Commentary: Effect of Skeletal Muscle Native Tropomyosin on the Interaction of Amoeba Actin with Heavy Meromyosin

Keywords: troponin, tropomyosin, cardiomyopathy, troponin T, mutations Troponin-tropomyosin inhibits skeletal and cardiac muscle contraction at low Ca rigor-type myosin S1 to actin-tropomyosin-troponin, particularly at saturating Ca2+, produces activation of myosin ATPase activity in excess of that seen in the absence of the regulatory proteins. The binding energy of S1 can overcome the inhibitory activity of troponin (Bremel et al., 1972) and may allow tropomyosin to move deep into the groove of actin. That particular arrangement of actin, tropomyosin, and troponin is a much better activator of ATP hydrolysis than actin alone. That active configuration of actin was called state 2 in the Hill model (Hill et al., 1980) and later named the M state because of its requirement for tight myosin binding

Fesselin, a Synaptopodin-like Protein, Stimulates Actin Nucleation and Polymerization

Fesselin is a proline-rich actin binding protein that has recently been isolated from smooth muscle [Leinweber, B. D., Fredricksen, R. S., Hoffman, D. R., and Chalovich, J. M. (1999) J. Muscle Res. Cell Motil. 20, 539–545]. Fesselin is similar to synaptopodin [Mundel, P., Heid, H. W., Mundel, T. M., Krüger, M., Reiser, J., and Kriz, W. (1997) J. Cell Biol. 139, 193–204] in terms of its size, isoelectric point, and sequence although synaptopodin is not present in smooth muscle. The function of fesselin is unknown. Evidence is presented here that fesselin accelerates the polymerization of actin. Fesselin was effective on actin isolated from either smooth or skeletal muscle at low ionic strength and in the presence of 100 mM KCl. At low ionic strength, fesselin decreased the time for 50% polymerization to about 1% of that in the absence of fesselin. The lag phase characteristic of the slow nucleation process of polymerization was eliminated as the fesselin concentration was increased from very low levels. Fesselin did not alter the critical concentration for actin but did increase the rate of elongation by ≈3-fold. The increase in elongation rate constant is insufficient to account for the total increase in polymerization rate. It is likely that fesselin stabilizes the formation of actin nuclei. Time courses of actin polymerization at varied fesselin concentrations and varied actin concentrations were simulated by increasing the rate of nucleation and both the forward and reverse rate constants for elongation.Originally published Biochemistry, Vol. 40, No. 47, Nov 200

Steric blocking upside down: a different way of thinking about the competition between myosin and tropomyosin

At low free Ca2+, the actin binding proteins tropomyosin, troponin I, troponin T and troponin C inhibit contraction in striated muscles. Ca2+ activation alters the position of tropomyosin on actin to uncover binding sites for high affinity forms of myosin (i.e., myosin-ADP). Inhibition of contraction is commonly thought to result from steric blocking of myosin binding to actin by tropomyosin. However, myosin-ADP binding to actin is energetically more favorable than localization of tropomyosin in the blocking position. Tropomyosin is an effective inhibitor of binding only at low levels of myosin-ADP. At low free Ca2+, troponin-tropomyosin also inhibits the rate of a step associated with Pi release to about 1% of the maximum rate. This results in accumulation of myosin with bound ATP and ADP-Pi. Such myosin binds weakly to actin. Ca2+ activation increases the rate of Pi release, but not to the maximum value, and increases the population of myosin-ADP. The high affinity binding of myosin-ADP to actin can displace tropomyosin into the fully active position in relation to the amount of myosin-ADP bound. It seems likely that an important outcome of the steric clash between myosin-ADP and tropomyosin is the dual activation by Ca2+ and myosin-ADP. The C-terminal region of troponin T (TnT) contributes to the incomplete activation by Ca2+ alone. Because this region of TnT is highly conserved, the ability of myosin-ADP to move tropomyosin may be more important than any restriction that tropomyosin may place on myosin binding

Characterization of a caldesmon fragment that competes with myosin-ATP binding to actin.

