Interaction of rat muscle AMP deaminase with myosin. II. Modification of the kinetic and regulatory properties of rat muscle AMP deaminase by myosin.

The problems of whether the kinetic and regulatory properties of AMP deaminase were modified by formation of a deaminase-myosin complex were investigated with an enzyme preparation from rat skeletal muscle. Results showed that AMP deaminase was activated by binding to myosin. Myosin-bound AMP deaminase showed a sigmoidal activity curve with respect to AMP concentration in the absence of ATP and ADP, but a hyperbolic curve in their presence. Addition of ATP and ADP doubled the V value, but did not affect the Km value. Myosin-bound AMP deaminase also gave a sigmoidal curve in the presence of alkali metal ions, whereas free AMP deaminase gave a hyperbolic curve. GTP abolished the activating effects of both myosin and ATP.     

Interaction of AMP deaminase with RNA

tRNA, 18 S and 28 S ribosomal RNAs were found to activate muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) but inhibit liver and heart AMP deaminases. The macromolecular structures are essential for modulation of enzyme activity, since the effects of RNA disappeared after RNAase treatment. Sucrose density centrifugation experiments clearly demonstrated the binding of purified muscle AMP deaminase to tRNA, 18 S and 28 S RNAs. The binding is reversible and responsive to alterations of pH and KCl concentration. The binding was stable at pH 5.1-7.0 in 0.1 M KCl, but most of the enzyme dissociated at pH 7.5. KCl below 0.1 M concentration had no effect on dissociation of enzyme-RNA complex, but in 0.15 M KCl the complex was partially dissociated and in 0.2 M KCl most of the enzyme was released. Various nucleotides were also effective in dissociation of the enzyme from complex. The binding is saturable and the maximum number of muscle AMP deaminase molecules bound per mol 28 S RNA was calculated to be approx. 30. Liver and heart AMP deaminases were also found to interact with RNA.     

Effects of various ligands on interaction of AMP deaminase with myosin.

Purified rat muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) binds tightly to rat myosin. The binding is abolished in the presence of low concentrations of various ligands. Pyrophosphate and GTP at concentrations as low as 0.1 micrometer were effective in abolishing the interaction between two proteins. Other nucleoside triphosphates were less effective than GTP and the concentrations required for 50% inhibition were approximately 0.3 to 0.7 micrometer. ADP and AMP are effective in inhibiting the interaction between two proteins, but they are less effective than the nucleoside triphosphates; 50% inhibition occurred at 34 micrometer with ADP and at 1 mM with AMP. Creatine phosphate and inorganic phosphate showed 50% inhibition at 5 to 6 mM. All of the compounds, which affected AMP deaminase activity, were effective in abolishing the interaction of the enzyme with myosin; however, the interaction-abolishing effects of the compounds are not parallel with their inhibitory effects on the deaminase activity. Although there exist three parental isozymes of AMP deaminase in the rat, all three enzymes interacted with myosin.     

Negative homotropic cooperativity in rat muscle AMP deaminase. A kinetic study on the inhibition of the enzyme by ATP

1. Rat skeletal muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) at optimal KCl concentrations shows a biphasic response to increasing levels of the allosteric inhibitor ATP. 2. Up to 10 micrometer, ATP appears to convert the enzyme to a form exhibiting sigmoidal kinetics while at higher concentrations its inhibitory effect is manifested by an alteration of AMP binding to AMP deaminase indicative of negative homotropic cooperativity at about 50% saturation. 3. AMP deaminase is inactivated by incubation with the periodate oxidation product of ATP. The (oxidized ATP)--AMP deaminase complex stabilized by NaBH4 reduction shows kinetic properties similar to those of the native enzyme in the presence of high ATP concentrations. 4. A plausible explanation of the observed cooperativity is that ATP induces different conformational state of AMP deaminase subunits, causing the substrate to follow a sequential mechanism of binding to enzyme. 5. Binding of the radioactive oxidized ATP shows that 3.2 mol of this reagent bind per mol AMP deaminase.     

