The effects of caldesmon on the ATPase activities of rabbit skeletal-muscle myosin.

We studied the effects of caldesmon, a major actin- and calmodulin-binding protein found in a variety of muscle and non-muscle tissues, on the various ATPase activities of skeletal-muscle myosin. Caldesmon inhibited the actin-activated myosin Mg2+-ATPase, and this inhibition was enhanced by tropomyosin. In the presence of the troponin complex and tropomyosin, caldesmon inhibited the Ca2+-dependent actomyosin Mg2+-ATPase; this inhibition could be partly overcome by Ca2+/calmodulin. Caldesmon, phosphorylated to the extent of approximately 4 mol of Pi/mol of caldesmon, inhibited the actin-activated myosin Mg2+-ATPase to the same extent as did non-phosphorylated caldesmon. Both inhibitions could be overcome by Ca2+/calmodulin. Caldesmon also inhibited the Mg2+-ATPase activity of skeletal-muscle myosin in the absence of actin; this inhibition also could be overcome by Ca2+/calmodulin. Caldesmon inhibited the Ca2+-ATPase activity of skeletal-muscle myosin in the presence or absence of actin, at both low (0.1 M-KCl) and high (0.3 M-KCl) ionic strength. Finally, caldesmon inhibited the skeletal-muscle myosin K+/EDTA-ATPase at 0.1 M-KCl, but not at 0.3 M-KCl. Addition of actin resulted in no inhibition of this ATPase by caldesmon at either 0.1 M- or 0.3 M-KCl. These observations suggest that caldesmon may function in the regulation of actin-myosin interactions in striated muscle and thereby modulate the contractile state of the muscle. The demonstration that caldesmon inhibits a variety of myosin ATPase activities in the absence of actin indicates a direct effect of caldesmon on myosin. The inhibition of the actin-activated Mg2+-ATPase activity of myosin (the physiological activity) may not be due therefore simply to the binding of caldesmon to the actin filament causing blockage of myosin-cross-bridge-actin interaction.     

The sulfhydryl groups involved in the active site of myosin B adenosinetriphosphatase. I. Relantionship of the sulfhydryl group responsible for Mg2+-ATPase activation to the S1 and S2 groups.

The reactivity of the sulfhydryl groups in myosin B to N-ethylmaleimide (NEM) was investigated under various conditions. Under the conditions where actin and myosin associate, i.e. at low ionic strength, only Mg2+-ATPase [EC 3.6.1.3] activity was markedly activated by NEM treatment, whereas coupling of EDTA-ATPase inhibition with Ca2+-ATPase activation, which was seen on blocking S1 of myosin A with NEM, was observed under conditions at which the dissociation of actomyosin occurs, i.e. at high ionic strength, suggesting the covering with actin of the S1 region of myosin. Nevertheless, APT accelerated the reactivity of S1 and S2 much more in the myosin B system than in myosin alone. NEM-modified myosin B ATPase exhibited a shift of the KCL dependence curve to high concentration, a shift of the maximum activation of ATPase activity to high Mg ion concentration and a suppression of substrate inhibition at high substrate concentrations. These all indicate that the blocking by NEM of Sa, the sulfhydryl group related to the activation of Mg2+-ATPase of myosin B, brings about an increase in the association of myosin and actin in the myosin B system, resulting in an activation of Mg2+-ATPase activity. In addition, the relationship between Sa and a sulfhydryl group(s) essential for Ca2+ sensitivity was discussed.     

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.     

Motor function and regulation of myosin X.

Myosin X is a member of the diverse myosin superfamily that is ubiquitously expressed in various mammalian tissues. Although its association with actin in cells has been shown, little is known about its biochemical and mechanoenzymatic function at the molecular level. We expressed bovine myosin X containing the entire head, neck, and coiled-coil domain and purified bovine myosin X in Sf9 cells. The Mg(2+)-ATPase activity of myosin X was significantly activated by actin with low K(ATP). The actin-activated ATPase activity was reduced at Ca(2+) concentrations above pCa 5 in which 1 mol of calmodulin light chain dissociates from the heavy chain. Myosin X translocates F-actin filaments with the velocity of 0.3 microm/s with the direction toward the barbed end. The actin translocating activity was inhibited at concentrations of Ca(2+) at pCa 6 in which no calmodulin dissociation takes place, suggesting that the calmodulin dissociation is not required for the inhibition of the motility. Unlike class V myosin, which shows a high affinity for F-actin in the presence of ATP, the K(actin) of the myosin X ATPase was much higher than that of myosin V. Consistently nearly all actin dissociated from myosin X in the presence of ATP. ADP did not significantly inhibit the actin-activated ATPase activity of myosin X, suggesting that the ADP release step is not rate-limiting. These results suggest that myosin X is a nonprocessive motor. Consistently myosin X failed to support the actin translocation at low density in an in vitro motility assay where myosin V, a processive motor, supports the actin filament movement.     

