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The CacyBP/SIP protein interacts with several targets, including actin. Since the majority of actin filaments are associated with tropomyosin, in this work we characterized binding of CacyBP/SIP to the actin–tropomyosin complex and examined the effects of CacyBP/SIP on actin filament functions. By using reconstituted filaments composed of actin and AEDANS-labeled tropomyosin, we observed that binding of CacyBP/SIP caused an increase in tropomyosin fluorescence intensity indicating the occurrence of conformational changes within the filament. We also found that CacyBP/SIP bound directly to tropomyosin and that these proteins did not compete with each other for binding to actin. Electron microscopy showed that in the absence of tropomyosin CacyBP/SIP destabilized actin filaments, but tropomyosin reversed this effect. Actin-activated myosin S1 ATPase activity assays, performed using a colorimetric method, indicated that CacyBP/SIP reduced ATPase activity and that the presence of tropomyosin enhanced this inhibitory effect. Thus, our results suggest that CacyBP/SIP, through its interaction with both actin and tropomyosin, regulates the organization and functional properties of the thin filament.

The ABC transporter LmrA from Lactococcus lactis has been intensively studied and a role in multidrug resistance was proposed. Here, we performed a comprehensive detergent screen to analyze the impact of detergents for a successful solubilization, purification and retention of functional properties of this ABC transporter. Our screen revealed the preference of LmrA for zwitterionic detergents. In detergent solution, LmrA purified with FC-16 was highly active with respect to ATPase activity, which could be stimulated by a substrate (rhodamine 123) of LmrA Both, high ATPase activity and substrate stimulation were not detected for LmrA solubilized in DDM. Interestingly, reconstituted LmrA showed an opposite behavior, with a high basal ATPase activity and stimulation by rhodamine 123 for a DDM-reconstituted, but only low ATPase activity and no substrate stimulation for a FC-16 reconstituted sample. (C) 2011 Elsevier B.V. All rights reserved.

A method is described for the isolation of subfractions from human liver plasma membranes, enriched in canalicular domains (cLPM) and basolateral domains (blLPM), respectively, and the results are compared to those obtained with rat liver. The studies were performed in 18 human livers. The cLPM (isolated at densities 1.103-1.127 for human and 1.036-1.127 for rat cLPM) from human as well as rat liver showed a lower density than the blLPM (1.141-1.161 for human and 1.151-1.172 for rat blLPM). Human and rat blLPM were characterized by increased levels of (Na+/K+)-ATPase (relative enrichment 33 and 21, respectively). Both human and rat cLPM showed high specific activities of leucine aminopeptidase; relative enrichment factors were 42 and 3 1, respectively. Mg 2+-ATPase and alkaline phosphatase, specific canalicular enzymes in rat liver, were only slightly enriched in the cLPM of human liver, which indicates that these enzymes are not suitable as marker enzymes for human liver cLPM. Both cLPM and blLPM of human and rat origin were only slightly contaminated with mitochondria, lysosomes, Golgi membranes and endoplasmic reticulum. Total recoveries of cLPM and blLPM were 0.02 mg protein/g liver each for the human membrane preparations, compared to 0.07 and 0.16 mg protein/g liver for the membranes prepared from rat liver. Analysis of membrane fluidity revealed that the human liver cLPM were more rigid than blLPM (mean difference in fluorescence polarization P(DPH)0.024). They contained more cholesterol (0.43 vs. 0.30-mu-mol/mg protein) and phospholipids (0.54 vs. 0.39-mu-mol/mg protein, respectively), which was compatible to rat liver plasma membrane fractions. This study shows that besides similarities, there are several differences between human and rat liver plasma membrane fractions.

Melittin, a cationic, amphiphilic polypeptide, has been reported to inhibit the ATPase activity of the catalytic portions of the mitochondrial (MF1) and chloroplast (CF1) ATP synthases. Gledhill and Walker [J.R. Gledhill, J.E. Walker. Inhibition sites in F1-ATPase from bovine heart mitochondria, Biochem. J. 386 (2005) 591-598.] suggested that melittin bound to the same site on MF1 as IF1, the endogenous inhibitor polypeptide. We have studied the inhibition of the ATPase activity of CF1 and of F1 from Escherichia coli (ECF1) by melittin and the cationic detergent, cetyltrimethylammonium bromide (CTAB). The Ca2+- and Mg2+-ATPase activities of CF1 deficient in its inhibitory epsilon subunit (CF1-epsilon) are sensitive to inhibition by melittin and by CTAB. The inhibition of Ca2+-ATPase activity by CTAB is irreversible. The Ca2+-ATPase activity of F1 from E. coli (ECF1) is inhibited by melittin and the detergent, but Mg2+-ATPase activity is much less sensitive to both reagents. The addition of CTAB or melittin to a solution of CF1-epsilon or ECF1 caused a large increase in the fluorescence of the hydrophobic probe, N-phenyl-1-naphthylamine, indicating that the detergent and melittin cause at least partial dissociation of the enzymes. ATP partially protects CF1-epsilon from inhibition by CTAB. We also show that ATP can cause the aggregation of melittin. This result complicates the interpretation of experiments in which ATP is shown to protect enzyme activity from inhibition by melittin. It is concluded that melittin and CTAB cause at least partial dissociation of the alpha/beta heterohexamer.

The following reaction cycle is observed in myofibrils from rabbit muscle treated with deoxycholate: In the absence of added nucleotides, ADP bound to myofibrils is phosphorylated in the presence of creatine phosphate and ATP-creatine trans-phosphorylase, resulting in the liberation of free creatine, and the ATP produced is hydrolyzed by the Mg-activated ATPase activity of the myofibrils. The same phenomenon also occurs with myofibrils isolated from glycerinated psoas fibers.

