Single actin filaments undergoing brownian movement in two dimensions were observed at 20 degrees C in fluorescence optical video microscopy. The persistence length (Lp) was derived from the analysis of either the cosine correlation function or the average transverse fluctuations of a series of recorded shapes of filaments assembled from rhodamine-action. Phalloidin-stabilized filaments had a persistence length of 18 +/- 1 micron, in agreement with recent observations. In the absence of phalloidin, rhodamine-labeled filaments could be observed under a variety of solution conditions once diluted in free unlabeled G-actin at the appropriate critical concentration. Such nonstabilized F-ADP-actin filaments had the same Lp of 9 +/- 0.5 microns, whether they had been assembled from ATP-G-actin or from ADP-G-actin, and independently of the tightly bound divalent metal ion. In the presence of BeF3-, which mimics the gamma-phosphate of ATP, F-ADP-BeF3-actin was appreciably more rigid, with Lp = 13.5 microns. Hence, newly formed F-ADP-Pi-actin filaments are more rigid than "old" F-ADP-actin filaments, a fact which has implications in actin-based motility processes. In the presence of skeletal tropomyosin and troponin, filaments were rigid (Lp = 20 +/- 1 micron) in the off state (-Ca2+), and flexible (Lp = 12 microns) in the on state (+Ca2+), consistent with the steric blocking model. In agreement with x-ray diffraction data, no appreciable difference was recorded between the off and on states using smooth muscle tropomyosin and caldesmon (Lp = 20 +/- 1 micron). In conclusion, this method allows accurate measurement of small (< or = 15%) changes in mechanical properties of actin filaments in correlation with their biological functions.

The topography of the rigor complex between F-actin and myosin heads (S1) has been investigated by carbodiimide zero-length cross-linking. The results demonstrate for the first time that the 95,000-molecular weight (95K) heavy chain of the myosin head enters into van der Waals contact with two neighbouring actin monomers; one is bound to the 50K domain and the other to the 20K domain of the myosin chain. The covalent F-actin-S1 complex can be isolated; it shows a vastly elevated Mg2+-ATPase. Each pair of actin subunits in the thin filament seems to act as a functional unit for specific binding of a myosin head and stimulation of its Mg2+-ATPase activity.

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1. Phenylglyoxal reacts rapidly with isolated myosin heads (subfragment 1) and induces two successive and distinguishable effects on their enzymic properties : first, a twofold activation of the Ca2+ and Mg2+-dependent ATPases with no effect on the K+-ATPase followed by inhibition of the K + , Ca2 + and actin-activated Mg2 +-ATPases. A specific protein-reagent complex is formed during the second phase of the modification reaction (Ki z 5 x lop3 M).2. ADP and ATP with or without cations provide efficient protection only against the loss of ATPase activities, suggesting that the second inhibitory process is occurring at or close to the active site.3. On the basis of ['4C]phenylglyoxal-labelling experiments and the composition of modified subfragment-1 derivatives, it is demonstrated that the sequential modification of two reactive arginyl residues is responsible for the observed activation-inhibition phenomena. Blocking of the first reactive residue produces a shift in the pH/activity curves related to the Ca2+ and Mg2+-dependent ATPases with an apparent activation effect. Modification of the second guanidino group does not destroy the affinity of the protein for the nucleotide substrates but does alter the nucleotide binding site as reflected in the inability of Mg2+. ATP to dissociate the modified subfragment-1 -actin complex. It is concluded that electrostatic interactions between this positively charged group and the negatively charged ATP and ADP molecules may be critical for the hydrolytic efficiency of myosin heads.4. After dissociation and separation of the polypeptide constituents of the protein in acetic acid medium, both labelled sites are found to reside in the heavy chain.During muscle contraction, myosin catalyses, in the presence of F-actin, the hydrolysis of ATP. While the kinetic features of this important biological reaction are well advanced [1,2], little is known about the identity and chemical reactivity of the functional groups involved in the hydrolytic action of myosin. In particular, the participation of the well-studied cysteinyl side-chains called SH1 and SH2 [3], which were thought to play a central role in myosin [4], appears now highly improbable [5]. As part of an effort initiated to elucidate the relationship between the chemical structure and enzymatic function of skeletal muscle myosin [6], we have investigated the chemical modification of arginyl residues in an attempt to assess their contributions to the ATPase reaction and to the association properties of myosin with F-actin. In this work we describe the interaction of the a-dicarbonyl reagent, phenylglyoxal, with isolated myosin heads which reveals the particular chemical reactivity of two arginyl groups; one of them appears as a functional residue located at the ATPase active site. EXPERIMENTAL PROCEDURE MaterialsRabbit skeletal myosin was prepared according to Offer [7] and stored in 50% glycerol at -30°C; subfragment 1 was prepared by digestion of myosin filaments [8,9] with chymotrypsin (Worthington) which has been pretr...