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.     

Ca2+ -dependent interaction of S100A1 with F1-ATPase leads to an increased ATP content in cardiomyocytes

S100A1, a Ca(2+)-sensing protein of the EF-hand family that is expressed predominantly in cardiac muscle, plays a pivotal role in cardiac contractility in vitro and in vivo. It has recently been demonstrated that by restoring Ca(2+) homeostasis, S100A1 was able to rescue contractile dysfunction in failing rat hearts. Myocardial contractility is regulated not only by Ca(2+) homeostasis but also by energy metabolism, in particular the production of ATP. Here, we report a novel interaction of S100A1 with mitochondrial F(1)-ATPase, which affects F(1)-ATPase activity and cellular ATP production. In particular, cardiomyocytes that overexpress S100A1 exhibited a higher ATP content than control cells, whereas knockdown of S100A1 expression decreased ATP levels. In pull-down experiments, we identified the alpha- and beta-chain of F(1)-ATPase to interact with S100A1 in a Ca(2+)-dependent manner. The interaction was confirmed by colocalization studies of S100A1 and F(1)-ATPase and the analysis of the S100A1-F(1)-ATPase complex by gel filtration chromatography. The functional impact of this association is highlighted by an S100A1-mediated increase of F(1)-ATPase activity. Consistently, ATP synthase activity is reduced in cardiomyocytes from S100A1 knockout mice. Our data indicate that S100A1 might play a key role in cardiac energy metabolism.     

Inhibition of actin-activated myosin Mg(2+)-ATPase in smooth muscle by ruthenium red.

Ruthenium red was found to inhibit actin-activated myosin Mg(2+)-ATPase in smooth muscle and to bind to myosin heavy chain, but not to F-actin. The inhibition by Ruthenium red of actin-activated Mg(2+)-ATPase was of the competitive type with respect to actin (Ki 4.4 microM) and of the non-competitive type with respect to ATP (Ki 6.6 microM). However, Ruthenium red scarcely dissociated the acto-heavy meromyosin complex during the ATPase reaction. These results suggest that Ruthenium red interacts directly with the binding site for F-actin on the myosin heavy chain. This site is considered to be necessary not for maintaining the binding affinity of myosin for F-actin, but for activation of the Mg(2+)-ATPase.     

Preparation of Monoclonal Antibodies against Human Ventricular Myosin Light Chain 1 （HVMLC1） for Functional Studies

Using purified recombinant human ventricular myosin light chain 1 (HVMLC 1) as the antigen,three monoclonal antibodies,designated C8,C9 and B 12,were prepared.Immunoblot experiments demonstratedthat all monoclonal antibodies could react with the ventricular myosin light chain 1 isolated from differentsources,such as human,rat or pig.It was also demonstrated that C8 was directed against the NN part of theN-fragment (amino acid 1-40) of HVMLC1,and both C9 and B12 against the C-fragment (amino acid 99-195).The affinity constants of C8,C9 and B12 were 3.20×10~8,8.60×10~7 and 1.77×10~8 M~(-1),respectively,determined by non-competitive ELISA.The isotype of B12 was determined as lgG2a,whereas the isotype ofboth C8 and C9 were IgG1.In the presence of C9 or B12,the actin-activated Mg~(2 )ATPase activity of myosinwas greatly inhibited,but there was almost no effect on the Mg~(2 )ATPase activity for C8.B12 and C9 alsoinhibited the superprecipitation of porcine cardiac native actomyosin (myosin B) and reconstituted actomyosin,but C8 did not.The results indicate that all three monoclonal antibodies could bind the intact myosin molecule,but B12 and C9 might more easily react with epitopes located in the C-fragment of HVMLC1.The inhibitoryeffects of B 12 and C9 on ATPase activity and superprecipitation assays show that light chain 1,particularlythe C-fragment domain,is involved in the modulation of the actin-activated Mg~(2 )ATPase activity of myosinand,as a consequence,plays an essential role in the interaction of actin and myosin.     

