Cooperative dependence of the actin-activated Mg2+-ATPase activity of Acanthamoeba myosin II on the extent of filament phosphorylation

The actin-activated Mg2+-ATPase of myosin II from Acanthamoeba castellanii is regulated by phosphorylation of 3 serine residues at the tip of the tail of each of its two heavy chains; only dephosphorylated myosin II is active, whereas the phosphorylated and dephosphorylated forms have identical Ca2+-ATPase activities and Mg2+-ATPase activities in the absence of F-actin. We have now chemically modified phosphorylated and dephosphorylated myosin II with N-ethylmaleimide (NEM). The modification occurred principally at a single site within the NH2-terminal 73,000 Da of the globular head of the heavy chain. NEM-myosin II bound to F-actin and formed filaments normally, but the Ca2+- and Mg2+-ATPase activities of phosphorylated and dephosphorylated myosin II and the actin-activated Mg2+-ATPase activity of NEM-dephosphorylated myosin II were inhibited. Only filamentous myosin II has actin-activated Mg2+-ATPase activity. Native phosphorylated myosin II acquired actin-activated Mg2+-ATPase activity when it was co-polymerized with NEM-inactivated dephosphorylated myosin II, and the increase in its activity was cooperatively dependent on the fraction of NEM-dephosphorylated myosin II in the filaments. From this result, we conclude that the specific activity of each molecule within a filament is independent of its own state of phosphorylation, but is highly cooperatively dependent upon the state of phosphorylation of the filament as a whole. This enables the actin-activated Mg2+-ATPase activity of myosin II filaments to respond rapidly and extensively to small changes in the level of their phosphorylation.     

Functional consequences of the proteolytic removal of regulatory serines from the nonhelical tailpiece of Acanthamoeba myosin II

The actin-activated Mg2(+)-ATPase activity of myosin II from Acanthamoeba castellanii is regulated by phosphorylation of 3 serines in its 29-residue, nonhelical, COOH-terminal tailpiece, i.e., serines-1489, -1494, and -1499 or, in reverse order, residues 11, 16, and 21 from the COOH terminus. To investigate the essential requirements for regulation, myosin II filaments in the presence of F-actin were digested by arginine-specific submaxillary gland protease. Two-dimensional peptide mapping of purified, cleaved myosin II showed that the two most terminal phosphorylation sites, serines-1494 and -1499, had been removed. Cleaved dephosphorylated myosin II retained full actin-activated Mg2(+)-ATPase activity (with no change in Vmax or Kapp) and the ability to form filaments similar to those of the native enzyme. However, higher Mg2+ concentrations were required for both filament formation and maximal ATPase activity. The one remaining regulatory serine in the cleaved myosin II was phosphorylatable by myosin II heavy-chain kinase, and phosphorylation inactivated the actin-activated Mg2(+)-ATPase activity, as in the case of the native myosin II. Also as in the case of the native myosin II, phosphorylated cleaved myosin II inhibited the actin-activated Mg2(+)-ATPase activity of dephosphorylated cleaved myosin II when the two were copolymerized. These results suggest that at least 18 of the 29 residues in the nonhelical tailpiece of the heavy chain are not required for either actin-activated Mg2(+)-ATPase activity or filament formation and that phosphorylation of Ser-1489 is sufficient to regulate the actin-activated Mg2(+)-ATPase activity of myosin II.     

The purification and characterization of a globular subfragment of Acanthamoeba myosin II that is fully active when cross-linked to F-actin

Acanthamoeba myosin II contains two heavy chains of Mr 185,000 and two pairs of light chains of Mr 17,500 and 17,000. We now report the purification of a globular proteolytic 103-kDa subfragment of myosin II which contained a 68-kDa NH2-terminal segment of the heavy chain and one pair of intact light chains. The myosin II head fragment expressed full Ca2+-ATPase activity but its actin-activated Mg2+-ATPase activity had a Vmax of only 0.07 s-1 compared to 1.9 s-1 (per head) for filaments of native unphosphorylated myosin II. The head fragment had a similar KATPase to that of filaments (5 versus 4 microM) and about 75% of the head fraction could bind to F-actin in the presence of ATP with a Kbinding of 5.6 microM. The Kbinding of the head fragment may be similar to that of individual heads in the native myosin II filaments although the experimentally determined apparent Kbinding for filaments is much lower, 0.3 microM. The head fragment was covalently cross-linked to F-actin in the absence of nucleotide using the zero length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The cross-linked actin-myosin head complex hydrolyzed MgATP at a rate equivalent to Vmax for the active dephosphorylated native myosin II. These data indicate that the isolated head fragment had intact catalytic and actin-binding domains but that it bound to F-actin in the presence of ATP in a relatively inactive conformation. When covalently cross-linked to F-actin the head fragment was apparently locked into a catalytically fully active conformation.     

