (in addition to the tightly bound ADP). Howeever, actin is not phosphorylated in presence of ATP