The protein caldesmon inhibits actin-activated ATP hydrolysis of myosin and inhibits the binding of myosin*ATP to actin. Afragment isolated from a chymotryptic digest of caldesmon contains features of the intact molecule that make it useful as a selective inhibitor of the binding of myosin ATP complexes to actin without having the complexity of binding to myosin. The COOH-terminal 20 kDa region of caldesmon binds to actin with one-sixth the affinity of caldesmon with a stoichiometry of binding of one fragment per two actin monomers. This contrasts with the 1:6-9 stoichiometry of intact caldesmon. The binding of the 20 kDa fragments to actin is totally reversed by Ca2+-calmodulin and, like intact caldesmon, the 20 kDa fragments are competitive with the binding of myosin subfragments to actin. This competition with myosin binding is largely responsible for the inhibition of ATP hydrolysis, although both the fragments and intact caldesmon also reverse the potentiation of ATPase activity caused by tropomyosin. These polypeptides are useful both in defining the function of caldesmon and in studying the role of weakly bound cross-bridges in muscle. Originally published Biophysical Journal, Vol. 65, No. 2, Aug 199

Localization and Characterization of a 7.3-kDa Region of Caldesmon Which Reversibly Inhibits Actomyosin ATPase Activity

Cleavage of caldesmon with chymotrypsin yields a series of fragments which bind both calmodulin and actin and inhibit the binding of myosin subfragments to actin and the subsequent stimulation of ATPase activity. Several of these fragments have been purified by cation exchange chromatography and their amino-terminal sequences determined. The smallest fragment has a molecular mass of about 7.3 kDa and extends from Leu597 to Phe665. This polypeptide inhibits the actin-activated ATPase of myosin S-1; this inhibition is augmented by smooth muscle tropomyosin and relieved by Ca2+- calmodulin. The binding of the 7.3-kDa fragment to actin is competitive with the binding of S - 1 to actin. Thus, this polypeptide has several of the important features characteristic of intact caldesmon. However, although an intact caldesmon molecule covers between six and nine actin monomers, the 7.3-kDa fragment binds to actin in a 1:1 complex. Comparison of this fragment with others suggests that a small region of caldesmon is responsible for at least part of the interaction with both calmodulin and actin. Originally published in the Journal of Biological Chemistry, vol. 267, No. 23, 199

The Interaction of Caldesmon with the COOH Terminus of Actin

Caldesmon interacts with the NH2-terminal region of actin. It is now shown in airfuge centrifugation experiments that modification of the penultimate cysteine residue of actin significantly weakens its binding to caldesmon both in the presence and absence of tropomyosin. Furthermore, as revealed by fluorescence measurements, caldesmon increases the exposure of the COOH-terminal region of actin to the solvent. This effect of caldesmon, like its inhibitory effect on actomyosin ATPase activity, is enhanced in the presence of tropomyosin. Proteolytic removal of the last three COOH-terminal residues of actin, containing the modified cysteine residue, restores the normal binding between caldesmon and actin. These results establish a correlation between the binding of caldesmon to actin and the conformation of the COOH-terminal region of actin and suggest an indirect rather than direct interaction between caldesmon and this part of actin. Originally published Journal of Biological Chemistry, Vol. 266, No. 30, Oct 199

Purification and Partial Characterization of Relaxin and Relaxin Precursors from the Hamster Placenta

