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PubMed1989

Caldesmon inhibits the cooperative turning-on of the smooth muscle heavy meromyosin by tropomyosin-actin.

The 38-kDa chymotryptic fragment of caldesmon, which possesses the actin/calmodulin binding domain, was purified and utilized to study the mechanism for the inhibition of acto-myosin ATPase by caldesmon. The intact caldesmon inhibited the acto-HMM ATPase although it caused an increase in the binding of HMM to actin, presumably due to the interaction between the S-2 region of HMM and the caldesmon located on the actin filament. The 38-kDa fragment, which lacks the S-2 binding domain, inhibited both the acto-HMM ATPase and the HMM binding to actin. The ATPase and the HMM binding to actin decreased in parallel on increasing the 38-kDa fragment bound to actin. In the presence of tropomyosin, the ATPase activity fell more rapidly than did the HMM binding to actin. Binding of intact caldesmon or 38-kDa fragment to actin inhibited the cooperative turning-on of tropomyosin-actin by NEM.S-1, which forms rigor complexes in the presence of ATP. The absence of cooperative turning-on of the acto-HMM ATPase by rigor complexes in the presence of 38-kDa fragment was associated with an inhibition of the binding of HMM to tropomyosin-actin. Addition of NEM.S-1 to tropomyosin-actin-caldesmon caused a gradual decrease in the caldesmon-induced binding of HMM to actin. The calmodulin restored the caldesmon-induced binding of HMM to tropomyosin-actin, but it had only a slight effect on the acto-HMM ATPase. These data suggest that the cooperative turning-on of the smooth muscle tropomyosin-actin by rigor bonds is modulated by the interaction of caldesmon, tropomyosin, and calmodulin on the thin filament.

Actins↗
PubMed1996

Innocuous labeling of the subfragment-2 region of skeletal muscle heavy meromyosin with a fluorescent polyacrylamide nanobead and visualization of individual heavy meromyosin molecules.

We have studied transglutaminase-catalyzed incorporation of monodansylcadaverine and monobiotincadaverine into rabbit skeletal muscle heavy meromyosin (HMM). The incorporation of dansylcadaverine reached saturation at 4 mol per 1 mol of HMM. An electrophoretogram of the chymotryptic digest of the dansyl-labeled HMM revealed that the labeling took place primarily in the S-2 region of HMM. Atomic force microscopic images and electron micrographs of the complexes of the biotinylated HMM and UltraAvidin-coated fluorescent polyacrylamide nanoparticles revealed that the biotinylated site on S-2 was very close to the C-terminus (near the S-2/light meromyosin junction). In keeping with this result, together with HMM's key sites being localized on the S-1 region, the enzymatic conjugation of biotincadaverine had no influence upon the actin-activated ATPase activity of HMM or upon the ability of HMM to actuate sliding of actin filaments in in vitro motility assay. Attachment of an UltraAvidin-coated fluorescent nanobead to the biotinylated HMM also did not alter the motile activity of HMM. Thus, we can optically pinpoint individual HMM molecules in a sample, which will facilitate handling and manipulation of single HMM molecules and observation of their functional behavior.

Acrylic Resins↗
PubMed1975

The amounts of adenosine di- and triphosphates bound to H-meromyosin and the adenosinetriphosphatase activity of the H-meromyosin-F-actin-relaxing protein system in the presence and absence of calcium ions. The physiological functions of the two routes of myosin adenosinetriphosphatase in muscle contraction.