Diadenosine tetraphosphate activates AMP deaminase from rat muscle

Diadenosine tetraphosphate, Ap4A, doubled the activity of AMP deaminase from rat muscle, with an activation constant of 0.005 mM, in the presence of 0.05 mM AMP. The presence of Ap4A appeared to induce Michaelian kinetic behavior. The activation by Ap4A was not dependent on the presence of either MgCl2 or KCl in the reaction mixture. Diguanosine tetraphosphate was inhibitor of the enzyme. Diadenosine and diguanosine triphosphates, adenylosuccinate and xanthosine monophosphate were neither inhibitors nor activators of the reaction.     

Identification of a noncatalytic domain in AMP deaminase that influences binding to myosin

AMP deaminase (AMP-D) plays a critical role in energy metabolism in skeletal muscle. Prior studies have demonstrated AMP-D binds to myosin heavy chain in vitro, and it decorates the end of the A band in the myofibril. The present study presents evidence that proteolytic removal of 14 kilodaltons, presumably from the carboxy terminus, of the native 80K peptide does not eliminate catalytic activity but this deletion has a pronounced influence on binding of AMP-D to myosin in the presence of ATP. Comparison of the sequence of the rat skeletal muscle form of AMP-D to that of yeast AMP-D demonstrates conservation of an ATP binding site in the carboxy-terminal domain of the rat protein. These results provide a mechanism for regulating binding of AMP-D to myosin heavy chain in response to changes in ATP concentration and suggest a potential function for AMP-D/myosin complex formation in myocytes.     

Adaptation of rat skeletal muscle to creatine depletion: AMP deaminase and AMP deamination.

AMP deaminase catalyzes deamination of the AMP formed in contracting muscles to inosine 5'-monophosphate (IMP). Slow-twitch muscle has only approximately 30% as high a level of AMP deaminase activity as fast-twitch muscle in the rat, and rates of IMP formation during intense contractile activity are much lower in slow-twitch muscle. We found that feeding the creatine analogue beta-guanidinopropionic acid (beta-GPA) to rats, which results in creatine depletion, causes a large decrease in muscle AMP deaminase. This adaptation was used to evaluate the role of AMP deaminase activity level in accounting for differences in IMP production in slow-twitch and fast-twitch muscles. beta-GPA feeding for 3 wk lowered AMP deaminase activity in fast-twitch epitrochlearis muscle to a level similar to that found in the normal slow-twitch soleus muscle but had no effect on the magnitude of the increase in IMP in response to intense contractile activity. Despite a similar decrease in ATP in the normal soleus and the epitrochlearis from beta-GPA-fed rats, the increase in IMP was only approximately 30% as great in the soleus in response to intense contractile activity. These results demonstrate that the accumulation of less IMP in slow- compared with fast-twitch skeletal muscle during contractile activity is not due to the lower level of AMP deaminase in slow-twitch muscle.     

Possible correlation between binding of muscle type AMP deaminase to myofibrils and ammoniagenesis in rat skeletal muscle on electrical stimulation

Contraction of rat skeletal muscle by electrical stimulation of the sciatic nerve caused remarkable increase in binding of AMP deaminase (EC 3.5.4.6) to myofibrils, but did not change the total enzyme activity. After 30 sec stimulation, the ratio of bound to free enzyme was about 5 times that in resting muscle. This treatment also increased the ammonia content of the muscle to 5 times that in resting state. From these findings, we suggest that there is a correlation between the binding of muscle type AMP deaminase to myofibrils and ammoniagenesis in the muscle.     

Immunofluorescent and histochemical localization of AMP deaminase in skeletal muscle

Fluorescent antibody staining experiments with both isolated myofibrils and muscle fibers grown in culture show that AMP deaminase is bound to the myofibril in the A band. The strongest staining occurs at each end of the A band. The approximate width of the fluorescent stripes and their relation to the A band remains constant as a function of sarcomere length. Removal of enzyme from the myofibrils leads to loss of staining, and readdition of purified enzyme restores the original staining pattern. A histoenzymatic method for the detection of AMP deaminase activity in cultured fibers gives comparable localization. The results are consistent with the previous observation (Ashby, B. and C. Frieden. 1977.J. Biol. Chem. 252:1869--1872) that AMP deaminase forms a tight complex in solution with subfragment-2 (S-2) of myosin or with heavy meromyosin (HMM).     

Studies on Ca2+-Mg2+ binding sites of frog skeletal muscle myosin.