The giant protein AHNAK is a specific target for the calcium- and zinc-binding S100B protein: potential implications for Ca2+ homeostasis regulation by S100B

Transformation of rat embryo fibroblast clone 6 cells by ras and temperature-sensitive p53val(135) is reverted by ectopic expression of the calcium- and zinc-binding protein S100B. In an attempt to define the molecular basis of the S100B action, we have identified the giant phosphoprotein AHNAK as the major and most specific Ca(2+)-dependent S100B target protein in rat embryo fibroblast cells. We next characterized AHNAK as a major Ca(2+)-dependent S100B target protein in the rat glial C6 and human U-87MG astrocytoma cell lines. AHNAK binds to S100B-Sepharose beads and is also recovered in anti-S100B immunoprecipitates in a strict Ca(2+)- and Zn(2+)-dependent manner. Using truncated AHNAK fragments, we demonstrated that the domains of AHNAK responsible for interaction with S100B correspond to repeated motifs that characterize the AHNAK molecule. These motifs show no binding to calmodulin or to S100A6 and S100A11. We also provide evidence that the binding of 2 Zn(2+) equivalents/mol S100B enhances Ca(2+)-dependent S100B-AHNAK interaction and that the effect of Zn(2+) relies on Zn(2+)-dependent regulation of S100B affinity for Ca(2+). Taking into consideration that AHNAK is a protein implicated in calcium flux regulation, we propose that the S100B-AHNAK interaction may participate in the S100B-mediated regulation of cellular Ca(2+) homeostasis.     

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.     

Calmodulin bound to the first IQ motif is responsible for calcium-dependent regulation of myosin 5a

Myosin 5a is as yet the best-characterized unconventional myosin motor involved in transport of organelles along actin filaments. It is well-established that myosin 5a is regulated by its tail in a Ca(2+)-dependent manner. The fact that the actin-activated ATPase activity of myosin 5a is stimulated by micromolar concentrations of Ca(2+) and that calmodulin (CaM) binds to IQ motifs of the myosin 5a heavy chain indicates that Ca(2+) regulates myosin 5a function via bound CaM. However, it is not known which IQ motif and bound CaM are responsible for the Ca(2+)-dependent regulation and how the head-tail interaction is affected by Ca(2+). Here, we found that the CaM in the first IQ motif (IQ1) is responsible for Ca(2+) regulation of myosin 5a. In addition, we demonstrate that the C-lobe fragment of CaM in IQ1 is necessary for mediating Ca(2+) regulation of myosin 5a, suggesting that the C-lobe fragment of CaM in IQ1 participates in the interaction between the head and the tail. We propose that Ca(2+) induces a conformational change of the C-lobe of CaM in IQ1 and prevents interaction between the head and the tail, thus activating motor function.     

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.     

Vascular smooth muscle caldesmon

Caldesmon, a major actin- and calmodulin-binding protein, has been identified in diverse bovine tissues, including smooth and striated muscles and various nonmuscle tissues, by denaturing polyacrylamide gel electrophoresis of tissue homogenates and immunoblotting using rabbit anti-chicken gizzard caldesmon. Caldesmon was purified from vascular smooth muscle (bovine aorta) by heat treatment of a tissue homogenate, ion-exchange chromatography, and affinity chromatography on a column of immobilized calmodulin. The isolated protein shared many properties in common with chicken gizzard caldesmon: immunological cross-reactivity, Ca2+-dependent interaction with calmodulin, Ca2+-independent interaction with F-actin, competition between actin and calmodulin for caldesmon binding only in the presence of Ca2+, and inhibition of the actin-activated Mg2+-ATPase activity of smooth muscle myosin without affecting the phosphorylation state of myosin. Maximal binding of aorta caldesmon to actin occurred at 1 mol of caldesmon: 9-10 mol of actin, and binding was unaffected by tropomyosin. Half-maximal inhibition of the actin-activated myosin Mg2+-ATPase occurred at approximately 1 mol of caldesmon: 12 mol of actin. This inhibition was also unaffected by tropomyosin. Caldesmon had no effect on the Mg2+-ATPase activity of smooth muscle myosin in the absence of actin. Bovine aorta and chicken gizzard caldesmons differed in several respects: Mr (149,000 for bovine aorta caldesmon and 141,000 for chicken gizzard caldesmon), extinction coefficient (E1%280nm = 19.5 and 5.0 for bovine aorta and chicken gizzard caldesmon, respectively), amino acid composition, and one-dimensional peptide maps obtained by limited chymotryptic and Staphylococcus aureus V8 protease digestion. In a competitive enzyme-linked immunosorbent assay, using anti-chicken gizzard caldesmon, a 174-fold molar excess of bovine aorta caldesmon relative to chicken gizzard caldesmon was required for half-maximal inhibition. These studies establish the widespread tissue and species distribution of caldesmon and indicate that vascular smooth muscle caldesmon exhibits physicochemical differences yet structural and functional similarities to caldesmon isolated from chicken gizzard.     