The rates of the ATPase [EC 3.6.1.3] reaction of the H-meromyosin-F-actin-relaxing protein system were measured in 2 mM MgCl2, 50mM KC1, and 10mM Tris-HC1 at pH 7.8 and 20 degrees in the presence and absence of 0.05-0.1 mM Ca2+ ions. The concentrations of H-meromyosin (HMM) and the F-actin-relaxing protein (F-A-PR) complex were 3.4 and 3 mg/ml, respectively, and the ATPase reaction was coupled with 4 mg/ml of pyruvate kinase [EC 2.7.1.40] and 1 or 20 mM phosphoenolpyruvate to regenerate ATP. The amount of ADP bound to HMM during the ATPase reaction was determined by measuring the amount of ADP remaining in the reaction mixture. The amount of ATP bound to HMM was determined by subtracting the amount of bound ADP from the total amount of nucleotides bound to HMM, which was measured by a rapid flow-dialysis method. The following results were obtained. 1. The ATPase activity of the HMM-F-A-RP system increased linearly with increase in the amount of ATP added, and was independent of the presence of 0.05 mM Ca2+, when the amount of ATP added was less than 1 mole/mole of HMM. In the presence of 0.05 mM Ca2+, the ATPase activity reached a maximal level when 1.2-1.5 mole of ATP was added per mole of HMM, and maintained this level even at 3 moles of added ATP/mole of HMM. In the presence of 3mM EGTA, the ATPase activity decreased with increase in the amount of ATP added, from 1.5 to 3 moles of ATP/mole of HMM, and reached the level of the HMM ATPase reaction at 3 moles of added ATP/mole of HMM. Similar results were observed when the concentration of HMM was maintained at 3.4 mg/ml and the concentration of the F-A-RP complex was decreased from 3 to 1 or 0.5 mg/ml.

Adenosine Diphosphate↗
PubMed1985

Mechanism of the phosphorylation-dependent regulation of smooth muscle heavy meromyosin.

Phosphorylation of smooth muscle heavy meromyosin (HMM) has been shown to result in about a 25-fold increase in the steady-state Vmax of the actin-activated MgATPase activity from 0.07 s-1 for unphosphorylated HMM to 1.9 s-1 for phosphorylated HMM. The steady-state MgATPase activity of unphosphorylated HMM in the absence of actin is 0.004 s-1. The true extent of regulation might be even larger since the actin activation of the MgATPase activity of unphosphorylated HMM (from 0.004 to 0.07 s-1 at Vmax) could be arising from a small fraction of modified HMM molecules which are no longer regulated and that truly regulated unphosphorylated HMM molecules are not activated by actin. To test this idea, a "limited turnover" experiment was used to measure the reassociation rate of acto-unphosphorylated HMM following addition of a 2-4-fold molar excess of ATP. The reassociation rate was very slow and was not significantly increased by raising the actin concentration from 10 to 75 microM or by addition of trace phosphorylated HMM. The rate constant was estimated to be about 0.002 s-1, which is in good agreement with the rate of product (both Pi and ADP) release estimated from unphosphorylated HMM alone measured by a gel filtration technique. These two experiments suggest that the rate of product release from unphosphorylated HMM may not be significantly affected by actin and that perhaps the true extent of regulation of HMM by phosphorylation is much greater than that determined by steady-state methods. It also suggests that phosphorylation may operate by increasing the forward rate constant for product release by approximately 1000-fold.

Adenosine Diphosphate↗
PubMed1987

Effect of phosphorylation on the binding of smooth muscle heavy meromyosin X ADP to actin.

Relaxation of both smooth and skeletal muscles appears to be caused primarily by inhibition of the step associated with Pi release in the actomyosin ATPase cycle, rather than by a block in the binding of the myosin X ATP and myosin X ADP X Pi complexes to actin. In skeletal muscle, troponin-tropomyosin not only causes marked inhibition of Pi release, but it also markedly inhibits the binding of myosin subfragment-1 X ADP to actin, raising the possibility that the two phenomena are coupled in some way. In the present study we determined whether phosphorylation of smooth muscle heavy meromyosin (HMM) also affects both the binding of HMM X ADP to actin and the Pi release step. This was done by having phosphorylated and unphosphorylated HMM X ADP compete for sites on F-actin. At mu = 30 mM, phosphorylation increased the affinity of the HMM molecule for actin about 12-fold and at mu = 170 mM, there was less than a 3-fold increase in the affinity of HMM. If phosphorylation affects the binding of each head of HMM to the same extent, then phosphorylation caused about a 4- and 2-fold increase in the affinity of each head of HMM for actin at mu = 30 and 170 mM, respectively. In contrast, at both ionic strengths, phosphorylation caused more than 100-fold actin activation of the ATPase activity of smooth muscle HMM. Therefore, the marked activation of Pi release in the acto X HMM ATPase cycle upon phosphorylation of HMM is not accompanied by a comparable increase in the affinity of HMM X ADP for actin. We have also found that phosphorylation increases by only 4-fold the rate of Pi release from HMM alone. These results suggest that in smooth muscle, phosphorylation accelerates the step associated with the release of Pi both in the forward and the reverse direction without correspondingly affecting the binding of myosin X ADP to actin.