Regulation of the actin-activated ATPase activity of Acanthamoeba myosin II by copolymerization with phosphorylated and dephosphorylated peptides derived from the carboxyl-terminal end of the heavy chain

Myosin II from Acanthamoeba castellanii is a conventional myosin composed of two heavy chains and two pairs of light chains. The amino-terminal approximately 90 kDa of each heavy chain form a globular head that contains the ATPase site and an ATP-sensitive actin-binding site. The carboxyl-terminal approximately 80 kDa of both heavy chains interact to form a coiled coil, helical rod (through which the molecules self-associate into bipolar filaments) ending in a short nonhelical tailpiece. Phosphorylation of 3 serine residues at the tip of the tail (at positions 11, 16, and 21 from the carboxyl terminus) inactivates the actin-activated Mg2(+)-ATPase activity of myosin II filaments. Previous work had indicated that the activity of each myosin II molecule in a filament reflects the global state of phosphorylation of the filament rather than the phosphorylation state of the molecule itself. We have now purified the approximately 28-kDa carboxyl-terminal region of the heavy chain lacking the last two phosphorylation sites, and we have shown that this peptide copolymerizes with and regulates the actin-activated Mg2(+)-ATPase activities of native dephosphorylated and phosphorylated myosin II. It can be concluded from these studies that the biologically relevant enzymatic activity of myosin II is regulated by a phosphorylation-dependent conformational change in the myosin filaments.     

Purification and characterization of a third isoform of myosin I from Acanthamoeba castellanii

A third isoform of myosin I has been isolated from Acanthamoeba and designated myosin IC. Peptide maps and immunoassays indicate that myosin IC is not a modified form of myosin IA, IB, or II. However, myosin IC has most of the distinctive properties of a myosin I. It is a globular protein of native Mr approximately 162,000, apparently composed of a single 130-kDa heavy chain and a pair of 14-kDa light chains. It is soluble in MgATP at low ionic strength, conditions favoring filament assembly by myosin II. Myosin IC has high Ca2+- and (K+,EDTA)-ATPase activities. Its low Mg2+-ATPase activity is stimulated to a maximum rate of 20 s-1 by the addition of F-actin if its heavy chain has been phosphorylated by myosin I heavy chain kinase. The dependence of the Mg2+-ATPase activity of myosin IC on F-actin concentration is triphasic; and, at fixed concentrations of F-action, this activity increases cooperatively as the concentration of myosin IC is increased. These unusual kinetics were first demonstrated for myosins IA and IB and shown to be due to the presence of two actin-binding sites on each heavy chain which enable those myosins I to cross-link actin filaments. Myosin IC is also capable of cross-linking F-actin, which, together with the kinetics of its actin-activated Mg2+-ATPase activity, suggests that it, like myosins IA and IB, possesses two independent actin-binding domains.     

Effects of limited tryptic cleavage on the physical and enzymatic properties of myosin II from Acanthamoeba castellanii

Limited digestion of Acanthamoeba myosin II by trypsin selectively cleaved the 185,000-Da heavy chains into a 73,000-Da peptide containing the catalytic and actin-binding sites and a 112,000-Da peptide containing the regulatory phosphorylatable sites. The light chains were unaffected. The proteolytic products remained associated and formed bipolar filaments that were very similar in appearance to filaments of native myosin by negative staining electron microscopy. Filaments of trypsin-cleaved, dephosphorylated myosin, however, had a smaller sedimentation coefficient than filaments of native dephosphorylated myosin. Trypsin-cleaved dephosphorylated myosin retained complete Ca2+-ATPase activity but had no actin-activated ATPase activity under conditions that are optimal for native, dephosphorylated myosin (pH 7.0, 4 mM MgCl2, 30 degrees C or pH 6.4, 1 mM MgCl2, 30 degrees C). Trypsin-cleaved dephosphorylated myosin had higher actin-activated ATPase activity at pH 6.0 and 1 mM MgCl2 than undigested dephosphorylated myosin which is appreciably inhibited under these conditions. Trypsin-cleaved, dephosphorylated myosin inhibited the actin-activated ATPase activity of native, dephosphorylated myosin when both were present in the same co-polymers, when enzymatic activity was assayed at pH 7.0, 4 mM MgCl2, and 30 degrees C, but this inhibition was overcome by raising the MgCl2 to 6 mM. These results provide additional evidence that regulation of acanthamoeba myosin II occurs at the filament level and that, under most conditions of assay, the heavy chains must be intact and the regulatory serines unphosphorylated for actin-activated ATPase activity to be maximally expressed.     

