Measurement Of ATPase Activity Research Articles

A highly sensitive method for the measurement of ATPase activity

A highly sensitive method for the measurement of ATPase activity

Molecular polymorphism and mechanisms of activation and deactivation of the hydrolytic function of the coupling factor of oxidative phosphorylation.

The 13S coupling factor of oxidative phosphorylation from Alcaligenes faecalis has a latent adenosine triphosphatase (ATPase) function that can be activated by heating at 55 degrees C for 10 min at pH 8.5 in 50% glycerol. The specific activity increases from 0.1 to 20--30 mumol min-1 mg-1. Adenosine 5'-triphosphate (ATP) is not required for stabilization at 55 degreesC when glycerol is present. Activation involves displacement of the endogenous ATPase inhibitor subunit (epsilon subunit), and readdition of this subunit results in deactivation. In the deactivation process the ATPase inhibitor subunit can be replaced by other cationic proteins such as protamine, histones, or poly(lysine). Mg2+ and H+ also are effective deactivators. The fact that every positively charged substance tested deactivated the enzyme suggests that the inhibitor subunit is complexed with the enzyme at a site containing a surplus of negative charges. The activated enzyme is not labile, but it is salt labile, having a half-life of 2-3 min in 0.1 M KI at either 25 or 0 degrees C. The activated ATPase is also inhibited by aurovertin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD), and by the cross-linking agent dimethyl suberimidate. Evidence for polymorphism comes from finding that the properties of the unactivated enzyme (intrinsic ATPase) are different in many ways from the properties of activated ATPase. With respect to the coupling factor's ability to hydrolyze ATP, the data in this study suggest that there are at least four distinct functional allomorphs of this enzyme: (1) the latent enzyme, which has no kinetically measurable ATPase activity, (2) intrinsic ATPase, which is catalyzed by a small percentage of the molecular population that has been activated by some natural mechanism, (3) activated ATPase, which has properties different from those of intrinsic ATPase, and (4) aged activated ATPase, in which some of the properties (Km for substrate, sensitivity to deactivation by Mg2+ and H+) spontaneously change within 30 min.

Identical behavior of the two active sites of myosin with respect to trinitrophenylation.

Myosin and its active subfragments were trinitrophenylated under conditions in which mainly the active site(s) was modified. Proteins modified at the active site(s) could be separated by affinity chromatography on agarose-ATP columns. By two independent methods, ATPase activity measurements and analysis of elution patterns on agarose-ATP columns, it was shown that the introduction of two trinitrophenyl groups per myosin or one per heavy meromyosin subfragment 1 molecule is responsible for the remarkable change in the ATPase activities. Heavy meromyosin subfragment 1 prepared from trinitrophenylated myosin retained the original degree of trinitrophenylation per "active head." The kinetic constant of trinitrophenylation of the epsilon-amino group of lysine at the active site was found to be 2000 S-1-M-1, whereas a much smaller constant of 2.2 S-1-M-1 was obtained for the trinitrophenylation of the unessential lysyl residues of myosin. By using affinity chromatography, we could follow the formation of mono- and ditrinitrophenyl myosin. The amounts of these myosin derivatives at various extents of the reaction corresponded approximately to the calculated amounts, assuming a random and independent trinitrophenylation of the two myosin "heads." It is concluded that in each of the two heads of myosin there is one ATPase active site and these two sites behave in an identical manner with respect to trinitrophenylation.

Binding of a solubilized membrane ATPase to phospholipid bilayers

Interaction of purified Streptococcus fecalis membrane ATPase with lipid bilayer membranes was investigated using aqueous dispersions of phospholipids. A liposome-ATPase complex was demonstrated by gel filtration analysis. Binding of the solubilized ATPase to liposomes was studied by differential ultracentrifugation and the determination of binding parameters was based on the measurement of ATPase activity in the liposome-ATPase complex and the concentration of free ATPase in equilibrium with the complex. ATPase combined in phosphatidylcholine liposomes was essentially as active as the solubilized enzyme, and the binding of the ATPase to the liposomes was not reduced by increasing the ionic strength. The ratio of phosphatidylcholine to ATPase in the liposome complex was approximately 11 1 (w/w). The intrinsic association constant for the interaction, determined from Scatchard plots was 7.4·10 7±1.4·10 7 M −1. Phosphatidylcholine liposomes containing stearylamine or cardiolipin were prepared to investigate the effects of surface charge on binding. Stearylamine increased the ATPase binding at low ionic strength. In contrast, cardiolipin did not appear to change the binding characteristics. Phosphatidylethanolamine also had little effect on the ATPase binding. Cholesterol incorporated into phosphatidylcholine liposomes lowered ATPase binding. ATPase binding to synthetic dimyristoyl phosphatidylcholine liposomes was lower than with natural phosphatidylcholine liposomes at 4 °C but not significantly different at 30 °C, above the gel-liquid crystalline phase-transition temperature.

(Na +-K +)-activated ATPase in isolated mucosal cells of toad bladder

1. 1. A method is described for reproducible measurement of ATPase activity in isolated mucosal cells of toad bladder. 47 ± 7 % (S.D.) of the total activity is Na +- and K +-dependent, and yields 22 ± 5 (S.D.) μmoles P i per mg protein per h. 2. 2. The curve depicting this enzymatic activity as a function of Na + concentration is sigmoid in shape, suggesting cooperative interaction between binding sites for cations. 3. 3. Graded doses of ouabain were preincubated without ATP with the enzyme for 10 min. In deoxycholate-treated preparations, half-maximal inhibition was seen at 10 −4 M and complete inhibition at ⩾ 10 −3 M. In the absence of deoxycholate, 39 % of the activity persisted even at 10 −2 M ouabain.

