Peter Dancker

Interaction of actin with phalloidin:: Polymerization and stabilization of F-actin

The cyclic peptide phalloidin, one of the toxic components of Amanita phalloides prevented the drop of viscosity of F-actin solutions after the addition of 0.6 M KI and inhibited the ATP splitting of F-actin during sonic vibration. The data concerning ATP splitting are consistent with the assumption (a) that only 1 out of every 3 actin units of the filaments needs to be combined with phalloidin in order to suppress the contribution of these 3 actins to the ATPase activity of the filament and (b) that all actin units of the filaments can combine with phalloidin with a very high affinity. -halloidin did not only stabilize the actin-actin bonds in the F-actin structure but it also increased the rate of polymerization of G-actin to F-actin. The ability of F-actin to activate myosin ATPase was not affected by phalloidin. The tropomyosin-troponin complex did not prevent the stabilizing effect of phalloidin on the F-actin structure.

Stabilization of F-actin by phalloidin reversal of the destabilizing effect of cytochalasin B

The cyclic peptide phalloidin, a toxic component of the mushroom Amanita phalloides prevents the reduction by KI of the high viscosity of F-actin and inhibits the ATPase activity of F-actin during ultrasonic vibration [ 1 ] . Cytochalasin B (CB), a metabolite of the fungus Helminthosporium dematioideum, on the other hand obviously weakens the F-actin structure. This can be concluded from the fact that CB inhibits cellular functions that are believed to be linked to actin-like microfilaments (for reviews see [2] and [3 ] ) and that it decreases the visosity of F-actin [4] . This communication describes that phalloidin is able to antagonize the weakening effect, which CB exerts on F-actin structure.

Dependence of actomyosin ATPase activity on ionic strength and its modification by thiol group substitution

ATP plays a dual role in muscular contraction: on the one hand, it is the substrate of an enzymatic interaction between actin and myosin providing the energy for contraction; on the other hand, it is able to dissociate actomyosin into its constituents actin and myosin, thus performing relaxation. Which of the two influences prevails depends most sensitively on the ionic strength. If it remains constant as in the sarcoplasm other factors must decide whether contraction or relaxation is induced. This communication presents evidence that modification of SH-groups of myosin activates actomyosin ATPase by reducing its sensitivity against ionic strength and, in addition, diminishes, under special conditions, the influence of the tropomyosin-troponin system which confers Ca-sensitivity on the contractile system [l] .

Dual effect of Ca2+ on ultrasonic ATPase activity and polymerization of muscle actin

Millimolar concentrations of Ca2+ stimulate actin polymerization whereas micromolar concentrations of Ca2+ depress polymerization. This latter effect leads to a reduction of ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity of actin during sonication at low Mg2+ concentrations and in the absence of KCl. In the presence of KCl (90 mM) there is activation of ATPase activity by micromolar Ca2+ concentrations. These Ca2+ effects are half-maximal at a Ca2+ concentration of 2-10(-7) M. They can be explained by assuming that that ATPase activity is optimal in a medium range of actin polymer stability and that micromolar Ca2+ concentrations tend to labilize and depolymerize F-actin.

Stabilization of actin filaments by ATP and inorganic phosphate.

Both inorganic orthophosphate and ATP stabilize actin filaments. This is reflected by a reduced nucleotide exchange and by a protection against filament breakdown by SDS or KI. When the filament-stabilizing effect of ATP was maximal, only about 15% of the actin subunits of the filament had bound one molecule of the nucleotide offered in the stabilizing solution.