Localization of the actin-binding sites of Acanthamoeba myosin IB and effect of limited proteolysis on its actin-activated Mg2+-ATPase activity

Acanthamoeba myosin IB contains a 125-kDa heavy chain that has high actin-activated Mg2+-ATPase activity when 1 serine residue is phosphorylated. The heavy chain contains two F-actin-binding sites, one associated with the catalytic site and a second which allows myosin IB to cross-link actin filaments but has no direct effect on catalytic activity. Tryptic digestion of the heavy chain initially produces an NH2-terminal 62-kDa peptide that contains the ATP-binding site and the regulatory phosphorylation site, and a COOH-terminal 68-kDa peptide. F-actin, in the absence of ATP, protects this site and tryptic cleavage then produces an NH2-terminal 80-kDa peptide. Both the 62- and the 80-kDa peptides retain the (NH+4,EDTA)-ATPase activity of native myosin IB and both bind to F-actin in an ATP-sensitive manner. However, only the 80-kDa peptide retains a major portion of the actin-activated Mg2+-ATPase activity. This activity requires phosphorylation of the 80-kDa peptide by myosin I heavy chain kinase but, in contrast to the activity of intact myosin IB, it has a simple, hyperbolic dependence on the concentration of F-actin. Also unlike myosin IB, the 80-kDa peptide cannot cross-link F-actin filaments indicating the presence of only a single actin-binding site. These results allow the assignment of the actin-binding site involved in catalytic activity to the region near, and possibly on both sides of, the tryptic cleavage site 62 kDa from the NH2 terminus, and the second actin-binding site to the COOH-terminal 45-kDa domain. Thus, the NH2-terminal 80 kDa of the myosin IB heavy chain is functionally similar to the 93-kDa subfragment 1 of muscle myosin and most likely has a similar organization of functional domains.     

Nonlinear dependence of actin-activated Mg2+-ATPase activity on the extent of phosphorylation of gizzard myosin and H-meromyosin

1. The actin-activated Mg2+-ATPase activity of gizzard HMM increased in proportion to the square of the extent of LC phosphorylation. This result indicates that the LCs of HMM are randomly phosphorylated, and the phosphorylation of both heads of HMM is required for the activation of HMM Mg2+-ATPase by F-actin. 2. In 75 mM KCl, the Mg2+-ATPase activity of gizzard myosin was activated by F-actin only slightly when a half of the total LC was phosphorylated. From 1 to 2 mol LC phosphorylation, the activity was enhanced by F-actin almost linearly. In 30 mM KCl, the activity of acto-gizzard myosin increased sigmoidally with increase in the extent of LC phosphorylation. On electron microscopy, side-by-side aggregates of myosin filaments were observed in 30 mM KCl, but not in 75 mM KCl. It was suggested that the activation of the Mg2+-ATPase activity of acto-gizzard myosin LC phosphorylation is modified by formation of myosin filaments and their aggregates. 3. The relationship between the actin-activated Mg2+-ATPase activity of HMM or myosin and the extent of LC phosphorylation was unaffected by tropomyosin.     