Actins↗
PubMed1985

Pharmacokinetics of hexamethylmelamine administered via the Ip route in an oil emulsion vehicle.

Saline and Intralipid were compared as vehicles for the ip administration of hexamethylmelamine (HMM) in mice. The drug proved stable over at least 7 weeks in solution and 2 years in crystal form. Fiftyfold higher concentrations of HMM could be achieved in Intralipid than in saline. The peritoneal pharmacokinetics were first-order and linear over the concentration range of HMM in Intralipid from 50 to 2000 micrograms/ml. The mean peritoneal half-life of HMM in Intralipid was 12.5-fold greater than the mean half-life of HMM in saline at the same concentration. Peritoneal concentration X time drug exposure, as measured by the area under the curve in single-dose elimination experiments, was 1200-fold greater for HMM at 2000 micrograms/ml in Intralipid than at 50 micrograms/ml, near saturation, in saline. The steady-state peritoneal to plasma concentration ratios were 96-104 for HMM in Intralipid at concentrations of 300-2000 micrograms/ml, and the peritoneal concentration that could be maintained by the constant ip infusion of HMM at 2000 micrograms/ml in Intralipid was at least 1600-fold greater than that maintainable with HMM in saline. The mean peritoneal clearance calculated by two independent methods and at three different concentrations of HMM in Intralipid was 0.112 +/- 0.016 ml/minute. HMM in Intralipid is a stable formulation that can be used to increase peritoneal exposure to HMM and may potentially be used iv as well.

Altretamine↗
PubMed1984

Protein kinase C modulates in vitro phosphorylation of the smooth muscle heavy meromyosin by myosin light chain kinase.

Protein kinase C phosphorylates different sites on the 20,000-Da light chain of smooth muscle heavy meromyosin (HMM) than did myosin light chain kinase (Nishikawa, M., Hidaka, H., and Adelstein, R. S. (1983) J. Biol. Chem. 258, 14069-14072). Although protein kinase C incorporates 1 mol of phosphate into 1 mol of 20,000-Da light chain when either HMM or the whole myosin molecule is used as a substrate, it catalyzes the incorporation of up to 3 mol of phosphate/mol of 20,000-Da light chain when the isolated light chains are used as a substrate. Threonine is the major phosphoamino acid resulting from phosphorylation of HMM by protein kinase C. Prephosphorylation of HMM by protein kinase C decreases the rate of phosphorylation of HMM by myosin light chain kinase due to a 9-fold increase of the Km for prephosphorylated HMM compared to that of unphosphorylated HMM. Prephosphorylation of HMM by myosin light chain kinase also results in a decrease of the rate of phosphorylation by protein kinase C due to a 2-fold increase of the Km for HMM. Both prephosphorylations have little or no effect on the maximum rate of phosphorylation. The sequential phosphorylation of HMM by myosin light chain kinase and protein kinase C results in a decrease in actin-activated MgATPase activity due to a 7-fold increase of the Km for actin over that observed with phosphorylated HMM by myosin light chain kinase but has little effect on the maximum rate of the actin-activated MgATPase activity. The decrease of the actin-activated MgATPase activity correlates well with the extent of the additional phosphorylation of HMM by protein kinase C following initial phosphorylation by myosin light chain kinase.

Animals↗
PubMed1991

Hexamethylmelamine: pharmacology and mechanism of action.

