An intact heavy chain at the actin-subfragment 1 interface is required for ATPase activity of scallop myosin

Summary Scallop S1 has a region sensitive to tryptic hydrolysis not found thus far in S1s of other species, located 65K from theN-terminus as determined by SDS/polyacrylamide-gel electrophoresis. In the presence of actin the S1 heavy chain is preferentially cleaved at this site. The high-salt EDTA and calcium ATPase activities of the nicked 65K–31K S1 are abolished. This inactivation is not due to denaturation, conformational effects of actin, or to light chain dissociation. The unique proteolytic site of scallop S1 is adjacent to a peptide involved in actin-S1 interaction in scallop and rabbit but it is far removed from the nucleotide-binding site in the linear amino acid sequence. We conclude that proteolysis inactivates the high-salt ATPase activities through a connection mediated by tertiary interactions. Such a connection provides a structural correlate for the known reciprocal relationship between the nucleotide and actin affinities of myosin.Abbreviations S1 papain subfragment 1 containing both light chains - S1^–LC papain subfragment 1 prepared in the absence of bound regulatory light chain containing only a part of the essential light chain and having a shorter heavy chain - HC heavy chain - LC light chain - DTT dithiothreitol - TLCK 7-amino-1-chloro-3-l-tosylamidoheptan-2-one - TPCK 1-chloro-4-phenyl-3-l-toluene-p- sulphonamidobutan-2-one - K kilodalton     

Effects of physical training on cardiac myosin ATPase activity.

Cardiac myosin from rats exercised 90 or 150 min daily for 8 wk was compared with the myosin from the hearts of matched sedentary controls. The Ca++-ATPase activity was increased 17 percent in rats exercised 90 min and 30 percent in rats exercised 150 min daily. In the exercised group 0.18 M KCl increased the myosin ATPase activity by 50 percent but had no effect in the control group. Ethylene glycol activated the Ca++-ATPase in control myosin preparations, but had no significant effect on myosin from conditioned hearts. Heavy meromyosin (HMM) from conditioned hearts had a higher Ca++-ATPase activity than from controls. Fluorescence with 8-anilinonaphthalene sulfonate (ANS) was increased 30 percent in HMM from conditioned hearts. The results suggest that the increased myosin ATPase activity in the hearts of exercised animals may be due to a local conformational change at or near the active site.     

Fesselin binds to actin and myosin and inhibits actin-activated ATPase activity

Fesselin is an actin binding protein that bundles actin filaments and accelerates nucleation of actin polymerization. The effect of fesselin on actin polymerization is regulated by Ca⁺⁺-calmodulin. Because actin filaments serve both structural and contractile functions we also examined the effect of fesselin on activation of myosin S1 ATPase activity. Fesselin inhibited the activation of S1-catalyzed ATP hydrolysis in a similar manner in both the presence and absence of tropomyosin. This inhibition was unaffected by Ca⁺⁺-calmodulin. Fesselin inhibited the binding of myosin-S1 to actin during steady-state ATP hydrolysis. Fesselin also displaced caldesmon from actin. S1 displaced fesselin from actin in the absence of nucleotide when the affinity of S1 for actin was much greater than the affinity of fesselin for actin. It is likely that fesselin and S1 share common binding sites on F-actin. We also observed that fesselin could bind to smooth muscle myosin with μM affinity. Fesselin shares some similarities to caldesmon in binding to several other proteins and having multiple potential functions. Parts of this work were presented in preliminary form at the 45th Annual Biophysical Society Meeting, Baltimore, MD, February 2004 and the 46th Annual Meeting, Long Beach, CA, February 2005.     

The relationship of mechanicalV max to myosin ATPase activity in rabbit and marmot ventricular muscle

Papillary muscle mechanics and ventricular myosin calcium-activated ATPase activity were measured in the same heart as a function of temperature (8–28°) in rabbits and marmots, in order to examine further the hypothesis that the velocity of cardiac muscle shortening at zero load (V _max) is correlated with myosin ATPase activity. There was a similarQ ₁₀ forV _max in each muscle type, as measured with isotonic afterloaded quick-releases at 30–33% time-to-peak tension; the calcium activated ATPase of myosin in the two muscle types also was similar. The least squares linear regression of rabbitV _max on calcium-activated myosin ATPase activity was the same as in the marmot, so all the data were pooled to yield a linear regression (Y=0.47+3.82X) with a high correlation between the two variables [r=0.95,P<0.01 (ANOV)]. Furthermore, the correlation proved to be predictive of cardiacV _max and myosin ATPase activity levels in other experiments where these two measurements decreased below normal as a result of hypertrophic growth. Consequently, the quantitative relationship betweenV _max and myosin ATPase defined here may prove to be predictive of the ability of cardiac muscle to release bond energy.This work was supported in part by National Science Foundation Grant No. 1971-122; American Heart Association Grant No. 71 1080; Vermont Heart Association Grant Nos. AG71, 1971–1973, 1973–1974; PHS Nos. 16858 and AM 15594, and University of Vermont, College of Medicine General Research Support Grant No. RR05429. B. B. Hamrell was the recipient of a Special Research Fellowship, National Institutes of Health, National Institute of General Medical Sciences, 1F3GM39, 62301.     