An ultracentrifugal study has been made on myosin treated with methylmercuric hydroxide. 1. 1. Myosin is transformed by tritration of its -SH groups with methylmerucuric hydroxide to a faster-sedimenting product. This occurs in the same range of methyl-mercuric hydroxide titration at which the calcium-activated ATPase activity is inhibited. A parallelism can be demonstrated between the loss of ATPhase activity and the loss of myosin due to the transformation process. 2. 2. Complete titration of the -SH groups of myosin with methylmercuric hydroxide causes the release of a small subunit with sedimentation rate of 1.6 S, at 25° but not at 15°. 3. 3. These results suggest that the -SH groups whose titration causes inactivation of ATPase activity are responsible for the configurational integrity of the active site, and that release of the small subunit is not related to titration of these -SH groups.

Effects of dioxane and p-chloromercuribenzoate (PCMB) on the optical-rotatory dispersion of myosin A were measured in 0.6 M KCl at pH 7.0 and compared with those on the ATPase activity. The α-helical content of myosin A estimated from the b0 term of the Moffitt-Yang plot was 57–61%. On addind 8–10 volume percent of dioxane, increase in the helical content by several percent and pronounced activation of ATPase were firstly observed and were followed by gradual decreases in the helical content and the ATPase activity. 2 h after the addition of dioxane, the helical content decreased only by a few percent, while the ATPase activity disappeared completely. Immediately after the addition of dioxane, specific rotatory power at 5000 A showed its maximum at about 10% of dioxane in accordance with the activation of ATPase. The helical content of the alkaline-inactivated myosin A, however, remained constant on the addition of dioxane. On adding PP1 before and at various times after the addition of dioxane, shift in the helical content caused by dioxane was depressed completely and the content remained constant during the measurements. On adding 3–4 moles PCMB per 105 g of myosin A, the maximum velocity and the Michaelis constant of ATPase at 20° were increased, respectively, from 0.22 to 0.44 mmoles P1/min/g and from 1.3 to 1.5·104M, and temperature dependence of the maximum velocity was increased significantly, while on adding 8 moles PCMB the ATPase activity was completely inhibited. The helical content of myosin A increased by several percent on addition of 4 moles PCMB and decreased by several percent on 8 moles PCMB per 105 g. On adding PCMB in the presence of ATP or PP1, the helical content fell in between those in the presence of either of the two. On the basis of these and other observations, it was suggested that conformation of the active site is very susceptible to influences of dioxane and PCMB and that, accordingly, a minute change in the helical content induces a pronounced change in the ATPase activity.

l -Myosin and actin have been separated from an actomyosin sol which had interacted with ATP. When an actomyosin sol was treated with ATP and centrifuged at 100, 00 g , the upper 40% of the supernatant contained about 20 to 30% of the total protein. The same actomyosin centrifuged in absence of ATP contained only 2 to 3% of the total protein in the corresponding portion of the supernatant. This protein in the supernatant was identified as l -myosin by the following characteristics: 1. 1. sedimentation constant after the dephosphorylation of ATP; 2. 2. the inhibiting action of Mg ++ on the ATPase activity, reversed by the addition of actin; 3. 3. the disappearance of opalescence at an ionic strength below 0.1 on the addition of ATP and the immediate precipitation after the addition of actin; 4. 4. an ATP sensitivity of zero, changing to one of 100 after the addition of an appropriate amount of actin. The pellet produced on centrifugation yielded actin on extraction with water containing ATP. From these experiments it is concluded that ATP effects the dissociation of an actomyosin sol.

K+ interactions with a rat brain (Na+ + K+)-dependent ATPase and the associated K+-dependent nitrophenyl phosphatase activity were examined. Classes of sites for K+ were distinguished, initially, on the basis of affinity estimated by kinetic analysis in terms of KO.5 (the concentration for half-maximal activation), and by K+-accelerated enzyme inactivation by F-minus, which permits evaluation of a dissociation constant for K+, KD. Moderate-affinity sites ("alpha sites"), with a KD near 1 mM, were demonstrable for the phosphatase activity and for the "free" enzyme. High-affinity sites ("beta sites"), with a KD near 0.1 mM, were seen for the overall ATPase activity and under conditions in which enzyme phosphorylation by substrate also occurs. Further differentiation between alpha and beta sites was made in terms of (i) the characteristic changes in affinity with pH, and (ii) the efficacy of Li+ relative to K+, Rb+, Cs+, and Tl+ at these two classes of sites. Low-affinity sites ("gamma sites") through which K+ inhibits enzymatic activity were also detectable, with a KD around 140 mM. These data are incorporated into a model for the reaction sequence to accommodate both transport processes and certain K+/ATP antagonisms.

1. 1. Heating of susceptible sugarcane leaves (4 h at 35°C) renders them resistant, for 24 h, to the effects of helminthosporoside. Membrane ATPase activity is reduced by 50% as a result of the heat treatment. When the leaves again become susceptible (after 24 h), membrane ATPase activity is fully restored. 2. 2. Inhibitors of membrane ATPase activity protect susceptible leaves from the effects of helminthosporoside (KF, EDTA, and octylguanidine). 3. 3. Helminthosporoside activates (30%) membrane ATPase in microsomes from susceptible, but not heat-treated (resistant) leaves. Once heat-treated leaves again become susceptible, helminthosporoside activation of membrane ATPase activity resumes. 4. 4. A plot of the production of helminthosporoside-induced symptoms, and membrane ATPase activity as a function of the reciprocal of the absolute temperature reveals that both have sharp breaks at 32°C. 5. 5. Protoplasts of susceptible cane are rendered insensitive to the effects of the toxin in a medium deficient in K+ and Mg2+. When these ions are added, cell sensitivity to the toxin is restored. Since K+ uptake in plants is mediated by membrane ATPase, a connection with this enzyme activity can be made to cell sensitivity to the toxin.