Several conclusions can be drawn from a review of HMM preclinical and clinical pharmacology data. The drug is extensively metabolized by animals and by man. The drug is well absorbed following oral administration to animals, but oral bioavailability is low due to first pass metabolism. Based on limited human data and more complete animal data, absorption of HMM following oral administration may be quite high in man. We do not yet know the oral bioavailability of HMM in patients, but again based primarily on animal studies, oral bioavailability is most likely low and variable due to extensive first pass metabolism. Systemic exposure to HMM and demethylated metabolites following oral administration varies greatly from patient to patient and is sometimes quite low. Most patients are, however, exposed to a substantial fraction of the administered dose when determined by urinary recovery of the total dose (based on parent drug and metabolites or total radioactivity) or by the total plasma AUC of parent drug and all metabolites. Systemic exposure to HMM following intravenous administration is clearly greater and less variable than following oral administration. An unresolved question is whether the highly variable and often low systemic exposure after oral administration compromise antitumor activity when compared to intravenous administration. A key issue is whether or not one accepts the hypothesis that metabolism is a prerequisite for antitumor activity. The metabolic activation studies do not rule out other mechanisms of HMM antitumor activity. Modest activity of HMM was observed after prolonged exposure to cells which did not metabolize the drug. However, most of the accumulated data are consistent with the metabolic activation hypothesis. Certainly HMM has clinical activity when administered by mouth. If metabolism is required, then exposure to the total dose (parent drug and metabolites) could be of significance even when exposure to HMM is low, since every demethylated metabolite must have come ultimately from the initial HMM demethylation. We do not know whether the initial metabolic reaction (occurring in the liver rather than in the tumor) provides sufficient exposure of tumor to reactive species. Specifically, does the variable HMM plasma AUC seen after oral administration lead to variable delivery of potentially reactive species to tumor (by rapid breakdown and/or further metabolism of MPMM before it leaves the gut and/or liver) or are quantities of MPMM delivered to tumor comparable to those delivered following intravenous administration. The issue of rate of MPMM formation compared to rate of breakdown and ultimate delivery to tumor has been noted by Judson and Rutty.(ABSTRACT TRUNCATED AT 400 WORDS)

Altretamine↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1989

Interaction of two heads of myosin with F-actin: binding of H-meromyosin with F-actin in the absence of nucleotide.

The bindings of S-1 and the two heads of HMM with pyrene-labeled F-actin were studied using the change in light-scattering intensity or that in the fluorescence intensity of the pyrenyl group. At low ionic strength (50 mM KCl), both S-1 and HMM became bound tightly with F-actin (Kd less than 0.1 microM) and both heads of HMM became bound to F-actin. The affinities of S-1 and HMM for F-actin decreased with increasing KCl concentration. In 1 M KCl, the Kd values of S-1 and HMM for F-actin were 11 and 0.58 microM, respectively. Thus, HMM was bound to F-actin 19 times more tightly than S-1. We compared the extent of binding of HMM to F-actin measured by a centrifugation method with that measured by the fluorescence change of pyrenyl-group, and found that even in 1 M KCl, HMM became bound to F-actin with a two-headed attachment. We measured the kinetics of binding and dissociation of acto-S-1 and acto-HMM from the time course of the change in light-scattering intensity after mixing S-1 or HMM with F-actin at 1 M KCl and that after mixing 1 M KCl with acto-S-1 or acto-HMM formed at low ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1976

Structure and function of the two heads of the myosin molecule. III. Cooperativity of the two heads of the myosin molecule, shown by the effect of modification of head A with rho-chloromercuribenzoate on the interaction of head B with F-actin.

Subfragment-1 of HMM was prepared by tryptic [EC 3.4.21.4] digestion of HMM, which had been modified with 1 mole of CMB per mole of HMM at a specific SH group, SHr. S-1(T) obtained from CMB-HMM retained almost all the CMB, and the amount of bound CMB was about 0.8-0.9 mole per 2 moles of S-1(T). S-2 of CMB-HMM contained no bound CMB. The ATPase [EC 3.6.1.3] activity of HMM increased gradually with increase in the concentration of FA, and the acto-HMM ATPase was inhibited by excess substrate or removal of Ca2+ ions in the presence of RP. The ATPase activity of CMB-HMM increased to a maximum level on adding a small amount of FA, and the acto-CMB-HMM ATPase showed neither substrate inhibition nor Ca2+ sensitivity in the presence of RP. On the other hand, the dependence on the concentration of FA of the ATPase activity of acto-S-1(T) was unaffected by modification of S-1 with CMB. The Ca2+ sensitivity of the ATPase activity of acto-S-1(T) in the presence of RP was also unaffected by the modification. Acto-S-1(T) dissociated almost completely, while acto-CMB-S-1(T) was only 50% dissociated on adding ATP. More than 80% of the bound CMB was contained in S-1(T) undissociated from FA. Furthermore, superprecipitation of actomyosin induced by ATP was completely inhibited by adding about 2 moles of CMB-S-1(T) per mole of actin monomer. On the other hand, about 90% of the burst size of Pi liberation was retained in S-1(T) dissociated from FA. It was concluded that the two heads of the myosin molecule are different: one shows the initial burst of Pi liberation, and does not contain the SHr group which binds CMB (head B), and the other does not show the initial burst and contains the SHr group (head A). It was also concluded that modification of head A of HMM or myosin with CMB increases its binding strength to FA, and consequently the substrate inhibition and Ca2+ sensitivity of acto-HMM or actomyosin ATPase at head B are lost on modification of head A with CMB. CMB-S-1(CT) was prepared by chymotryptic [EC 3.4.21.1] digestion of CMB-myosin, and separated into two fractions by ultracentrifugation of acto-CMB-S-1(CT) in the presence of ATP. Three components of CMB-S-1(CT) with molecular weights of 9, 2.4, and 1.2 X 10(4) were separated by SDS-polyacrylamide gel electrophoresis. The ratios of the peak areas of the three components in electrophoretograms were the same in CMB-S-1(CT) and in the two fractions (1 : 0.18 : 0.09), indicating that heads A and B have the same subunit structure.