Requirement of phosphorylation of Physarum myosin heavy chain for thick filament formation, actin activation of Mg2+-ATPase activity, and Ca2+-inhibitory superprecipitation

In an attempt to elucidate the Ca2+-regulated mechanism of motility in Physarum plasmodia, we improved the preparation method for myosin B and pure myosin. The obtained results are as follows: 1. We obtained two types of myosin B which are distinguishable from each other with respect to their sensitivity to Ca2+. The inactive type of myosin B had low superprecipitation activities both in the presence and in the absence of Ca2+. The active type showed very high superprecipitation activity in EGTA, and the activity was conspicuously inhibited by Ca2+. The active type was converted into the inactive type by treatment with potato acid phosphatase. Also the inactive type or the phosphatase-treated active type was converted into the active type upon reacting with ATP-gamma-S. 2. In the reaction with ATP-gamma-S, only the myosin HC of myosin B was phosphorylated. The phosphorylation was independent of Ca2+ and calmodulin, and the extent was about 1 mol/mol HC. 3. The Ca2+ sensitivity in the superprecipitation of the active type was not decreased by adding an excess amount of F-actin. Besides, the actin-activated Mg2+-ATPase activity of purified phosphorylated myosin was not Ca2+-sensitive. Therefore, presence of a Ca2+-dependent inhibitory factor(s) that could bind to myosin was suggested. 4. The Mg2+-ATPase activity of purified phosphorylated myosin was 7-8 times enhanced by F-actin, but that of dephosphorylated myosin was hardly activated at all. 5. In a gel filtration in 0.5 M KCl, phosphorylated myosin was eluted behind dephosphorylated myosin. Electron microscopy applying the rotary-shadow method showed significant difference in flexibility in the tail between phosphorylated and dephosphorylated myosin molecules. 6. In 40 mM KCl and 5-10 mM MgCl2, phosphorylated myosin formed thick filaments, but dephosphorylated myosin did not, whether there was ATP or not. The above results clearly show that the phosphorylation of myosin HC is indispensable to ATP-induced superprecipitation, the actin-activated Mg2+-ATPase activity, and the formation of thick filaments of myosin. A myosin-linked factor(s) that inhibits an actin-myosin interaction in a Ca2+-dependent manner may exist.     

Regulation of the actin-activated ATPase and in vitro motility activities of monomeric and filamentous Acanthamoeba myosin II.

The actin-activated Mg(2+)-ATPase activity of filamentous Acanthamoeba myosin II is inhibited by phosphorylation of 3 serine residues at the tip of the tail of each heavy chain. From previous studies, it had been concluded that the activity of each molecule in the filament was regulated by the global state of phosphorylation of the filament and was independent of its own phosphorylation state. The actin-activated Mg(2+)-ATPase activity of monomeric phosphorylated myosin II was not known because it polymerizes under the ionic conditions necessary for the expression of this activity. We have now found conditions to maintain myosin II monomeric and active during the enzyme assay. The actin-activated Mg(2+)-ATPase activities of monomeric dephosphorylated and phosphorylated myosin II were found to be the same as the activity of filamentous dephosphorylated myosin II. These results support the conclusion that phosphorylation regulates filamentous myosin II by affecting filament conformation. Consistent with their equivalent enzymatic activities, monomeric and filamentous dephosphorylated myosin II were equally active in an in vitro motility assay in which myosin adsorbed to a surface drives the movement of F-actin. In contrast to their very different enzymatic activities, however, filamentous and monomeric phosphorylated myosin II had similar activities in the in vitro motility assay; both were much less active than monomeric and filamentous dephosphorylated myosin II. One interpretation of these results is that the rate-limiting steps in the two assays are different and that, while the rate-limiting step for actin-activated Mg(2+)-ATPase activity is regulated only at the level of the filament, the rate-limiting step for motility can also be regulated at the level of the monomer.     

ATPase activities and actin-binding properties of subfragments of Acanthamoeba myosin IA

Previous studies had led to the conclusion that the globular, single-headed myosins IA and IB from Acanthamoeba castellanii contain two actin-binding sites: one associated with the catalytic site and whose binding to F-actin activates the Mg2+-ATPase activity and a second site whose binding results in the cross-linking of actin filaments and makes the actin-activated ATPase activity positively cooperative with respect to myosin I concentration. We have now prepared a 100,000-Da NH2-terminal peptide and a 30,000-Da COOH-terminal peptide by alpha-chymotryptic digestion of the myosin IA heavy chain. The intact 17,000-Da light chain remained associated with the 100,000-Da fragment, which also contained the serine residue that must be phosphorylated for expression of actin-activated ATPase activity by native myosin IA. The 30,000-Da peptide, which contained 34% glycine and 21% proline, bound to F-actin with a KD less than 0.5 microM in the presence or absence of ATP but had no ATPase activity. The 100,000-Da peptide bound to F-actin with KD = 0.4-0.8 microM in the presence of 2 mM MgATP and KD less than 0.01 microM in the absence of MgATP. In contrast to native myosin IA, neither peptide cross-linked actin filaments. The phosphorylated 100,000-Da peptide had actin-activated ATPase activity with the same Vmax as that of native phosphorylated myosin IA but this activity displayed simple, noncooperative hyperbolic dependence on the actin concentration in contrast to the complex cooperative kinetics observed with native myosin IA. These results provide direct experimental evidence for the presence of two actin-binding sites on myosin IA, as was suggested by enzyme kinetic and filament cross-linking data, and also for the previously proposed mechanism by which monomeric myosins I could support contractile activities.     

