Photoaffinity labeling of the (Ca+Mg)ATPase of skeletal and cardiac sarcoplasmic reticulum with [γ-32P]-8-azido-ATP

8-azido-ATP, when used in the 0.2–5 μM concentration range, fulfills the criteria for a specific photoaffinity label for the (Ca+Mg)ATPase of sarcoplasmic reticulum. It is a substrate for the enzyme. It is a mixed inhibitor of ATPase activity. When photolyzed at 0° it is an inhibitor of ATPase activity. The photoinduced binding of 8-azido-ATP to the (Ca+Mg)ATPase is promoted by Ca²⁺. The dependence of the labeling of the (Ca+Mg)ATPase on 8-azido-ATP, Ca²⁺ and Mg²⁺ concentrations strongly suggests that 2 classes of sites are labeled. When 10–60 μM 8-azido-ATP was used to label sarcoplasmic reticulum, proteins in addition to the (Ca+Mg)ATPase were labeled.     

(Ca + Mg)-stimulated ATPase activity of a rat brain microsomal preparation.

ATPase activity of freshly prepared brain microsomes was stimulated 20% when 0.1 mm CaCl₂ was added in the presence of a “saturating” concentration of MgCl₂ (4 mm). This (Ca + Mg)-stimulated activity declined rapidly on storage. Treatment of the microsomes with 0.12% deoxycholate in 0.15 m KCl, followed by centrifugation and resuspension in sucrose, produced a preparation both stable on storage at ?15 °C and with an increased stimulation in the presence of CaCl₂. SrCl₂ was more effective than CaCl₂, but BaCl₂ was a poor activator. KCl and NaCl stimulated the (Ca + Mg)-ATPase activity by reducing substrate (ATP) inhibition. The K_m for ATP was 0.1 mm, a third that of the Mg-ATPase. CTP, ITP, and GTP could not substitute for ATP, although they were fair substrates for the Mg-ATPase. The energy of activation of the (Ca + Mg)-ATPase was 21 kcal, nearly twice that of the Mg-ATPase. After sucrose density-gradient centrifugation of the microsomal preparation, the (Ca + Mg)-ATPase activity was distributed with the (Na + K)-ATPase and not with the mitochondrial marker succinic dehydrogenase. Studies with ouabain, oligomycin, and azide distinguished the (Ca + Mg)-stimulated ATPase from (Na + K)- and mitochondrial ATPases. Sensitivity to ruthenium red suggested a link to Ca transport, although the microsomal ⁴⁵Ca accumulating system was much more sensitive to the inhibitor than was this ATPase activity.     

Cyclic GMP-dependent protein kinase stimulates the plasmalemmal Ca2+ pump of smooth muscle via phosphorylation of phosphatidylinositol.

The effect of phosphorylation by cyclic GMP-dependent protein kinase (G-kinase) on the activity of the plasmalemmal Ca2+-transport ATPase was studied on isolated plasma membranes and on the ATPase purified from pig erythrocytes and from the smooth muscle of pig stomach and pig aorta. Incubation with G-kinase resulted, in both smooth-muscle preparations, but not in the erythrocyte ATPase, in a higher Ca2+ affinity and in an increase in the maximal rate of Ca2+ uptake. Cyclic AMP-dependent protein kinase (A-kinase) did not exert such an effect. The stimulation of the (Ca2+ + Mg2+)-dependent ATPase activity of the purified Ca2+ pump reconstituted in liposomes depended on the phospholipid used for reconstitution. The stimulation of the (Ca2+ + Mg2+)-ATPase activity by G-kinase was only observed in the presence of phosphatidylinositol (PI). G-kinase, but not A-kinase, stimulated the phosphorylation of PI to phosphatidylinositol phosphate (PIP) in a preparation of (Ca2+ + Mg2+)-ATPase obtained by calmodulin affinity chromatography from smooth muscle, but not in a similar preparation from erythrocytes. Adenosine inhibited both the phosphorylation of PI and the stimulation of the (Ca2+ + Mg2+)-ATPase by G-kinase. In the absence of G-kinase the (Ca2+ + Mg2+)-ATPase was stimulated by the addition of PIP, but not by PI. In contrast with previous results of Furukawa & Nakamura [(1987) J. Biochem (Tokyo) 101, 287-290], no convincing evidence for a phosphorylation of the (Ca2+ + Mg2+)-ATPase was found. Evidence is presented showing that the apparent phosphorylation occurs in a contaminant protein, possibly myosin light-chain kinase. It is proposed that G-kinase stimulates the plasmalemmal Ca2+ pump of smooth-muscle cells indirectly via the phosphorylation of an associated PI kinase.     

Inhibitory antibodies to plasmalemmal Ca2+-transporting ATPases. Their use in subcellular localization of (Ca2+ + Mg2+)-dependent ATPase activity in smooth muscle.