Actins↗
PubMed1979

Heavy meromyosin from skipjack tuna, Euthynus pelamis. Preparation and enzymic properties.

A method was developed to obtain heavy meromyosin (HMM) from the tryptic digest of skipjack tuna dorsal myosin. The tuna HMM thus obtained was shown to be homogeneous on gel filtration-gel electrophoresis, and on ultracentrifugation. The sedimentation constant (S20,w) was estimated to be 6.1S for tuna HMM. The ATPase activity of tuna dorsal HMM was found to be very similar to that of rabbit skeletal HMM in many respects: KCl concentration dependence, pH dependence, effect of pCMB, kinetic parameters (Vmax and Ka) in actin activation, and Arrhenius activation energy. The only difference found between tuna HMM and rabbit HMM was in heat denaturation behavior: the ATPase activities of tuna HMM were approximately four times as sensitive to heat inactivation as those of rabbit HMM. Thus, tuna HMM should represent a good experimental material for investigations of the molecular basis of susceptibility to denaturation, and of the characteristics of fish myosins in general. A new type of heat denaturation of myosin was observed. It occurred in a very early stage of heat treatment of either tuna dorsal myosin or rabbit skeletal myosin; however, it did not occur upon heat treatment of HMM of either tuna or rabbit, and it was detectable in terms of the Mg-ATPase activity only when the activity was measured in the presence of untreated actin.

Actins↗
PubMed1986

Intracellular immunoglobulins in human milk macrophages. Ultrastructural localization and factors affecting the kinetics of immunoglobulin release.

Immunoglobulins (Ig) were localized by immunofluorescence and immunoperoxidase techniques inside human milk macrophages (HMM phi). Immunoelectron microscopy was employed to study the distribution and localization of Ig within HMM phi. IgA and IgM were detected inside phagocytic vacuoles of different sizes in the vicinity of the cellular membrane and in the periphery of larger vacuoles with a dense and homogeneous content. The intramacrophagic IgA represents 5-10% of the total milk IgA. The in vitro release of IgA into the medium was quantitated using an enzyme-linked immunoabsorbent assay. The amount of IgA released was in most samples around 1,720-5,200 ng/10(6) HMM phi. The concentrations of IgM and IgG released by HMM phi were approximately 50-600 and 20-40 ng/10(6) HMM phi, respectively. The concentrations of Ig determined in the lysated pellets of HMM phi ranged between 2 and 100 micrograms/10(6) HMM phi for IgA and around 500 ng/10(6) HMM phi for IgM. Increased IgA release occurred when HMM phi were cultured in medium with a pH lower than 3. IgA concentrations were not very different between pH 5 and pH 7 and alkalinization of the medium did not produce any significant effect on IgA release. Release of IgA and IgM was not significantly increased when HMM phi were cultured in the presence of phorbol myristate acetate, formylated peptides, latex particles, lymphocyte-derived chemotactic factor or cholate. The kinetic study of IgA release by HMM phi, both with or without adding these stimuli to in vitro culture, indicates that IgA release depends on time and that these substances do not have any effect on it.