The dependence of calcium efflux from cardiac muscle on temperature and external ion composition

1. Exchangeable Ca in guinea-pig auricles and ventricular trabeculae of sheep and calf hearts was labelled with (45)Ca and the loss of radioactivity into inactive rinsing solutions of different ion composition was measured for periods up to 6 hr. At no time did the decrease of radioactivity in the muscle follow a single exponential course, while the rate coefficient k (= fraction of (45)Ca lost per minute from muscle into rinsing solution) decreased slightly with time.2. On the basis of the temperature-sensitivity of Ca efflux from auricles the activation energy has been calculated to have a value of 5900 cal/mole, corresponding to a Q(10) of 1.35. 2,4-Dinitrophenol (5.5 x 10(-5) - 5.5 x 10(-4)M) had either no effect on Ca efflux or increased it slightly.3. Compared to control efflux in 1.8 mM [Ca](o) Ca efflux decreased to 70% in Ca-free solution, to 20% in Ca-free, Na-free solution and to 65% in Ca-containing, Na-free solution, NaCl being replaced by either sucrose or LiCl. Quantitatively, Ca efflux from auricles has been shown to depend to a large extent on the ratio [Ca(2+)](o)/[Na(+)](o) (2). The affinity for Na of the activation site for Ca efflux (carrier) is much less than for Ca.4. The efflux from muscles soaked for about 2 hr in Ca-free solution was not linearly related to Ca-concentration in the tissue but followed a square law.5. While Ca content in auricles increased in Ca-containing, Na-poor solution it decreased again when Tyrode solution was readmitted indicating a Na-sensitive Ca net transport in cardiac muscle.6. The results are interpreted in terms of a modified exchange diffusion mechanism (Ussing, 1947) which is responsible for Ca extrusion from mammalian cardiac muscle.     

Myofibrillar myosin ATPase activity in hindlimb muscles from young and aged rats

We tested the hypothesis that Ca(2+)-activated myosin ATPase activity is lower in muscles of aged rats relative to muscles of young rats, independent of changes in myosin isoform expression. Myofibrils were prepared from permeabilized fibers of soleus, plantaris, and semimembranosus muscles of young (8-12 months) and aged (32-38 months) F344 x BN rats and assayed for resting myosin ATPase, Ca(2+)-activated myosin ATPase, and myosin heavy chain (MHC) and myosin light chain (MLC) isoform compositions. Resting myosin ATPases were not affected by age in any muscle (P > or = 0.42). Ca(2+)-activated myosin ATPases of soleus and plantaris myofibrils were not affected by age (P > or = 0.31) but were 16% lower in semimembranosus myofibrils from aged rats (0.448 +/- 0.019 micromol P(i)/min/mg) compared to young rats (0.533 +/- 0.031 micromol P(i)/min/mg; P = 0.03). Correspondingly, maximal unloaded shortening velocity of single semimembranosus fibers from aged rats was slow (4.6 +/- 0.2 fiber lengths/s) compared with fibers from young rats (5.8 +/- 0.3 fiber lengths/s; P < 0.01). No age-related changes in MHC or regulatory MLC isoforms were detected in any muscle (P > or = 0.08) but changes in the essential MLC occurred in plantaris and semimembranosus muscles. The data indicate that Ca(2+)-activated myosin ATPase activity is reduced with age in semimembranosus muscle, independent of age-related changes in MHC isoform expression, and is one mechanism contributing to age-related slowing of contraction in that muscle.     

The higher myosin ATPase activity in the right heart ventricle of the rat, proved by histophotometry

Comparing the myosin-ATPase activity in the left and right ventricular wall of the hearts from 6 rats by scanning histophotometry, a higher enzyme activity in the right ventricle was found. These findings are in a good agreement with the higher numbers of myofilaments per myofibril in the right ventricle, reported in literature.     

The ATPase kinetics of insect fibrillar flight muscle myosin subfragment-1

Summary Myosin subfragment-1 (S1) has been prepared from the fibrillar flight muscles of the giant water bugLethocerus by chymotryptic digestion of myofibrillar suspensions in the absence of magnesium ions. The S1 obtained has a single light chain and a heavy chain with molecular weights of about 18 kDa and 90 kDa respectively.The kinetics of the elementary steps of the magnesium-dependent ATPase of insect S1 and rabbit S1 are similar, both with ATP and with ATP analogues as substrates. However, the presence of variable amounts of inactive protein within our preparation means that several rate constants cannot be obtained with as much precision in the case of insect S1. The most striking differences between the rabbit and insect S1 are values for the V_max and the K_m of actin during actin-activation of the MgATPase activity, which are up to an order of magnitude lower and greater in the insect than in the rabbit, respectively.The mechanical properties of strain activation and of capacity to do extended oscillatory work are unique to insect fibrillar flight muscle and distinguish it from vertebrate striated muscle. It is likely that these properties reflect differences in the organization of actin and myosin within the respective filament lattices rather than intrinsic differences in the ATPase mechanisms of the isolated myosin molecules from the two types of muscle.     