From skeletal muscle myosin light chains readily dissociate from the myosin oligomer in the absence of divalent cations, and unlike rabbit skeletal muscle myosin light chains, the released light chains of frog skeletal muscle myosin have a high Ca2+ binding affinity. Whereas each Ca2+ binding light chain of frog skeletal muscle myosin, when in association with the heavy chains bound 1 mol of Ca2+, when in the dissociated state bound 0.5 mol of Ca2+; the latter were readily displaced with low Mg2+ concentrations. Whereas 10(-5) M Mg2+ displaced all of the Ca2+ binding sites on the released light chains at Ca2+ concentration ranges of 10(-7) to 10(-4) M, there was negligible displacement of the Ca2+ binding sites with native frog skeletal muscle myosin under these same conditions.     

Regulatory properties of 14-day embryo and adult hen skeletal muscle AMP deaminase

Chromatography on phosphocellulose column revealed changes in the elution profile of 14 day-old chicken embryo and adult hen skeletal muscle AMP deaminase. In the presence of 5 mM potassium the enzyme from embryo muscle exhibited a sigmoid-shaped plot of the reaction rate versus substrate concentration. The increase of KCl concentration up to 100 mM diminished distinctly sigmoidicity of the plot. Micromolar concentrations of ADP or ATP activated, whereas GTP at the same concentrations inhibited the embryo and hen skeletal muscle AMP deaminase while 5 mM KCl was present in the incubation medium. 100 mM potassium concentration diminished the effect of ADP and ATP but not of GTP. Palmitoyl-CoA inhibited strongly the embryo skeletal muscle adenylate deaminase but had no effect on the activity of the hen enzyme. Alanine inhibited only the adult hen enzyme. The embryo and hen AMP deaminase differed also in the specificity to adenylate analogues and exhibited a different dAMP/AMP ratio. The data presented indicate that kinetic and regulatory properties of the two developmental forms of AMP deaminase are different.     

Molecular forms of human heart muscle AMP deaminase.

Chromatography on phosphocellulose revealed the presence of two, kinetically different forms of human heart AMP deaminase. The main portion of the activity eluted from the column at about 1.1 M KCl, and the enzyme present in this eluate manifested a sigmoidal type of substrate saturation kinetics. The results from gel filtration indicate that human heart AMP deaminase is a tetrameric protein capable of aggregating in more complex active structures.     

Effects of storage on activity and subunit structure of rabbit skeletal-muscle AMP deaminase.

On storage, AMP deaminase is converted into a form exhibiting hyperbolic kinetics even at low KCl concentration. This effect results from cleavage of the enzyme subunit (mol.wt. 79 000) to a product of similar size to the component of approx. mol.wt. 70 000 present in trace amounts in AMP deaminase just prepared from fresh muscle.     

Effect of pH and KCl on aggregation state and sulphydryl groups reactivity of rat skeletal muscle AMP deaminase

The effects of pH and KCl on sedimentation properties and SH groups reactivity of rat skeletal muscle AMP deaminase have been investigated. The values obtained for apparent molecular weight are consistent with an association of AMP deaminase subunits in response to increasing KCl concentration. Increasing pH value from 6.0 to 8.0 causes a reduction in the apparent molecular weight of the enzyme at high KCl concentration, which can be interpreted as due to a deprotonation-induced isomerization process. Removal of Zn2+ from AMP deaminase has effect similar to alkalinization in modifying the sedimentation properties of the enzyme. In the native enzyme at high K+ concentration about 7, 9 and 12 SH groups can be titrated with Nbs2, approximately 1, 2 and 4 SH groups reacting as fast sets, at pH 6.0, 7.0 and 8.0, respectively. Substitution of the 12 SH groups reactive with Nbs2 at pH 8.0 has no effect on the pH-dependent allosteric behaviour of the enzyme. Removal of K+ causes considerable changes in the reactivity of AMP deaminase towards Nbs2, unmasking a class of additional SH groups, so that the total number of titratable SH groups approaches that of 30 determined in denaturing conditions. In the enzyme previously treated with N-ethylmaleimide to alkylate the fast reacting class of SH groups, the class of additional SH groups are substituted by Nbs2 at basic pH, but not at acidic pH, with a concomitant reduction of the enzyme activity.     