Animals↗
PubMed1998

Calorimetric studies of the thermal unfolding of smooth muscle myosin fragments and their complexes with ADP and phosphate analogs.

The thermal unfolding of turkey gizzard smooth muscle myosin subfragment 1 (S1) and heavy meromyosin (HMM) in the absence of added nucleotides, in the presence of ADP, and in S1 or HMM ternary complexes with ADP and Pi analogs, orthovanadate (Vi), beryllium fluoride (BeFx), or aluminum fluoride (AlF4-), have been studied by differential scanning calorimetry (DSC). It has been shown that the formation of these ternary complexes causes significant structural changes in S1 or in the heads of HMM which are reflected in a pronounced increase of the protein thermal stability. The effect of BeFx was less distinct than that of AlF4- or Vi. Phosphorylation of regulatory light chains (RLC) in S1 or in HMM had practically no influence on these effects. In general, the changes caused by various Pi analogs in smooth muscle S1 or HMM were similar to those observed earlier with skeletal muscle S1 devoid of RLC. It is concluded that RLC and their phosphorylation do not significantly affect the character of structural changes induced in motor domains of the HMM heads by the formation of ternary complexes HMM--ADP--Vi, HMM--ADP--AlF4-, and HMM--ADP--BeFx--stable analogs of the intermediate states of the HMM ATPase reaction, HMM.ADP.Pi and HMM. ATP.

Adenosine Diphosphate↗
PubMed1995

Macrophage colony-stimulating factor (M-CSF) induction of enhanced anticryptococcal activity in human monocyte-derived macrophages: synergy with fluconazole for killing.

Induction of enhanced anticryptococcal activity in human monocyte-derived macrophages (HMM) by macrophage colony-stimulating factor (M-CSF) and possible synergy with fluconazole (FCZ) for killing of Cryptococcus neoformans (CN) was studied. Fungistasis by HMM cultured in medium for 3, 5, or 7 days was minimal, 0-17%. The fungistasis of HMM cocultured with M-CSF at 1000, 5000, or 20,000 U/ml for 3, 5, or 7 days was increased significantly (P < 0.02) at all study times and by all concentrations. The optimal M-CSF concentration for HMM treatment for enhanced fungistasis was 5000 U/ml for Day 3 (84%), whereas 1000 U/ml was sufficient with more prolonged HMM culture and M-CSF treatment (Days 5-7). The enhancement by M-CSF was seen with four different donors and three patient isolates of CN. FCZ at 5 micrograms/ml was fungicidal, 28 +/- 17% (n = 8). Killing by FCZ was enhanced by HMM treated with M-CSF 5000 or 20,000 U/ml for 5 days compared to control HMM, 58% (P = 0.001) and 60% (P = 0.002) vs 48%, respectively. This was also seen with HMM cultured with 1000 U/ml M-CSF for 7 days (P < 0.05). M-CSF also induced in HMM enhancement of fungistasis by lower, fungistatic, concentrations of FCZ. These results demonstrate enhancement of anticryptococcal activity by HMM treated with M-CSF and synergy with FCZ for inhibition and killing. These findings may provide a rationale for combined treatment of FCZ and M-CSF against cryptococcosis.

Cells, Cultured↗
PubMed2002

Active site control of myosin cross-bridge zeta potential.