The Changes of “Myosin B” during Storage of Rabbit Muscle

The denaturation process of actomyosin during storage in 0.6 m KCl at pH 5.7 and 25°C was studied by measuring ATPase activity, turbidity and solubility. During the storage of actomyosin the inactivation rate of myosin A ATPase was similar to that of the ATPase activating ability of F-actin. While the inactivation rate of isolated F-actin was largely dependent on the initial protein concentration, that of F-actin in actomyosin was less dependent. When F-actin was stored alone, inactivation of the ATPase activating ability was accompanied with a remarkable increase in turbidity and a decrease in solubility. However, this was not true when F-actin was stored with myosin A. The denaturation process of actomyosin during storage in 0.6 m KCl at pH 5.7 and 25°C was studied with regard to changes in the binding ability of myosin A and actin. When F-actin was stored alone, it lost the ability to bind with myosin A in pararell to the decrease in the ATPase activating ability. When myosin A was stored alone for 3 hr, it maintained 60% of the initial ability to bind with F-actin, even if it lost 85% of the initial ATPase activity. Thus, the ATPase-less myosin A forms a myosin A-actin complex when it is mixed with intact F-actin. F-Actin was not stabilized in this modified myosin A-actin complex. This complex was dissociated with ATP into ATPase-less myosin A and intact F-actin. The modified myosin A-actin complex did not occur in the stored actomyosin. During the storage of actomyosin, the interaction between myosin A and F-actin did not break and became less dissociable by ATP. This feature is in contrast with the one observed previously on the actomyosin stored in 0.6 m KCl at pH 5.7 and 3°C.

The Change of “Myosin B” during Storage of Rabbit Muscle

The denaturation process of actomyosin during storage in 0.6M KCl at pH 5.7 and 25°C was studied by measuring ATPase activity, turbidity and solubility. During the storage of actomyosin the inactivation rate of myosin A ATPase was similar to that of the ATPase activating ability of F-actin. While the inactivation rate of isolated Factin was largely dependent on the initial protein concentration, that of F-actin in actomyosin was less dependent. When F-actin was stored alone, inactivation of the ATPase activating ability was accompanied with a remarkable increase in turbidity and a decrease in solubility. However, this was not true when F-actin was stored with myosin A.

The Interaction of Actin with Myosin and Heavy Meromyosin in Solution at Low Ionic Strength

Abstract The interaction of actin with myosin and heavy meromyosin was studied in the presence of Mg++ ion and adenosine triphosphate at low ionic strength. When the heavy meromyosin ATPase was 17-fold activated by actin, the actoheavy meromyosin complex responsible for this activation did not give rise to a measurable increase in the viscosity of the sample above that predicted for a noninteracting mixture of actin and myosin. In the case of myosin, simultaneous measurements of turbidity, viscosity, and ATPase activity in the presence of actin showed, first, that considerable activation of the myosin ATPase could occur in the absence of any measurable precipitation of the actomyosin, and second, that in contrast to the behavior of the acto-heavy meromyosin system, ATPase activation was always accompanied by a corresponding viscosity increase indicating the formation of a relatively stable actomyosin complex in the cleared state. Comparison of the ATPase activity of acto-heavy meromyosin and actomyosin, under conditions where both systems were in solution (0.1 m KCl), indicated a distinct difference in the response of the actomyosin and acto-heavy meromyosin ATPases to falling ATP concentration.

The Changes of “Myosin B” during Storage of Rabbit Muscle

The differences between myosin B extracted from fresh muscle with Weber-Edsall solution and myosin B type protein from the muscle stored at 4°C post-mortem were investigated by the measurement of ATPase activity, ATP-sensitivity, salting-out curves, chromatographic patterns on triethylaminoethyl (TEAE)-cellulose, viscosity and ultra-centrifugation diagrams. As compared with myosin B from fresh muscle and myosin B type protein from post-rigor muscle, myosin B type protein from rigor muscle showed relatively high values of ATPase activity, ATP-sensitivity and reduced viscosity, and moreover, was remarkably different in salting-out curves, precipitating gradually from ammonium sulfate of lower concentration than that in others. The differences between myosin B from fresh muscle and myosin B type protein from post-rigor muscle were clearly demonstrated by chromatography and sedimentation data.

FORMATION OF ACTOMYOSIN DURING THE EXTRACTION OF MUSCLE MINCE WITH WEBER-EDSTALL SOLUTION.

Abstract The process of transformation of myosin A to myosin B during the prolonged extraction of muscle mince was investigated in detail by various physico-chemical techniques, viz. , viscosity, turbidity, flow birefringence, ultracentrifugation and ATPase activity measurements. All the determinations have shown that the formation of actomyosin started at around 8–10 h after the start of extraction and was completed at about 20 h. Flow birefringence measurements suggested that actomyosin particles of fairly uniform size-1–2 μ in length-were formed from the beginning and the amount increased during further extraction of muscle mince.

Preservation of washed bovine spermatozoa in a synthetic medium at room temperature

1. 1. Development is described of a completely synthetic medium that permitted storage at room temperature of washed bovine spermatozoa for one week with 10 per cent loss of initial motility. 2. 2. The respiratory ability and the ATP and ADP content of the stored cells declined rapidly with time. The relationship of these measurements to the state of “health” of the cells is not clear. 3. 3. Measurement of DNP-stimulated ATP-ase activity of stored cells indicated that the ability to couple oxidation and phosphorylation may not be impaired.