Contrary to earlier reports, we have been able to demonstrate the presence of (Na++K+)-activated, ouabain-inhibited ATPase activity in rabbit polymorphonuclear leukocyte membranes. These results coupled with others suggest that (Na++K+)-ATPase and not cation-sensitive phosphatases are responsible for the regulation of the electrolyte content in these cells.

We present a revised method for the preparation, storage and assay of the Cl−-ATPase activity responsible for salt secretion in Limonium vulgare. Altering the centrifugation step improved the yield and a linked-enzyme assay for ADP provided a sensitive method for continuous monitoring of Cl−-ATPase activity. The activity of untreated membranes was low but fairly stable. Treatment with detergent gave strong stimulation of Cl−-ATPase activity but caused a rapid decline in activity with time. V app was approx. 6.5 μmoles/h per mg protein and K m, app for ATP between 0.1 and 0.2 mM. Cl− stimulated the activity up to a maximum at 0.13 M Cl−, and the pH optimum was around 6.3 to 6.4.

1. 1. Human erythrocyte membranes were preincubated with ethyleneglycolbis( β- aminoethyl )-N,N′ tetraacetate (EGTA) and subsequently labelled for short periods with micromolar concentrations of [8- 3 H , γ- 32 P]ATP . Under these conditions, and at temperatures ≦ 22 ° C , both ATP hydrolysis and membrane phosphorylation were stimulated by Ca2+ 2. 2. The properties of the Ca2+-stimulated ATP hydrolysis and associated phosphorylation of a 150 000 molecular weight protein component, previously described (Knauf, P. A., Proverbio, F. and Hoffman, J. F. (1974) J. Gen. Physiol. 63, 324–336), have been studied. The behavior of the phosphorylated component, ECaP, has properties consistent with its role as a phosphorylated intermediate of Ca2+ATPase activity, including: (1) similar dependence of the steady-state level of ECaP and Ca2+-ATPase on ATP concentration; (2) rapid turnover apparent upon the addition of excess non-radioactive ATP; and (3) good correlation between the steady-state levels of Ca2+-dependent phosphorylation and Ca2+-ATPase activity in separate preparations possessing variable specific activity. Addition of excess EGTA to ECaP caused only partial dephosphorylation. Sensitivity of Ca2+-stimulated ATP hydrolysis and associated phosphorylation to micromolar concentrations of Ca2+ implicates this activity in the “high-affinity” Ca2+-pump system of the human erythrocyte (Schatzmann, H. J. (1973) J. Physiol. London 235, 551–569).

The concentrations of n-butanol and tetracaine required for 50% inhibition of the ATPase activity of F1 particles isolated from bovine heart mitochondria were 160 mM and 1.1 mM, respectively. The results are offered as evidence that the physiological effects of these anesthetics may be due to direct interaction with membrane proteins rather than with the lipids.

Myosin and subfragment-1 were prepared from rabbit hearts hypertrophied secondary to pulmonary artery constriction. The Ca2+ -stimulated ATPase activity was reduced while the potassium/EDTA-stimulated ATPase activity was unchanged in both the myosin and subfragment 1 (S-1) from hypertrophied hearts. When hypertrophy myosin was mixed with an equal quantity of control myosin, the ATPase activity of the mixed protein fell halfway between control and hypertrophy values. Similar results were obtained with control and hypertrophy S-1. The actin-stimulated ATPase activity of hypertrophy S-1 was slightly depressed but unlike hypertrophy myosin this depression was not significant when compared to normal S-1. This suggests that papain cleavage may have removed part of the conformational difference that exists between control and hypertrophy myosins.

1. The lag time before maximum velocity of ATP hydrolysis is reached upon mixing ATP with F1 is much greater than can be explained by a simple Michaelis-Menten mechanism, and must be due to an activation reaction. The lag time is dependent on the concentration of MgATP (half-maximal at 30μM) and is equal to 30 ms at infinite MgATP concentration. The initial rate of hydrolysis by nucleotide-depleted F1 is much greater than with normal F1. It is tentatively suggested that the activation reaction with normal preparations is due to replacement of firmly bound ADP by MgATP. 2. After the initial time lag, the reaction follows very closely first-order kinetics provided that the concentration of MgATP is much less than the Km and the reaction is completed within 2 s. This is not expected if the dissociation constant of the enzyme-MgADP complex, an intermediate in the enzymic reaction, is much lower than the Km as has been reported in the literature. The value of V/Km, calculated from the exponential decay, is very close to that calculated from independent measurements of V and Km. 3. The low values for Ki(ADP) reported in the literature were found to be due to a slow (in the order of seconds) formation of an inhibited MgADP-enzyme complex. Dissipation of this inhibited complex by ATP requires seconds. The dissociation constant of the MgADP-enzyme complex that is an intermediate in the enzyme reaction was found to be 150 μM. 4. ADP but not ATP becomes firmly bound to nucleotide-depleted F1 in the absence of Mg2+.