Previous immunological studies have indicated that the molecular structure of hamster relaxin is quite different from that of porcine relaxin. In the present study, hamster relaxin was purified from placentas and characterized in order to investigate its biochemical properties. Placentas from Days 14 and 15 of gestation were homogenized in 0.26 N HCl-62.5% acetone containing protease inhibitors. After centrifugation, soluble proteins were acetone precipitated. Soluble proteins were applied to a carboxymethyl cellulose ion-exchange column and bound proteins were eluted with 0.1 and 0.3 M NaCl. Western blot analysis detected 16.5-, 18.7-, and 36.0-kDa relaxin-immunoreactive (IR) proteins within the 0.1 M NaCl eluant and detected a 5.6-kDa relaxin-IR protein within the 0.3 M NaCl eluant. The 5.6-kDa protein was purified to homogeneity by gel filtration (Sephadex G-50), ion-exchange HPLC, and C18 -HPLC. Reduction of the 5.6-kDa protein prior to electrophoresis resulted in a single band of lower molecular mass, suggesting that hamster relaxin consists of two chains of approximately equal molecular mass. Isoelectric point of the 5.6-kDa protein was 7.78. The 16.5- and 18.7-kDa IR proteins were copurified by gel filtration and ion-exchange HPLC. At least five isoelectric point variants were observed for the 16.5- and 18.7-kDa proteins. The N-terminal amino acid for the 5.6 and 18.7 relaxin-IR proteins was arginine, and subsequent cycles indicated an identical partial sequence that was consistent with that for relaxins from other species. Originally published Biology of Reproduction, Vol. 49, No. 1, July 199

Tropomyosin Dynamics in Cardiac Thin Filaments: A Multisite Förster Resonance Energy Transfer and Anisotropy Study

Cryoelectron microscopy studies have identified distinct locations of tropomyosin (Tm) within the Ca21-free, Ca21-saturated, and myosin-S1-saturated states of the thin filament. On the other hand, steady-state Förster resonance energy transfer (FRET) studies using functional, reconstituted thin filaments under physiological conditions of temperature and solvent have failed to detect any movement of Tm upon Ca21 binding. In this investigation, an optimized system for FRET and anisotropy analyses of cardiac tropomyosin (cTm) dynamics was developed that employed a single tethered donor probe within a Tm dimer. Multisite FRET and fluorescence anisotropy analyses showed that S1 binding to Ca21 thin filaments triggered a uniform displacement of cTm toward F-actin but that Ca21 binding alone did not change FRET efficiency, most likely due to thermally driven fluctuations of cTm on the thin filament that decreased the effective separation of the donor probe between the blocked and closed states. Although Ca21 binding to the thin filament did not significantly change FRET efficiency, such a change was demonstrated when the thin filament was partially saturated with S1. FRET was also used to show that stoichiometric binding of S1 to Ca21-activated thin filaments decreased the amplitude of Tm fluctuations and revealed a strong correlation between the cooperative binding of S1 to the closed state and the movement of cTm. Originally published Biophysical Journal, Vol. 94, No. 11, June 200

Deoxyguanosine-resistant Leukemia L1210 Cells: Loss of Specific Deoxyribonecleoside Kinase Activity

A mouse leukemia L1210 cell line was selected for resistance to deoxyguanosine. The deoxyguanosine-resistant cells (dGuo-R) were 126-fold less sensitive to deoxyguanosine than the wild-type cells. The IC50 values for araC and araG were increased, but only 10-12-fold in the dGuo- R cells when compared with the wild-type cells. The dGuo-R cell line showed an increased level of resistance to 2-fluoro-2'-deoxyadenosine and 2-fluoroadenine arabinoside (11-14-fold), but essentially no increase in resistance to deoxyadenosine or adenine arabinoside. Deoxyribonucleoside kinase activity was decreased only slightly (19%) when deoxycytidine was utilized as substrate; when cytosine arabinoside or deoxyguanosine was used as the substrate, the kinase activity in the extracts from the dGuo-R cells was only 10% of the enzyme activity in the extracts from the wild-type cells. The determination of the kinetic parameters, Km and Vmax, indicated that there were marked decreases in the Vmax values for deoxyguanosine and cytosine arabinoside as substrates, but not for deoxycytidine as substrate; the Km values for deoxycytidine and cytosine arabinoside were increased in the extracts from the dGuo-R cells. By use of high-performance liquid chromatography, the kinase activities in the extracts from the wild-type and resistant cells could be resolved. There was the specific loss of kinase activity toward cytosine arabinoside and deoxyguanosine as substrates. These data indicate that the dGuo-R cells have decreased levels of a specific deoxyribonucleoside kinase activity. Originally published Journal of Biological Chemistry, Vol. 268, No. 1, Jan 199