Determination of AMP in the rat heart using skeletal muscle AMP deaminase

A new simple enzymatic method for measuring AMP content in freeze-clamped rat heart is presented. The method is based on the ammonia estimation after the deamination of 5'-AMP by muscle 5'-adenylic acid deaminase. The minimum detectable amount of AMP was about 1.5 nmol. The recovery of AMP added to the tissue homogenate was 94%. The variance coefficient evaluated by assaying five samples from one tissue extract was equal to 5%. AMP content of rat heart (0.28 mumol/g wet tissue) is comparable with the values reported by others.     

Effect of F-actin upon the binding of ADP to myosin and its fragments.

The effect of F-actin upon the binding of ADP to rabbit skeletal muscle myosin, heavy meromyosin, and subfragment 1 was studied by equilibrium dialysis, ultracentrifuge transport, and light scattering techniques. Both myosin and H-meromyosin (HMM) bind a maximum of approximately 1.6 mol of ADP/mol of protein, while S-1 binds approximately 0.9 mol of ADP/mol of protein. The affinity for ADP of all three preparations was similar at a given ionic strength (approximately 10(6) M-1 at 0.05 M KCl) and decreased with increasing ionic strength. Under conditions similar to those used for the measurement of ADP binding, the binding sites of myosin, HMM, and subfragment 1 (S-1) are saturated with actin at molar ratios of 2, 2, and 1 mol of actin monomer/mol of protein, respectively, as determined by light scattering, ultracentrifuge transport, and in the case of myosin by ATPase measurements. F-actin was found to inhibit ADP binding, but even at an actin concentration at least twice that required for saturation of myosin, HMM, or S-1, significant ADP binding remained. This ADP binding was inhibited by 10(-4) M pyrophosphate. The observations are consistent with the formation of an actomyosin-ADP complex in which actin and ADP are bound to myosin at distinct but interacting sites.     

Properties of porcine platelet myosin. I. Similarity between vertebrate smooth muscle and nonmuscle myosins in their binding properties with F-actin

The mechanism of the ATPase [EC 3.6.1.3] reaction of porcine platelet myosin and the binding properties of platelet myosin with rabbit skeletal muscle F-actin were investigated. The kinetic properties of the platelet myosin ATPase reaction, that is, the rate, the extent of fluorescence enhancement of myosin, the size of the initial P1 burst of myosin, and the amount of nucleotides bound to myosin during the ATPase reaction, were very similar to those found for other myosins. Strong binding of platelet myosin with rabbit skeletal muscle F-actin, as found for smooth muscle myosin, was suggested by the following results. The rate of the ATP-induced dissociation of hybrid actomyosin, reconstituted from platelet myosin and skeletal muscle F-actin, was very slow. The amount of ATP necessary for complete dissociation of hybrid actomyosin was 2 mol/mol of myosin, although skeletal muscle actomyosin is known to dissociate completely upon addition of 1 mol ATP per mol of myosin. Unlike skeletal muscle myosin, the EDTA(K+)-ATPase activity of platelet myosin was inhibited by skeletal muscle F-actin. These observations indicate that ATP hydrolysis by vertebrate nonmuscle myosin follows the same mechanism as with other myosins and that the binding properties of nonmuscle myosin with F-actin are similar to those of smooth muscle myosin but not to those of skeletal muscle myosin.     

Purification and characterization of myosin from the clonal rat glial cell strain C-6.

A myosin was isolated from the clonal rat glial cell strain C-6 and compared with rat skeletal muscle myosin. After cell extracts were subjected to gel filtration chromatography in the presence of KI and magnesium pyrophosphate the C-6 myosin was rapidly purified by a procedure similar to that used for skeletal muscle myosin. The C-6 myosin resembles muscle myosin both physically and enzymatically. It contains heavy chains of 200,000 daltons and two classes of light chains of 17,000 and 19,000 daltons in approximately equal molar ratios. This myosin forms bipolar thick filaments in 0.1 M KCl and binds reversibly to skeletal muscle F-actin, the binding being inhibited by MgATP. Skeletal muscle F-actin stimulates the C-6 myosin adenosine triphosphatase 2- to 3-fold in the presence of KCl and Mg2+. The action activation of muscle myosin ATPase at low ionic strength is 10-fold greater than that of C-6 myosin. Ca2+ and EDTA stimulated the ATPase activities of both enzymes. When assayed in the presence of 0.6 M KCl and 1 mM EDTA the skeletal muscle myocin ATPase demonstrates substrate saturation while the C-6 myosin enzyme activity is stimulated by ATP concentrations above 2.5 mM.     