1. 1. In the presence of 150 mM Na+, increasing K+ levels initially stimulated (K+ < 5 mM) and then inhibited ouabain-sensitive ATPase activity in cattle erythrocyte ghosts. 2. 2. Ouabain-sensitive K+ uptake measurements in high Na+ medium showed that the external affinity for K+ was about the same for high or low K+ cells. 3. 3. K+ inhibition of ATPase was greatest in ghosts derived from low K+ cells, and least in ghosts from high K+ cells, but a spectrum of sensitivities was found, correlating with the original red cell K+ level. 4. 4. Sensitization with sheep anti-L reagent gave an increased overall ouabain-sensitive ATPase activity, with a small decrease in the apparent affinity for the K+-inhibition component.

1. 1. Preincubation of purified ( Na + + K + )- ATPase (ATP phsophohydrolase, EC 3.6.1.3) preparations from rabbit kidney outer medulla with 5,5′-dithiobis-(2-nitrobenzoic acid) inhibits the ( Na + + K + )- ATPase and K+-stimulated 4-nitrophenylphosphatase activities. Phosphorylaton of the enzyme by ATP and the Na+-stimulated ATPase activity are inhibited to the same extent as the ( Na + + K + )- ATPase activity, whereas the K+-stimulated 4-nitrophenylphosphatase activity is inhibited much less. 2. 2. Titration with 5,5′-dithiobis-(2-nitrobenzoic acid) in sodium dodecyl sulphate shows the presence of 36 reactive sulfhydryl groups per molecule ( Na + + K + )- ATPase ( M r = 250 000 ). 3. 3. Treatment with N- ethylmaleimide , resulting in complete inhibition of ( Na + + K + )- ATPase activity, leads to modification of 26 sulfhydryl groups, whereas treatment with 5,5′-dithiobis-(2-nitrobenzoic acid) results in modification of 12 sulfhydryl groups under the same conditions. 4. 4. The reaction of N- ethymaleimide with an essential SH-group is not prevented by previous blocking of sulfhydryl groups with 5,5′-dithiobis-(2-nitrobenzoic acid). 5. 5. These findings indicate the existence of at least two classes of sulfhydryl groups on the enzyme, each containing at least one vital group. The difference between these classes consists in their differnt reactivity towards 5,5′-dithiobis-(2-nitrobenzic acid) and N- ethylmaleimide .

Gastric microsomes do not contain any significant Ca2+-stimulated ATPase activity. Trypsinization of pig gastric microsomes in presence of ATP results in a significant (2–3-fold) increase in the basal (with Mg2+ as the only cation) ATPase activity, with virtual elimination of the K+-stimulated component. Such treatment causes unmaksing of a latent Mg2+-dependent Ca2+-stimulated ATPase. Other divalent cations such as Sr2+, Ba2+, Zn2+ and Mn2+ were found ineffective as a substitute for Ca2+. Moreover, those divalent cations acted as inhibitors of the Ca2+-stimulated ATPase activity. The pH optimum of the enzyme is around 6.8. The enzyme has a K m of 70 μM for ATP and the K a values for Mg2+ and Ca2+ are about 4 · 10 −4 M and 10−7 M, respectively. Studies with inhibitors suggest the involvement of sulfhydryl and primary amino groups in the operation of the enzyme. Possible roles of the enzyme in gastric H+ transport have been discussed.

The K inhibition of ouabain-sensitive ATPase activity of LK goat red cell membranes is greatly reduced at high pH. This effect is reversible, and specific, since the apparent affinities for ATP, ouabain or external K do not alter. Anti-L-treated membranes show a similar alkali-induced affinity change, but have a lower pH optimum.

Under conditions in which palmityl-CoA inhibits adenine nucleotide translocase of heart and liver mitochondria the following energy-dependent functions of mitochondria were unaffected: reversed-electron flow, Sr 2+ accumulation, and low-amplitude swelling. Intramitochondrial phosphorylations accompanying α-ketoglutarate oxidation also appeared to remain unaffected by added palmityl-CoA. Evidence obtained suggests that the weak inhibition of adenine nucleotide translocase by (−)-palmitylcarnitine results due to its convertibility to palmityl-CoA because under identical conditions (+)-palmitylcarnitine was without much effect. Of several acyl-CoA esters tested palmityl-CoA was the most inhibitory for the 2,4-dinitrophenol-stimulated ATPase activity of rat heart mitochondria. The palmityl-CoA inhibition was more marked at 10° C than at 28° C, presumably because the adenine nucleotide translocase limited the ATPase activity at the lower temperature only.

1. (1) The response of the Ca 2+ -ATPase activity from human red cell membranes to ATP concentrations can be represented by the sum of two Michaelislike curves: one with a K m of 2.5 μM and the other with a K m of 145 μM. 2. (2) The maximum Ca 2+ -ATPase activity elicited by occupation of the site with lower K m represents about 10% of the activity attainable at non-limiting ATP concentrations. 3. (3) 30–50% of the Ca 2+ -ATPase activity with lower K m remains in the absence of Mg 2+ . Mg 2+ increases V and the maximum effect of Ca 2+ , having no effect on the apparent affinities for ATP and Ca 2+ . 4. (4) The large increase in Ca 2+ -ATPase activity which results from the occupation of the site with higher K m only takes place when Mg 2+ is present. 5. (5) Results are compatible with the idea that the Ca 2+ -ATPase from human red cell membranes has two classes of site for ATP binding, both of which are occupied when the enzyme catalyzes the hydrolysis of ATP at maximum rate. 6. (6) The properties of the high affinity site suggest that this is the catalytic site of the Ca 2+ -ATPase. It is proposed that binding of ATP at the low affinity site regulates the turnover of the system.