S100A1 and S100B interactions with annexins

Members of the annexin protein family interact with members of the S100 protein family thereby forming heterotetramers in which an S100 homodimer crossbridges two copies of the pertinent annexin. Previous work has shown that S100A1 and S100B bind annexin VI in a Ca(2+)-dependent manner and that annexin VI, but not annexin V, blocks the inhibitory effect of S100A1 and S100B on intermediate filament assembly. We show here that both halves of annexin VI (i.e., the N-terminal half or annexin VI-a and the C-terminal half or annexin VI-b) bind individual S100s on unique sites and that annexin VI-b, but not annexin VI-a, blocks the ability of S100A1 and S100B to inhibit intermediate filament assembly. We also show that the C-terminal extension of S100A1 (and, by analogy, S100B), that was previously demonstrated to be critical for S100A1 and S100B binding to several target proteins including intermediate filament subunits, is not part of the S100 surface implicated in the recognition of annexin VI, annexin VI-a, or annexin VI-b. Evaluation of functional properties with a liposome stability and a calcium influx assay reveals the ability of both S100 proteins to permeabilize the membrane bilayer in a similar fashion like annexins. When tested in combinations with different annexin proteins both S100 proteins mostly lead to a decrease in the calcium influx activity although not all annexin/S100 combinations behave in the same manner. Latter observation supports the hypothesis that the S100-annexin interactions differ mechanistically depending on the particular protein partners.     

Ca2+ dependence of the ATPase activity of phosphorylated smooth muscle myosin: effects of tropomyosin and actin

Calcium ions produce a 3-4-fold stimulation of the actin-activated ATPase activities of phosphorylated myosin from bovine pulmonary artery or chicken gizzard at 37 degrees C and at physiological ionic strengths, 0.12-0.16 M. Actins from either chicken gizzard or rabbit skeletal muscle stimulate the activity of phosphorylated myosin in a Ca2+-dependent manner, indicating that the Ca2+ sensitivity involves myosin or a protein associated with it. Partial loss of Ca2+ sensitivity upon treatment of phosphorylated gizzard myosin with low concentrations of chymotrypsin and the lack of any change on similar treatment of actin supports the above conclusion. Although both actins enhance ATPase activity, activation by gizzard actin exhibits Ca2+ dependence at higher temperatures or lower ionic strengths than does activation by skeletal muscle actin. The Ca2+ dependence of the activity of phosphorylated heavy meromyosin is about half that of myosin and is affected differently by temperature, ionic strength and Mg2+, being independent of temperature and optimal at lower concentrations of NaCl. Raising the concentration of Mg2+ above 2-3 mM inhibits the activity of heavy meromyosin but stimulates that of myosin, indicating that Mg2+ and Ca2+ activate myosin at different binding sites.     

Stimulation of the interaction between actin and myosin by Physarum caldesmon-like protein and smooth muscle caldesmon.