Interaction of Actin and Myosin in the Absence and Presence of ATP

Abstract The binding between F-actin and myosin has been studied by analyzing the amount of radio-actively labelled (by means of 14C-NEM) F-actin and myosin in the formed unsoluble actomyosin pellet after centrifugation of the reaction mixture. In the absence of ATP, the amount of actin bound to myosin varies, depending on the amounts of actin and myosin present, between 0.18 mg actin/mg myosin (2 actin units per 1 myosin molecule) and about 2 mg actin/mg myosin (each actin fibril only uncompletely saturated with myosin). In 0.03 ᴍ KCl ATP decreases, if at all, the affinity of actin towards myosin only slightly; but less actin is bound to myosin in the presence of MgATP and in low ionic strength, indicating that myosin is now more densily distributed over fewer actin fibrils leaving the rest of fibrils free. For precipitation of the total amount of myosin (myosin alone is soluble in MgATP) incomplete saturation of myosin with actin suffices; obviously actin promotes filament formation of myosin. The activation of myosin ATPase by actin depends in a similar manner as actin binding does on actin concentration, hence the enzymatic interaction between actin and myosin is accompanied by true actin-myosin binding. An actin-tropomyosin-troponin preparation, whose reduced viscosity is lower than that of F-actin and which consists of about 30% actin, activates myosin ATPase to the same extent as F-actin does. It competes with F-actin for the same binding sites on myosin and can in the presence of MgATP be displaced from myosin by those preparations of F-actin which have a strong tendency to become fully saturated with myosin. The activation of myosin ATPase by actin-tropomyosin-troponin is reduced after tryptic digestion of actin-tropomyosin-troponin, which affects, according to SDS gel electro phoresis, mainly two components of troponin with molecular weights of about 32 000 and 25 000, respectively.

The binding of calcium and magnesium to actomyosin and its modification by natural troposmyosin

Summary1.Artificial actomyosin (reconstituted from its isolated components myosin and actin without natural tropomyosin) takes up ∼3 μmoles calcium/g actomyosin if one increases the concentration of free Ca++ ions from ∼10−9 M to 10−6 M.2.In the presence of magnesium, however, no calcium is taken up by artificial actomyosin.3.The results 1. and 2. are independent of the actin content of actomyosin; accordingly, they hold true for pure myosin too.4.Natural tropomyosin increases the calcium content of actomyosin independent of the free Ca++ at a constant amount; the concentration dependent increment remains in the same order as without tropomyosin.5.In the presence of natural tropomyosin the calcium increment is independent of the magnesium concentration. Magnesium, however, decreases the total amount of bound calcium at all concentrations of free Ca++.6.Bound calcium and bound magnesium are negatively correlated: The sum of bound calcium plus magnesium is 8 μmoles/g actomyosin.7.Consequence: it is myosin that binds the calcium that regulates muscular activity.

Dependence of actomyosin NTPase activity on ionic strength and its modification by α-actinin

In the foregoing communication [ 1 J evidence has been presented which shows that the enzymatic properties of actomyosin are influenced by modification of SH-groups of myosin. The present communication draws attention to the fact that analogous results as those obtained by SH-group modification can be demonstrated by treating actomyosin with the muscle protein cw-actinin. This protein was first described in [2-41. It promotes gelation of actin and superprecipitation of actomyosin and was recently shown to activate actomyosin ATPase [5] . Its functional role is until now unclear although Ebashi and Ebashi [3 J have already mentioned that it may counteract the dissociating influence of ATP on actomyosin. The common view is now that it is a constituent of the Z-line of the myotibril.

The competition between adenosine triphosphate and inorganic pyrophosphate for myosin and its suppression by substoichiometric actin concentrations

Inorganic pyrophosphate (PPi) inhibits not only Mg2+-ATPase activity of myosin subfragment 1 (S-1) but abolishes also the ATP-induced increment of tryptophan fluorescence of subfragment 1. At the concentrations used (25-50 micron ATP, 12 mm PPi) these effects of PPi were abolished by substoichiometric actin concentrations (approx. 0.1 microM actin vs. approx. 1 microM S-1), where ATPase activity was barely stimulated by actin.

Natural tropomyosin as a physiological inhibitor of the contractile activity of actomyosin

SummaryThe contractile ATPase of artificial actomyosin (reconstituted from its purified components actin and myosin) is fully active even at low concentrations (∼10−9 M) of ionized calcium. Natural tropomyosin, the physiological inhibitor, however, inhibits the contractile ATPase under these conditions. Natural tropomyosin which is not further purified by ammonium sulfate precipitation inhibits also at higher concentrations (10−6 M) of Ca++. 2.Natural tropomyosin which is prepared by ammonium sulfate precipitation inhibits at high concentrations of Ca++ if it is briefly digested by trypsin.3.Natural tropomyosin influences the interaction between actomyosin and MgATP in such a way that the affinity of actomyosin for MgATP increases, the maximal splitting rate, however, decreases. Hence, substrate inhibition can already be seen at concentrations that are otherwise optimal.4.Natural tropomyosin affects the ITPase of actomyosin in a similar manner if the inhibitory activity of tropomyosin is mediated by the concentration of free Ca++. The ITPase activity of the actomyosin-tropomyosin complex, however, is even at high concentrations of free Ca++ lower than the ITPase activity of pure actomyosin.