Monospecific antisera were prepared against the most prominent arylamidase (alpha-aminoacyl-peptide hydrolase (microsomal), EC 3.4.11.2) active antigen in plasma membranes (the plasma membrane arylamidase) and lysomal content (the lysosomal content arylamidase), respectively. Plasma membrane extract and lysosomal content were allowed to react in crossed immunoelectrophoresis against their homologous antisera. The electrophoretic plates were washed extensively, dried and subsequently stained for arylamidase activity. The particular immunoprecipitates were thus identified and could be excised to be used for immunizations. The two resulting antisera precipitated the arylamidase used for immunization, but failed to be monospecific as they precipitated additional antigens. These antisera with restricted specificity against some plasma membrane and lysosomal content antigens, respectively, were used to produce immunoprecipitates intended for new attempts to prepare monospecific antisera by a second cycle of immunizations. A monospecific antiserum against the plasma membrane arylamidase was thus obtained, while a third cycle of immunizations was needed to get a monospecific anti-lysosomal content antiserum. The plasma membrane arylamidase showed ATPase activity also after precipitation with the monospecific antiserum, thus still retaining its characteristics as a multienzyme complex.

The active-streaming experiments of Oplatka et al. (Oplatka, A. and Tirosh, R. (1973) Biochim. Biophys. Acta 305, 684-688 and Oplatka, A. Gadasi, H., Tirosh, R. Lamed, Y., Muhlrad, A. and Liron, N. (1974) J. Mechanochem. Cell Motil. 2, 295-306) with actomyosin solution in a glass microcapillary is reexamined under various conditions with several kinds of reference material. It is found that vigorous streaming took place in the actomyosin solution as reported by Oplatka et al. However, streaming which is indistinguishable from that observed in the actomyosin solution in the presence of actomyosin ATPase activity also occurred, even when the ATPase activity was blocked. The streaming also cannot be confirmed as being active when using acto-heavy meromyosin solution. There is a possibility that the streaming experiment provides interesting information on the microscopic state of solutions which is not directly related to the chemo-mechanical conversion.

The migration of intestinal epithelial cells from the crypt area to the villus tip is associated with progressive differentiation of these cells. The distribution of (Na+K+) stimulated adenosinetriphosphatase ((Na+K+)-ATPase; EC 3.6.1.3) along the intestinal villus may have functional as well as developmental implications. To define this distribution, rat jejunal and ileal segments were incubated in vitro with a citrate solution that dissociates epithelial cells sequentially from villus tip to crypt area. ATPase activity in cell collections from villus tips and crypt areas were compared. The specific activity of (Na+K+)-ATPase was higher in the villus tip than in the crypt cells of both jejunum and ileum. Crypt cell (Na+K+)-ATPase activity in the jejunum and ileum were similar. Thus, (Na+K+)-ATPase activity of villus tip cells in the jejunum was greater than in the ileum. There was no difference in villus tip and crypt cell Mg2+-ATPase activity in either jejunum or ileum. The steep gradient for (Na+K+)-ATPase along the intestinal villus may signify an improtant difference in Na+ transport between the villus tip and crypt area. The higher level of (Na+K+)-ATPase activity in the jejunal villi is consistent with the more important role of the jejunum in Na+ and substrate-linked Na+ transport.

1. 1. Platelet actomyosin superprecipitation performed in 0.09 M to 0.12 M KCl showed a biphasic curve with clearing and turbidity increase after addition of Mg-ATP. 2. 2. Addition of ADP produced inhibition of the duration and extent of the clearing phase (Km of 4 · 10−5 M), and activation of the superprecipitation phase (Km of 1.96 · 10−5 M). 3. 3. The effect of ADP was observed with no significant increase of ATPase activity and was apparently specific for ADP and not for GDP, UDP, or IDP when superprecipitation was initiated only with Mg-ATP. 4. 4. The effect of ADP was observed when superprecipitation occurred in the presence of EGTA. These data suggest that ADP may form part of a control mechanism of platelet contractile activity.

Stimulation of the Escherichia coli ATPase activity by urea and trypsin shows that there is a latent ATPase activity in particles and in a crude coupling factor of E. coli . Moreover, crude coupling factor, completely dissociated by treatment with 7 M urea, can inhibit the ATPase activity of the crude coupling factor. It is suggested that the latency of the ATPase activity of the coupling factor is due to the presence of a protein, the ATPase-inhibitor.

An ATPase activity specifically stimulated by micromolar Ca2+ concentrations has been identified in association with rabbit neurophil membranes. These studies provide the basis of further characterization of the Ca2+-ATPase activity with regard to neutrophil function.

At a free Mg 2+ concentration of 1.0 mM, myosin binds one Ca 2+ per molecule when the Ca 2+ concentration is 20 μM, a value in the concentration range expected during contraction of skeletal muscle. Mg 2+ alters Ca 2+ binding in a complex manner, not by simple competition. In the range from 20 to 100 μM Mg 2+ it produces positive cooperativity between the high-affinity Ca 2+ binding sites, in addition to shifting binding to higher Ca 2+ concentrations. High-affinity Ca 2+ binding is not significantly affected by the addition of ATP, increase in ionic strength to 0.1 and changes in temperature. Ca 2+ binding did not increase actin-activated ATPase activity in the absence of regulatory proteins, but rather inhibited it.

By trypsin treatment of highly purified ATPase (EC 3.6.1.3) from Micrococcus sp. ATCC 398E, two enzyme modifications have been obtained. 1. (i) ATPase Ta, which has about the same activity as untreated ATPase. 2. (ii) A protein complex Ti, which lacks ATPase activity, but nevertheless binds ATP as shown by affinity chromatography. Trypsin primarily shortens the α-chains of the “native” enzyme to α′-chains and removes the γ-subunit, thus yielding ATPase Ta. The formation of the protein complex Ti appears to be due to additional cleavage of one α′-chain into at least two more fractions.