Binding and location of AMP deaminase in rabbit psoas muscle myofibrils

It is shown that an interaction exists between AMP deaminase (EC 3.5.4.6) and myofibrils that is sufficiently strong (Kd congruent to 10(-10) M) for more than 99% of the binding sites for the enzyme to be filled in vivo. The binding is not strong enough, however, to stop removal of the enzyme during the extensive washing normally used in the preparation of myofibrils. Fluorescent antibodies to the enzyme label myofibrils close to the junction of the A- and I-bands. The invariance of the position of the antibody stripes at this site, over a range of sarcomere lengths, indicates that the enzyme is attached to the A-band. The intensity of the fluorescence declines in parallel with dissociation of the enzyme. In this muscle, the number of AMP deaminase binding sites per thick filament is approximately six, suggesting that the enzyme is located at a single axial position in each half A-band. Electron microscopy of negatively stained, antibody-labelled myofibrils reveals the distance between the AMP deaminase sites at opposite ends of an A-band to be 1.69(+/- 0.02 micron). Since the length of the A-band is 1.57 micron, the binding site for the enzyme must be significantly beyond where thick filaments have previously been thought to end.     

Isolation and properties of actin, myosin, and a new actinbinding protein in rabbit alveolar macrophages.

Actin, myosin, and a high molecular weight actin-binding protein were extracted from rabbit alveolar macrophages with low ionic strength sucrose solutions containing ATP, EDTA, and dithiothreitol, pH 7.0. Addition of KCl, 75 to 100 mM, to sucrose extracts of macrophages stirred at 25 degrees caused actin to polymerize and bind to a protein of high molecualr weight. The complex precipitated and sedimented at low centrifugal forces. Macrophage actin was dissociated from the binding protein with 0.6 M KCl, and purified by repetitive depolymerization and polymerization. Purified macrophage actin migrated as a polypeptide of molecular weight 45,000 on polyacrylamide gels with dodecyl sulfate, formed extended filaments in 0.1 M KCl, bound rabbit skeletal muscle myosin in the absence of Mg-2+ATP and activated its Mg-2+ATPase activity. Macrophage myosin was bound to actin remaining in the macrophage extracts after removal of the actin precipitated with the high molecular weight protein by KCl. The myosin-actin complex and other proteins were collected by ultracentrifugation. Macrophage myosin was purified from this complex or from a 20 to 50% saturated ammonium sulfate fraction of macrophage extracts by gel filtration on agarose columns in 0.6 M Kl and 0.6 M Kl solutions. Purified macrophage myosin had high specific K-+- and EDTA- and K-+- and Ca-2+ATPase activities and low specific Mg-2+ATPase activity. It had subunits of 200,000, 20,000, and 15,000 molecular weight, and formed bipolar filaments in 0.1 M KCl, both in the presence and absence of divalent cations. The high molecular weight protein that precipitated with actin in the sucrose extracts of macrophages was purified by gel filtration in 0.6 M Kl-0.6 M KCl solutions. It was designated a macrophage actin-binding protein, because of its association with actin at physiological pH and ionic strength. On polyacrylamide gels in dodecyl sulfate, the purified high molecular weight protein contained one band which co-migrated with the lighter polypeptide (molecular weight 220,000) of the doublet comprising purified rabbit erythrocyte spectrin. The macrophage protein, like rabbit erythrocyte spectrin, was soluble in 2 mM EDTA and 80% ethanol as well as in 0.6 M KCl solutions, and precipitated in 2 mM CaCl2 or 0.075 to 0.1 M KCl solutions. The macrophage actin-binding protein and rabbit erythrocyte spectrin eluted from agarose columns with a KAV of 0.24 and in the excluded volumes. The protein did not form filaments in 0.1 M KCl and had no detectable ATPase activity under the conditions tested.