The electrical properties of contractile proteins contribute to muscle structure and perhaps function but have not been characterized adequately. Electrophoretic mobility, mu(e), is sensitive to the net electric charge and hydrodynamic size of a molecule in solution. Zeta potential, zeta, particle charge, Q(e), and particle charge-to-mass ratio are proportional to mu(e). We measured mu(e) for nucleotide complexes of skeletal muscle heavy meromyosin (HMM) and subfragment 1 (S1). The results indicate that mu(e) for HMM changes depending on the ligand bound in the active site. The changes in electric charge appear to occur mainly on the S1 moieties. For HMM(MgATPgammaS)(2) and HMM(MgADP.P(i))(2) the values of mu(e) are -0.077 and -0.17 (microm/s)/(V/cm), respectively. For these complexes, mu(e) is independent of [ATP], [ADP], and [P(i)]. When P(i) dissociates from HMM(MgADP.P(i))(2) to form HMM(MgADP)(2), mu(e) decreases to -0.61 (microm/s)/(V/cm). This large decrease in mu(e) is independent of free [ADP] or [ATP]. Increasing [P(i)], on the other hand, increases mu(e) for HMM(MgADP)(2) to values near those observed for the steady-state intermediate. For HMM, mu(e) = -0.34 and is independent of P(i). MgADP binding to HMM decreases mu(e) to -0.57 (microm/s)/(V/cm), and the dissociation constant is 9 microM. Taken together, these data indicate that mu(e) and, thus, zeta are controlled by ligand binding to the active site. The magnitudes of the particle charge-to-mass ratios for the HMM complexes are all in a range that falls within published values determined for a variety of other proteins. Possible roles that the observed nucleotide-dependent changes in cross-bridge electric charge might have in the contractile cycle in muscle are considered.

Adenosine Diphosphate↗
PubMed2003

Two functional heads are required for full activation of smooth muscle myosin.

The motor activity of smooth muscle myosin II is regulated by the regulatory light chain phosphorylation, but it is not understood how phosphorylation activates motor activity. To address this question, we produced asymmetric heavy meromyosin (HMM), which is composed of a wild-type (WT) heavy chain and a mutant heavy chain having no motor activity (i.e. S236T or G457A). The actin-activated ATPase activities (Vmax) of asymmetric HMMs were only 21.8 and 8.4% of the wild-type HMM for S236A/WT HMM and G456A/WT HMM, respectively. If the two heads of HMM are independent for their ATPase activities, asymmetric HMM should show 50% of the activity of wild-type HMM; however, the activity of asymmetric HMM was much lower than the expected value. The results suggest that the activity of the wild-type head is attenuated by the presence of inactive head. Consistently, the actin-gliding velocity of the asymmetric HMM (i.e. S236T/WT or G457A/WT) was less than one-fifth of the wild-type HMM. The present study supports an idea that the two heads of smooth muscle myosin II interact with each other and the presence of two active heads is required for full activation.

Actins↗
PubMed1979

Chicken gizzard heavy meromyosin that retains the two light-chain components, including a phosphorylatable one.

A method was developed to obtain a preparation of chicken gizzard heavy meromyosin (HMM) that retains the two light-chain components of parent myosin: the 20,000-dalton and 17,000-dalton light-chains. The HMM preparation was also shown to retain two characteristics of the ATPase activity of the parent myosin: the characteristic effect of phosphorylation of the 20,000-dalton light-chain component on the ATPase activity, and the characteristic dependence of the ATPase activity on the KCl concentration. 1. Two distinct stages were observed in the Mg-ATPase reaction catalyzed by gizzard HMM and rabbit skeletal actin in the presence of gizzard "native" tropomyosin (NTM) and Ca2+ ions: an early lag phase, in which the reaction rate gradually increased, and a subsequent steady state, in which the reaction proceeded at a high, constant rate. Urea-gel electrophoresis revealed that the 20,000-dalton light-chain component was gradually phosphorylated in the lag phase, and was fully phosphorylated in the steady state. It was also observed that addition of EGTA (to remove Ca2+ ions) at various times in the lag phase caused neither a further increase nor a decrease in the reaction rate, and that addition of EGTA in the steady state caused no change in the reaction rate. These observations imply that the ATPase activity increased as the amount of phosphorylated 20,000-dalton light-chain component increased, and also that Mg-ATPase of acto-phosphorylated HMM was no longer calcium-sensitive. 2. The Mg-ATPase activity of HMM in the presence of gizzard NTM and Ca2+ ions or EGTA was studied as a function of the concentration of rabbit skeletal actin. The maximal activity (Vmax) and the apparent affinity constant of acto-HMM (KA) were thus estimated from the double-reciprocal plot of Eisenberg-Moos: the Vmax and KA values for phosphorylated HMM (in the presence of Ca2+ ions) were 5 S(-1) and 5.5 mg/ml actin, respectively, and the Vmax value for unphosphorylated HMM (in the presence of EGTA) was 0.3 S(-1), assuming that the KA value with unphosphorylated HMM is equal to that with phosphorylated HMM.

Actins↗