The antitumour antibiotic, adriamycin, inhibited oxidative phosphorylation in freshly prepared mitochondria from the heart, liver and kidney of the rat. It abolished respiratory control and stimulated ATPase activity. Succinate oxidation by heart mitochondria was extremely sensitive to the drug when hexokinase was present in the reaction medium. The sensitive site has been identified to lie in the region between the succinate dehydrogenase flavoprotein and ubiquinone of the respiratory chain.

α-Actinin increases the ATPase activity of actin by up to 84%, depending on pH, divalent cations present and the added Mg 2+ : ATP ratio. Dithiothreitol decreases actin ATPase activity approx. 20% but does not reduce the ability of α-actinin to increase actin ATPase activity. Increasing amounts of added α-actinin up to 1 mol α-actinin to 49 mol actin cause an increasing increment in actin ATPase activity, but adding α-actinin beyond 1 mol α-actinin to 49 mol actin elicits only small additional increments in activity. Actin ATPase activity ranges from approx 100 nmol P i /mg actin per h (4.3 mol P i /mol actin per h) at high levels (10 mM) of ATP in the presence of lower amounts (1 mM) of added Mg 2+ to approx. 12.5 nmol P i /mg actin per h (0.52 mol P i /mol actin per h) at high pH (8.5) or at low levels (0.5–1.0 mM) of ATP in the presence of higher amounts (10 mM) of added Mg 2+ . ATP uncomplexed with Mg 2+ inhibits the ability of α-actinin to increase F-actin ATPase activity. Activities with different divalent cations showed that the actin ATPase in these studies, which was 1/100 as great as Mg 2+ -modified actomyosin ATPase activity, was not due to trace amounts of myosin contaminating the actin preparations. The results are consistent with the concept that α-actinin can alter the structure of actin monomers.

Volume changes in glycerol-extracted myofibrils were studied. The effect of pH, ion content and ATP concentration on the change in volume was determined. A volume change greater than that attributed to ATP dephosphorylation was observed. The magnitude of the volume change of the entire system was 10−3 cm3 whereas that due to dephosphorylation was 10−6 cm3. The ratio of ATPase activity in a volume decrease to that in a volume increase was found to be approx. 10:1. The total amount of volume decrease measured approx. 0.028 cm3/g of muscle with a fresh myo fibril preparation. The volume increase which was observed upon the addition of large amounts of ATP amounted to approx. 0.036 cm3/g of muscle. The relationship between the volume changes and molecular reorganization of the myofibril proteins was discussed. It was concluded that either a large configurational change occurs in the myofibril proteins during an ATP-induced contraction or relaxation, or that the volume changes are related to the dissociation and association of actin and myosin filaments

Sarcolemma tubes isolated from rabbit skeletal muscle exhibit a predominantly radial contraction which can be induced by ATP, Zn2+, and Cd2+ and to a smaller extent by ADP, SCN−, Ca2+ and Mg2+. The contraction is an intrinsic property of the sarcolemma, since it cannot be attributed to osmotic effects or to the activity of adhering myofibrils. Parallel studies on the surface ATPase activity of isolated semitendinosus muscles of the frog show that this enzymatic activity is inhibited reversibly by Zn2+ or Cd2+. The results are related to the twitch potentiating effects of some of the substances tested.

1. 1. Highly pure myelin fractions have been obtained from whole rat brains of various developmental stages. They contained 0.13–0.26% nucleic acids and a total ATPase activity of 0.3–0.8 μmole P per mg per h. 2. 2. These fractions contained a relatively constant amount of total ganglioside N -acetylneuraminic acid at all ages, but the distribution of individual major gangliosides changed drastically as the animal matured. The proportion of the normal major monosialoganglioside in myelin was higher than that in whole brain even at 15 days of age. With increasing age, its proportion increased rapidly reaching 90 mole % of total ganglioside in myelin by the age of 5 months. 3. 3. It is not known whether the gangliosides in the myelin fractions are intrinsic myelin components, or present in contaminating subcellular structures.

1. 1. The effect of Ca2+ on the ATPase activity of pig kidney alkaline phosphatase has been studied under a number of conditions. 2. 2. Contrary to an earlier report, it was found that Ca2+ and Mg2+ did not prevent substrate inhibition by ATP at either pH 8.0 or 9.0. At inhibitory levels of ATP however, activity was always greater when Mg2+ or Ca2+ was present. 3. 3. The effect of Ca2+ on ATPase activity was pH-dependent. At pH 8.0, increasing cation concentrations up to 50 mM brought about a doubling in enzymic activity. The inclusion of 1 mM Mg2+ together with Ca2+ resulted in further activation. At pH 9.0 ATPase was stimulated by Ca2+ at concentrations up to 10 mM but higher levels resulted in a fall in activity. The activity at a concentration of 50 mM Ca2+ was still greater, however, than with ATP alone. Because of Ca2 is inhibitory only at higher pH values, Ca2+ appears to lower the pH optimum of the ATPase reaction. 4. 4. The presence of 5 mM Ca2+ overcomes the inhibition of ATPase by Mg2+ at pH 9.0, but at pH 8.0, Ca2+ had little effect on the response of reaction velocity to Mg2+ concentration. 5. 5. These findings are discussed in terms of the proposed identity of alkaline phosphatase with a Ca2+-stimulated ATPase important in the transport of Ca2+ across biological membranes.