Action of peptide-B from bovine fibrinogen on ATPase activity and superprecipitation of myosin B.

Bovine peptide-B from fibrinogen was capable of accelerating the structural and enzymatic effects associated with superprecipitation of myosin B. The rate of superprecipitation coupled with the hydrolysis of ATP are increased during the structural transformation. In the concentration range from 10-8 to 10-4 M peptide-B, the rate of superprecipitation is increased 12-fold while the hydrolysis of ATP doubles and the time to reach the final extent of superprecipitation is decreased 68%. Under these same conditions, the hydrolysis of ATP by myosin A was unaffected. The concentrations of magnesium and calcium were between 10 and 20 muM, and no additional divalent metal ions were added to the system. Superprecipitation was treated as a model for muscle contraction to explain the in vivo studies of bovine peptide-B in which the peptide behaves as a vasopressor substance producing vascular vasoconstriction. A possible mechanism for the participation of bovine peptide-B in the model for muscle contraction, based on the polarizing interaction of the highly charged density of negativity of the peptide with the actomyosin complex, is presented. Furthermore, bovine peptide-B is speculated as participating in vasoconstriction via attachment to some smooth muscle receptor.     

The effect of low ATP concentrations on relaxation in the myosin regulated myofibrils from scallop

Summary Troponin-tropomyosin-regulated myofibrils show a significant increase in ATPase activity and contract in the absence of calcium when the ATP concentration falls significantly below the saturation level. By contrast, the ATPase of the myosin-regulated myofibrils of scallop striated muscle was not activated in the absence of calcium when the ATP concentration was lowered to 10 mm. Nevertheless, a very small fraction of crossbridges were active at 10 mm ATP resulting in very slow myofibrillar shortening. In contrast to the behaviour of rabbit contractile proteins there was no correlation between myofibrillar shortening and ATP induced turbidity changes of actomyosin taken from scallop.     

The myosin ATPase mechanism does not require a conformationally sensitive aromatic residue

Summary The effect of nucleotides or calcium ([Ca²⁺]_free=1.0×10^–4 m) or both on the near-u.v. absorption spectrum of myosins purified from two species of scallop,Aequipecten irradians andPlacopecten magellanicus, has been examined. No change in tyrosyl or tryptophanyl absorption was detected.The near-ultraviolet (u.v.) circular dichroism (CD) spectra of myosins from these two species of scallop were examined on a high sensitivity (Jasco J41C) spectropolarimeter.Aequipecten andPlacopecten myosin near-u.v. CD spectra were qualitatively distinct. The near-u.v. CD spectrum ofPlacopecten myosin was not perturbed by addition of a ten-fold molar excess of ADP±Ca²⁺, 0.1m PP_i or 1.0×10^–4 m calcium alone. ADP and PP_i did induce small, qualitatively distinct changes in the near-u.v. CD spectrum ofAequipecten myosin but no perturbation was observed with calcium alone.These data, together with those from an earlier study, indicate that the conformation of aromatic residues are not necessarily perturbed in the myosin ATPase mechanism as has previously been suggested.     

ATPase kinetics of the Dictyostelium discoideum myosin II motor domain

Structural characterization of the mode of interaction of nucleotides bound to myosin has relied upon the crystal structure of the Dictyostelium discoideum myosin II motor domain. This fragment, denoted S1dC, lacks the regulatory domain and light chain subunits and may therefore fail to display the normal ATPase activity of the intact myosin molecule. Here we show that the elementary steps of the S1dC ATPase pathway and the effects of actin are similar to those of the complete myosin head fragment. This indicates that truncation at residue E759, with the removal of the light chain binding sites, is not crucial to catalytic activity. In particular, S1dC does not show the anomolous tight binding of ADP displayed by the slightly shorter M754 construct reported elsewhere. We also show that the fluorescent analogue Cy3-EDA-ATP is a good substrate for S1dC and demonstrate the use of fluorescence correlation spectroscopy to determine the affinity of Cy3-EDA-ADP using microgram quantities of proteins. This revised version was published online in July 2006 with corrections to the Cover Date.     

Role of sarco/endoplasmic reticulum calcium content and calcium ATPase activity in the control of cell growth and proliferation

Ca²⁺, the main second messenger, is central to the regulation of cellular growth. There is increasing evidence that cellular growth and proliferation are supported by a continuous store-operated Ca²⁺ influx. By controlling store refilling, the sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) also controls store-operated calcium entry and, thus, cell growth. In this review, we discuss data showing the involvement of SERCA in the regulation of proliferation and hypertrophy. First, we describe the Ca²⁺-related signaling pathways involved in cell growth. Then, we present evidence that SERCA controls proliferation of differentiated cells and hypertrophic growth of cardiomyocytes, and discuss the role of SERCA isoforms. Last, we consider the potential therapeutic applications of increasing SERCA activity for the treatment of cardiovascular diseases and of modulating SERCA and SR content for the treatment of cancer.