Inhibition of Acanthamoeba myosin I heavy chain kinase by Ca(2+)-calmodulin.

The actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosins I depends on phosphorylation of their single heavy chains by myosin I heavy chain kinase. Kinase activity is enhanced > 50-fold by autophosphorylation at multiple sites. The rate of kinase autophosphorylation is increased approximately 20-fold by acidic phospholipids independent of the presence of Ca2+ and diglycerides. We show in this paper that Ca(2+)-calmodulin inhibits phospholipid-stimulated autophosphorylation of myosin I heavy chain kinase and hence also inhibits the catalytic activity of unphosphorylated kinase in the presence of phospholipid. Ca(2+)-calmodulin does not inhibit kinase activity in the absence of phospholipid. Micromolar Ca(2+)-calmodulin also inhibits binding of myosin I heavy chain kinase to phospholipid vesicles and purified plasma membranes. Proteolytic removal of a 7-kDa NH2-terminal segment from the 97-kDa kinase prevents binding of both calmodulin and phospholipid; therefore, we propose that they bind to the same or overlapping sites. These data provide a mechanism by which Ca2+ could inhibit the actin-activated Mg(2+)-ATPase activity of the myosin I isozymes in vivo and thus regulate myosin I-dependent motile activities.     

Identification of three phosphorylation sites on each heavy chain of Acanthamoeba myosin II

It has been previously demonstrated that the actin-activated Mg2+-ATPase activity of Acanthamoeba myosin II is inhibited by phosphorylation of its two heavy chains (Collins, J. H., and Korn, E. D. (1980) J. Biol. Chem. 255, 8011-8014). In this paper, it is shown that a partially purified kinase preparation from Acanthamoeba catalyzes the incorporation of 3 mol of phosphate into each mole of myosin II heavy chain. Tryptic digestion of the 32P-myosin, followed by two-dimensional peptide mapping, indicates that two of the three sites phosphorylated by the kinase in vitro correspond to the two major phosphorylation sites on the myosin heavy chain in vivo. Phosphorylation of myosin II in vitro by the kinase fraction completely inhibits the actin-activated Mg2+-ATPase activity of myosin II. Myosin II can be isolated in a highly phosphorylated, enzymatically inactive form, then dephosphorylated to an active form, and finally rephosphorylated to an inactive form. The Acanthamoeba kinase fraction catalyzes the phosphorylation of all three sites on the heavy chain of myosin II at virtually the same rate. From a comparison of the decrease in actin-activated Mg2+-ATPase activity with the amount of phosphate incorporated into myosin II, and from the results obtained previously by dephosphorylating myosin II (Collins, J. H., and Korn, E. D., (1980) J. Biol. Chem. 255, 8011-8014), it can be inferred that two of the sites phosphorylated in vitro act in a synergistic manner to inhibit the actin-activated myosin II Mg2+-ATPase.     

Acanthamoeba cofactor protein is a heavy chain kinase required for actin activation of the Mg2+-ATPase activity of Acanthamoeba myosin I.

We have purified a cofactor protein previously shown (Pollard, T. D., and Korn, E. D. (1973) J. Biol. Chem. 248, 4691-4697) to be required for actin activation of the Mg2+-ATPase activity of Acanthamoeba myosin I. The purified cofactor protein is a novel myosin kinase that phosphorylates the single heavy chain, but neither of the two light chains, of Acanthamoeba myosin I. Phosphorylation of Acanthamoeba myosin I by the purified cofactor protein requires ATP and Mg2+ but is Ca2+-independent. The Mg2+-ATPase activity of phosphorylated Acanthamoeba myosin I is highly activated by F-actin in the absence of cofactor protein. Actin-activated Mg2+-ATPase activity is lost when phosphorylated Acanthamoeba myosin I is dephosphorylated by platelet phosphatase. Phosphorylation and dephosphorylation have no effect on the (K+,EDTA)-ATPase and Ca2+-ATPase activities of Acanthamoeba myosin I. These results show that cofactor protein is an Acanthamoeba myosin I heavy chain kinase and that phosphorylation of the heavy chain of this myosin is required for actin activation of its Mg2+-ATPase activity.     