1. 1. Active K + transport and the distribution of the L and M antigens have been investigated in sheep and goat red cells. 2. 2. Low K − (LK) type goat red cells have a significantly lower rate of active K + uptake than high K + (HK) type cells. Active K + transport and the associated (Na + - K + -activated ATPase activity in LK goat red cells was stimulated 1.5-8-fold by sensitization with sheep anti-L, even though the presence of this antibody could not be detected by complement lysis. 3. 3. Ether and acid eluates of anti-L from sensitized goat and sheep red cells haemolysed sheep but not goat red cells and stimulated active K + transport in both goat and sheep red cells. There was no apparent correlation between the presence of the M antigen and K + types in goats as there is in sheep. 4. 4. It is suggested that there may be two specificities of L substance, one associated specifically with the K + pump in sheep and goat red cells and not readily detectable serologically, and the other, found only on sheep red cells which may or may not be associated with the pump but which is easily detected by haemolytic tests.

1. 1. Myosin was extracted from dog hearts and isolated by three methods which utilize ammonium sulfate fractionation and by two modifications of SZENT-GYORGYI'S dilution-precipitation method. 2. 2. The molecular weight of the myosin was determined by the YPHANTIS meniscus-depletion equilibrium method. 3. 3. Velocity sedimentation of the myosin using schlieren optics never revealed aggregation. Calculation of the molecular weight by the YPHANTIS method, however, revealed that several myosin samples were disperse. There is some evidence that dispersity may be avoided by using a method which includes a final high speed centrifugation. 4. 4. A statistically significant difference (P < 0.025) was demonstrated between the mean molecular weight, 4.76·105, of myosin prepared by MUELLER'S modification of SZENT-GYORGYI'S method and the mean molecular weight, 5.37·105, of the myosin prepared by all the methods which utilized ammonium sulfate fractionation. 5. 5. The best estimate of the molecular weight of myosin is 4.76·105. 6. 6. The calcium-activated ATPase activity was unaffected by the method of preparation.

1. 1. The effects of oligomycin and dicyclohexylcarbodiimide on particulate mitochondrial ATPase from rat liver and on several preparations with Na+,K+-stimulated ATPase activity were compared. 2. 2. In contrast to oligomycin, dicyclohexylcarbodiimide produced a weak inhibition of Na+,K+-stimulated ATPase from rabbit kidney, calf heart and human erythrocyte ghosts. Moreover, it was unable to reproduce the stimulating effect of oligomycin on p-nitrophenylphosphatase activity in the presence of limiting amounts of K+. 3. 3. The Na+,K+-stimulated ATPase preparations were found to be more sensitive to oligomycin than generally assumed; the levels for half-maximal inhibition were only 4–40 times higher than those needed for mitochondrial ATPase. 4. 4. Similarly to oligomycin, dicyclohexylcarbodiimide scarcely inhibited particulate mitochondrial ATPase if phospholipids or triolein were included in the incubation medium. 5. 5. It is concluded that oligomycin in a comparable range of concentrations is able to influence several mammalian ATPases associated with membranes, whereas the effect of dicyclohexylcarbodiimide is much more specific for mitochondrial ATPase. Inhibition of other ATPase systems from mammalian cells is produced at concentrations about 1000 times higher. On the basis of the antagonism by phospholipids it is proposed that the effect of both inhibitors is made possible by the presence of phospholipids in these structurally organized ATPase systems.

1. 1. The influence of pH on the ATPase activities of NaI-treated microsomes from pig brain has been investigated with Mg2+, Mn2+, Ca2+ or Zn2+ as divalent metal cofactor. Some ouabain-inhibitable ATPase has been found with each of these cations even without addition of alkali metal cations. 2. 2. The pH profile of ouabain-sensitive Mg2+-ATPase varied in different preparations of NaI-microsomes. Some preparations had three pH optima at pH values 5.5, 7.4 and 9.0, corresponding to the pH optima for the K+-, (Na+-K+)- and Na+-ATPases of the same preparation. 30 mM K+ inhibited Mg2+-ATPase activity to an extent and with a pH dependence similar to ouabain. 3. 3. ATPase activities with Mn2+ were usually similar to those with Mg2+, except that at alkaline pH Mn2+ was inhibited by 100 mM Na+. Na+ also reduced ouabain-insensitive Mn2+-ATPase activity at high pH. 4. 4. Ca2+-ATPase was optimal at pH 9 and was slightly inhibited by ouabain. Low concentrations of Na+ stimulated Ca2+-ATPase at pH 7.5, while joint addition of 100 mM Na+ and 30 mM K+ stimulated ouabain-inhibitable ATPase activity with a pH optimum of 7.7. 100 mM Na+ alone inhibited Ca2+-ATPase but the ouabain-sensitivity of Ca2+-ATPase activity was increased in the presence of Na+. 5. 5. Zn2+-ATPase activity was greatest at pH 5.5 and was partially inhibited by ouabain. 30 mM K+ inhibited Zn2+-ATPase with a pH-dependence and extent very similar to ouabain at pH values up to 7. Both 4 mM Na+ and joint addition of 100 mM Na+ and 30 mM K+ stimulated ouabain-inhibitable Zn2+-ATPase activity with pH optima of 7.4 and 6–6.5 respectively. 6. 6. NaI-microsomes also had Mg2+- and Ca2+-ITPase activities, but Zn2+-ITPase was very low. Mg2+-ITPase was activated by Na+ but not by K+.

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 Pi 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.