We have purified an actin-binding protein from the plasmodia of a lower eukaryote, Physarum polycephalum, with an apparent molecular mass of 210,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein bound to actin filaments with a stoichiometry of 1:7-8 in a Ca(2+)-calmodulin-dependent manner. Antibody raised against caldesmon from smooth muscle cross-reacted with the 210-kDa protein. In vitro motility assay revealed that the 210-kDa protein increased the sliding velocity of actin filaments on Physarum myosin. The 210-kDa protein more than doubled the actin-activated ATPase activity of Physarum myosin under comparative conditions of in vitro motility assay. Further increases in the concentration of the 210-kDa protein decreased its stimulatory effects. Ca(2+)-calmodulin prevented the stimulatory effects of the 210-kDa protein. Unexpectedly, smooth muscle caldesmon also increased the sliding velocity of actin filaments on smooth muscle myosin at lower concentrations. The well-known inhibitory effect of smooth muscle caldesmon on the actin-myosin interaction was observed with this motility assay when the concentration of the caldesmon was increased further. The stimulatory and inhibitory effects were confirmed by measurements of actin-activated ATPase activity of smooth muscle myosin. From estimations of the intracellular concentrations of the 210-kDa protein and smooth muscle caldesmon in vivo, it appears that effects of the former and the latter on actin-myosin interactions in vivo are stimulatory and inhibitory, respectively.     

Increase in the actin-activated Mg2+-ATPase activity of smooth muscle myosin by increasing myosin concentration

The actin-activated Mg2+-ATPase activity of smooth muscle myosin was measured in 85 mM KCl, 6 mM MgCl2 in the absence of tropomyosin. The activity was dependent on myosin concentration. Vmax increased as myosin concentration was increased, while the Ka (the apparent dissociation constant for actin) remained the same. The extent of filament formation was also correlated with myosin concentration and most of the myosin monomers existed in 10S conformation. These results suggest that myosin concentration influences the actin-activated Mg2+-ATPase activity by changing the 10S-6S-filaments equilibrium.     

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.     

SH1 (cysteine 717) of smooth muscle myosin: its role in motor function.

To determine if a thiol group called SH1 has an important role in myosin's motor function, we made a mutant heavy meromyosin (HMM) without the thiol group and analyzed its properties. In chicken gizzard myosin, SH1 is located on the cysteine residue at position 717. By using genetic engineering techniques, this cysteine was substituted with threonine in chicken gizzard HMM, and that mutant HMM and unmutated HMM were expressed in biochemical quantities using a baculovirus system. The basal EDTA-, Ca(2+)-, and Mg(2+)-ATPase activities of the mutant were similar to those of HMM whose SH1 was modified by N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS). However, while the chemically modified HMM lost the function of the light chain phosphorylation-dependent regulation of the actin-activated ATPase activity, the mutant HMM exhibited the normal light chain-regulated actin-activated ATPase activity. Using an in vitro motility assay system, we found that the IAEDANS-modified HMM was unable to propel actin filaments but that the mutant HMM was able to move actin filaments in a manner indistinguishable from filament sliding generated by unmutated HMM. These results indicate that SH1 itself is not essential for the motor function of myosin and suggest that various effects observed with HMM modified by thiol reagents such as IAEDANS are caused by the bulkiness of the attached probes, which interferes with the swinging motion generated during ATP hydrolysis.     

A monoclonal antibody to the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle inhibits plasmalemmal (Ca2+ + Mg2+)-dependent ATPase activity.

A monoclonal antibody (2B3) directed against the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle was prepared. This antibody reacts with a 130,000-Mr protein that co-migrates on SDS/polyacrylamide-gel electrophoresis with the calmodulin-binding (Ca2+ + Mg2+)-ATPase purified from smooth muscle by calmodulin affinity chromatography. The antibody causes partial inhibition of the (Ca2+ + Mg2+)-ATPase activity in plasma membranes from pig stomach smooth muscle, in pig erythrocytes and human erythrocytes. It appears to be directed against a specific functionally important site of the plasmalemmal Ca2+-transport ATPase and acts as a competitive inhibitor of ATP binding. Binding of the antibody does not change the Km of the ATPase for Ca2+ and its inhibitory effect is not altered by the presence of calmodulin. No inhibition of (Ca2+ + Mg2+)-ATPase activity or of the oxalate-stimulated Ca2+ uptake was observed in a pig smooth-muscle vesicle preparation enriched in endoplasmic reticulum. These results confirm the existence in smooth muscle of two different types of Ca2+-transport ATPase: a calmodulin-binding (Ca2+ + Mg2+)-ATPase located in the plasma membrane and a second one confined to the endoplasmic reticulum.