Purification and characterization of a myosin heavy chain kinase from Dictyostelium discoideum

A Dictyostelium discoideum myosin heavy chain kinase has been purified 14,000-fold to near homogeneity. The enzyme has a Mr = 130,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and greater than 700,000 as determined by gel filtration on Bio-Gel A-1.5m. The enzyme has a specific activity of 1 mumol/min X mg when assayed at a Dictyostelium myosin concentration of 0.3 mg/ml. A maximum of 2 mol of phosphate/mol of myosin is incorporated by the kinase, and the phosphorylated amino acid is threonine. Phosphate is incorporated only into the myosin heavy chains, not into the light chains. The actin-activated Mg2+-ATPase of Dictyostelium myosin is inhibited 70-80% following maximal phosphorylation with the kinase. The myosin heavy chain kinase requires 1-2 mM Mg2+ for activity and is most active at pH 7.0-7.5. The activity of the enzyme is not significantly altered by the presence of Ca2+, Ca2+ and calmodulin, EGTA, cAMP, or cGMP. When incubated with Mg2+ and ATP, phosphate is incorporated into the myosin heavy chain kinase, perhaps by autophosphorylation.     

A kinetic model for the molecular basis of the contractile activity of Acanthamoeba myosins IA and IB

Acanthamoeba myosins IA and IB are single-headed, monomeric molecules consisting of one heavy chain and one light chain. Both have high actin-activated Mg2+-ATPase activity, when the heavy chain is phosphorylated, but neither seems to be able to form the bipolar filaments that are generally thought to be required for actomyosin-dependent contractility. In this paper, we show that, at fixed F-actin concentration, the actin-activated Mg2+-ATPase activities of myosins IA and IB increase about 5-fold in specific activity in a cooperative manner as the myosin concentration is increased. The myosin concentration range over which this cooperative change occurs depends on the actin concentration. More myosin I is required for the cooperative increase in activity at high concentrations of F-actin. The cooperative increase in specific activity at limiting actin concentrations is caused by a decrease in the KATPase for F-actin. The high and low KATPase states of the myosin have about the same Vmax at infinite actin concentration. Both myosins are completely bound to the F-actin long before the Vmax values are reached. Therefore, much of the actin activation must be the result of interactions between F-actin and actomyosin. These kinetic data can be explained by a model in which the cooperative shift of myosin I from the high KATPase to the low KATPase state results from the cross-linking of actin filaments by myosin I. Cross-linking might occur either through two actin-binding sites on a single molecule or by dimers or oligomers of myosin I induced to form by the interaction of myosin I monomers with the actin filaments. The ability of Acanthamoeba myosins IA and IB to cross-link actin filaments is demonstrated in the accompanying paper (Fujisaki, H., Albanesi, J.P., and Korn, E.D. (1985) J. Biol. Chem. 260, 11183-11189).     

Supramolecular regulation of the actin-activated ATPase activity of filaments of Acanthamoeba Myosin II

Acanthamoeba myosin II has three phosphorylation sites clustered near the end of the tail of each of its two heavy chains (six phosphorylation sites/molecule). Myosin II has little or no actin-activated ATPase activity when four to six of these sites are phosphorylated. Maximal actin-activated ATPase activity is obtained when all six sites are dephosphorylated. Under assay conditions, both phosphorylated and dephosphorylated myosin II form bipolar filaments. Filaments of dephosphorylated myosin II have larger sedimentation coefficients than filaments of phosphorylated myosin II but this difference does not explain the difference in their actin-activated ATPase activities. Heteropolymers, formed by mixing soluble dephosphorylated and phosphorylated myosins and then diluting the mixture into low ionic strength buffer containing MgCl2, have sedimentation coefficients close to those of the homopolymer of phosphorylated myosin. The actin-activated ATPase activities of heteropolymers are, under most conditions, lower than the equivalent mixtures of homopolymers of dephosphorylated and phosphorylated myosins. It is concluded, therefore, that the phosphorylation of myosin tails regulates the actin-activated ATPase activity of Acanthamoeba myosin II by affecting the myosin filament as a whole rather than specifically affecting the heads of the phosphorylated myosin molecules only.     