1. 1. Methods have been developed for the isolation of myosin A-like protein (plasmodium myosin A) from a myxomycete plasmodium. 2. 2. Plasmodium myosin A is soluble in 0.5 M KCl solution at pH 7.0. Its molecular characteristics are examined and compared with those of myosin A from rabbit striated muscle. The sedimentation coefficient ( s 20, w ) is 6.05 S which is the same order as that of muscle myosin A. However, the viscosity ([ η ] = 1.60 dl/g) is only about two-thirds of that of muscle myosin A. 3. 3. Generally speaking, amino acid composition of plasmodium myosin A is very similar to that of muscle myosin A, but cysteine is not detected in plasmodium myosin A as far as we have examined. 4. 4. ATPase activity of plasmodium myosin A is as high as that of muscle myosin A, and it is activated by Ca 2+ and inhibited by Mg 2+ . Plasmodium actin as well as muscle actin activates ATPase of plasmodium myosin A. ATPase activity of plasmodium myosin A is always inhibited by EDTA and p -chloromercuribenzoate, even at low concentrations, whereas ATPase of muscle myosin A is greatly activated at such low concentrations of EDTA and p -chloromercuribenzoate. 5. 5. Plasmodium myosin A is completely soluble and forms only small aggregates with sedimentation coefficients which are about 9 S at low salt concentrations, including the physiological salt concentration of plasmodium (0.03 M KCl) at pH 7.0.

A study has been made of the consequences of the substitution of about half of the sulfhydryl groups in myosin or actin (or both) by reaction with 2-aminoethyl isothiuronium (AET). With respect to myosin, this substitution makes the ATPase activity specifically dependent on the presence of calcium ions, and independent of the suppression normally encountered at high ionic strength. AET-substitution of myosin makes the resulting actomyosin unresponsive to α-actinin; that of actin does not affect the responses to α-actinin. Either substitution diminishes the binding of α-actinin to actomyosin. Neither substitution abolishes contractility as measured in terms of gel synaeresis.

Some properties of the Mg 2+ -dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) system of pig red-cell ghosts are described. When the membrane structure had not been much damaged during the preparative procedure, this activity was weak but could be stimulated up to 3-fold by 2.4-dinitrophenol and up to 5-fold by Sr 2+ . Various kinds of injury to the membrane structure such as severe disruption or action of detergent led to a strong enhancement (‘unmasking’) of the normally latent ATPase activity. This was always accompanied by a loss of ability of the ATPase to be stimulated by 2,4-dinitrophenol and Sr 2+ . The possible relationship of this ATPase activity to energy-linked functions of the red-cell membrane are discussed.

1. 1. Ammonium sulphate salting-out curves of myofibrillar proteins prepared from muscles in contracture after thawing show that most of Dubuisson 's γ-myosin corresponds to a component which precipitates at 12–18% of saturation. The same results have been obtained in muscles in rigor mortis. 2. 2. The presence of increased amounts of γ-myosin in the myofibrillar-protein solutions causes a decrease of ATPase activity and a lower viscosity. 3. 3. The extractability of the myofibrillar proteins is strongly decreased in contracture after thawing; nevertheless, the increase of γ-myosin seems to be only relative.

1. The naturally occurring mitochondrial ATPase inhibitor inhibits the mitochondrial ATPase (F1) non-competitively. 2. The interaction between inhibitor and inhibitor-depleted F1 or submitochondrial particles is diminished when the ratio of ATP/ADP is low or when energy is generated by substrate oxidation. 3. The dissociation of the inhibitor from coupled Mg-ATP particles is promoted when substrates are being oxidized. This results in the appearance of a large uncoupler-stimulated ATPase activity. Activation of the uncoupler-stimulated ATPase activity is also achieved by incubation of the particles with ADP. 4. The ATPase activity of Mg-ATP particles is determined by the turnover capacity of F1. When endogenous inhibitor is removed, energy dissipation becomes the rate-limiting step. This energy dissipation can be activated by an uncoupler. 5. Evidence is presented for the existence of a non-inhibited intermediate F1-inhibitor complex.

The Mg2+-activated ATPase activity of reconstituted actomyosin at high ionic strengths (about 0.10 M KCl) is stimulated when Na+ is substituted for K+. This phenomenon does not appear to reflect the non-specific structure-disrupting effects seen with higher concentrations of these alkali metal ions because inhibition of actomyosin ATPase followed the order Cs+ > Li+ > Rb+ = K+ > Na+. Alkali metal sensitivity was similar in the case of actomyosins reconstituted from tropomyosin-free actin preparations and from actin preparations that included the tropomyosin-containing protein complex that sensitizes actomyosin to Ca2+. Modification of the myosin, effected by blocking some of the more reactive SH groups, abolished the sensitivity of the subsequently reconstituted actomyosin to substitution of K+ by Na+. Sensitivity to replacement of K+ by Na+ was seen only during the clearing phase at higher ionic strengths, and no significant differences between the effects of these alkali metal ions were seen at lower ionic strengths or after superprecipitation. These different actions of the alkali metal ions thus appear to be upon the association of actin and myosin in the presence of Mg2+.

Angiotensin increased microsomal (Na + K + )-activated ATPase from rat hypothalamus, mucosa of colon and bovine adrenal cortex but not brain cortex. Angiotensin did not change significantly Mg 2+ -ATPase in these tissues. Enhancement by angiotensin was evident at concentrations of 10 −8 − 10 −12 M. In bovine adrenal cortex angiotensin increased ATPase activity in the outer and not in the inner layer. Angiotensin increased ATPase activity with Na + above 20 mM and K + above 5 mM present simultaneously, but not with either ion alone.