Ricin B chain converts a non-cytotoxic antibody-ricin A chain conjugate into a potent and specific cytotoxic agent

A method is described for the preparation of partially and fully phosphorylated chicken gizzard myosin. When fully phosphorylated it possessed an actin-activated Mg2+-ATPase of similar specific activity to that of mammalian skeletal muscle myosin. The Mg2+-ATPase activity of these preparations was related in a non-linear fashion to increasing phosphorylation of the P light chain. When P light chain phosphorylation occurred during enzymic assay the Mg2+-ATPase activity remained constant. Fully phosphorylated preparations of gizzard myosin possessed an actin-activated Mg2+-ATPase that was not Ca2+-sensitive, whereas the Mg2+-ATPase of partially phosphorylated myosin preparations was Ca2+-sensitive.     

Physical, enzymatic, and contractile properties of brain myosin with anti-brain myosin Fab fragment bound on its tail

An antibody obtained by immunizing a rabbit with purified bovine brain myosin was found to react with the tail portion of the myosin heavy chain. An Fab fragment obtained by limited papain digestion of the antibody was allowed to bind to brain myosin, and the complex of the Fab fragment and brain myosin (Fab-myosin) was isolated. On examination of the rotary-shadowed Fab-myosin by electron microscopy, most of the Fab fragment was located on the middle to C-terminal regions of the tails of the myosin molecules. The solubility of Fab-myosin in low salt solutions was higher than that of control brain myosin. Fab-myosin was found to form small irregular aggregates in low salt solutions instead of regular bipolar filaments, and the relative population of the monomeric form of myosin molecules observed for the Fab-myosin was much larger than that observed for the control myosin. The actin-activated Mg2+-ATPase activity of Fab-myosin was stimulated two- to threefold by phosphorylation of the light chains with myosin light chain kinase, as observed for the control brain myosin. Furthermore, the levels of the ATPase activity of the phosphorylated and dephosphorylated Fab-myosins were similar to those of the phosphorylated and dephosphorylated control myosins, respectively. The superprecipitation activity of Fab-myosin was also highly dependent on phosphorylation of the light chains. Although control brain myosin formed a large superprecipitate network which contracted to a dense particle, Fab-myosin generated only numerous tiny superprecipitates under the same conditions. From these results it was deduced that a regular filamentous state of brain myosin was not prerequisite for its actin-activated Mg2+-ATPase and superprecipitation activities but was indispensable for the formation of a large and well contractible superprecipitate.     

Inhibition of conformational change in smooth muscle myosin by a monoclonal antibody against the 17-kDa light chain

Monoclonal antibodies against gizzard smooth muscle myosin were generated and characterized. One of these antibodies, designated MM-2, recognized the 17-kDa light chain and modulated the ATPase activities and hydrodynamic properties of smooth muscle myosin. Rotary shadowing electron microscopy showed that MM-2 binds 51 (+/- 25) A from the head-rod junction. The depression of Ca2+- and Mg2+-ATPase activities of myosin and Ca2+-ATPase activity of heavy meromyosin at low KCl concentration were abolished by MM-2. Viscosity measurement indicated that MM-2 inhibits the transition of 6 S myosin to 10 S myosin. While the rate of the production of subfragment-1 by papain proteolysis of 6 S myosin was inhibited by MM-2, the rate of proteolysis of the heavy chain of 10 S myosin was enhanced by MM-2 and reached the same rate as that of 6 S myosin plus MM-2. These results suggest that MM-2 inhibits the formation of 10 S myosin by binding to the 17-kDa light chain which is localized at the head-neck region of the myosin molecule. MM-2 increased the Vmax of actin-activated Mg2+-ATPase activities of both dephosphorylated myosin and dephosphorylated heavy meromyosin about 10- and 20-fold, respectively. MM-2 also activated the actin-activated Mg2+-ATPase activity of phosphorylated myosin at a low MgCl2 concentration and thus abolished the Mg2+-dependence of acto phosphorylated myosin ATPase activity. These results suggest that MM-2 inhibits the formation of 10 S myosin, and this results in the activation of actin-activated Mg2+-ATPase activity even in the absence of phosphorylation.