Antibodies directed against the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase [(Ca2+ + Mg2+)-dependent ATPase] from pig erythrocytes and from smooth muscle of pig stomach (antral part) were raised in rabbits. Both the IgGs against the erythrocyte (Ca2+ + Mg2+)-ATPase and against the smooth-muscle (Ca2+ + Mg2+)-ATPase inhibited the activity of the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase from smooth muscle. Up to 85% of the total (Ca2+ + Mg2+)-ATPase activity in a preparation of KCl-extracted smooth-muscle membranes was inhibited by these antibodies. The (Ca2+ + Mg2+)-ATPase activity and the Ca2+ uptake in a plasma-membrane-enriched fraction from this smooth muscle were inhibited to the same extent, whereas in an endoplasmic-reticulum-enriched membrane fraction the (Ca2+ + Mg2+)-ATPase activity was inhibited by only 25% and no effect was observed on the oxalate-stimulated Ca2+ uptake. This supports the hypothesis that, in pig stomach smooth muscle, two separate types of Ca2+-transport ATPase exist: a calmodulin-binding ATPase located in the plasma membrane and a calmodulin-independent one present in the endoplasmic reticulum. The antibodies did not affect the stimulation of the (Ca2+ + Mg2+)-ATPase activity by calmodulin.     

Cytoplasmic and plasma membrane adenosine triphosphatase of polymorphonuclear neutrophils: comparison of their enzymatic properties and attempt for a direct determination of myosin ATPase activity using polymorphonuclear neutrophil extract

Enzymatic properties of the ATPase of the plasma membrane and cytoplasmic myosin B from guinea-pig polymorphonuclear neutrophils were compared. In the plasma membrane, Mg2+- and Ca2+-activated ATPases showed the same dependence pattern on KCl concentration and pH, i.e., both ATPases increased with decreasing KCl concentration and with rising pH until pH 9.0. The maximum activation of Mg2+-ATPase was observed at 1 . 10(-3) M Mg2+. On the other hand, EDTA-activated ATPase activity was so low that no clear dependence curve was obtained. In myosin B, Mg2+-ATPase activity was below one-tenth that of the plasma membrane ATPase with the maximum activation at 1 . 10(-2) M Mg2+ and pH 9.0 EDTA- and Ca2+-activated ATPase exhibited almost the same activity and the same KCl-dependence curve, i.e., both ATPases increased and increasing KCl concentration. With regard to pH-dependence, Ca2+-ATPase showed a U-shaped curve with the minimum at pH 7.0, wherease EDTA-activated ATPase indicated a bell-shaped curve with the maximum at pH 9.0. Based on the findings that the EDTA-activated ATPase activity was hardly detected in the plasma membrane but high in myosin B, the distribution of ATPase activity on subcellular fractions was studied and the results obtained that the myosin-ATPase activity could be directly measured using the polymorphonuclear neutrophil extract if the EDTA-activated ATPase activity was used as an enzymatic marker for myosin.     

Effects of Ca2+ and Mg2+ on the actomyosin adenosine-5'-triphosphatase of stably phosphorylated gizzard myosin

There are conflicting reports on the effect of Ca2+ on actin activation of myosin adenosine-triphosphatase (ATPase) once the light chain is fully phosphorylated by a calcium calmodulin dependent kinase. Using thiophosphorylated gizzard myosin, Sherry et al. [Sherry, J. M. F., Gorecka, A., Aksoy, M. O., Dabrowska, R., & Hartshorne, D. J. (1978) Biochemistry 17, 4417-4418] observed that the actin activation of ATPase was not inhibited by the removal of Ca2+. Hence, it was suggested that the regulation of actomyosin ATPase activity of gizzard myosin by calcium occurs only via phosphorylation. In the present study, phosphorylated and thiophosphorylated myosins were prepared free of kinase and phosphatase activity; hence, the ATPase activity could be measured at various concentrations of Ca2+ and Mg2+ without affecting the level of phosphorylation. The ATPase activity of myosin was activated either by skeletal muscle or by gizzard actin at various concentrations of Mg2+ and either at pCa 5 or at pCa 8. The activation was sensitive to Ca2+ at low Mg2+ concentrations with both actins. Tropomyosin potentiated the actin-activated ATPase activity at all Mg2+ and Ca2+ concentrations. The calcium sensitivity of phosphorylated and thiophosphorylated myosin reconstituted with actin and tropomyosin was most pronounced at a free Mg2+ concentration of about 3 mM. The binding of 125I-tropomyosin to actin showed that the calcium sensitivity of ATPase observed at low Mg2+ concentration is not due to a calcium-mediated binding of tropomyosin to F-actin. The actin activation of both myosins was insensitive to Ca2+ when the Mg2+ concentration was increased above 5 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

DDT inhibition of Ca-Mg ATPase from peripheral nerves and muscles of lobster, Homarus americanus

Sensitivity of CaMg ATPase from axonic plasma membrane (APM) and sarcoplasmic reticulum (SR) of lobster, $\text{Homarus}\text{,}\text{americanus}$, to DDT was studied. The CaMg ATPase found in SR with the high Ca²⁺ affinity is sensitive to DDT while the portion of ATPase related to the low Ca²⁺ affinity site is not inhibited by DDT. Also, DDT is more inhibitory against the CaMg ATPase prepared from APM than the one obtained from SR. The relationship between inhibition of the CaMg ATPase by DDT in the axonic nerve membrane and $\text{in}\text{,}\text{vivo}$ poisoning symptoms of the nervous system is discussed.     

H-ATPase Activity from Storage Tissue of Beta vulgaris: III. Modulation of ATPase Activity by Reaction Substrates and Products

Two distinct membrane fractions containing H⁺-ATPase activity were prepared from red beet. One fraction contained a H⁺-ATPase activity that was inhibited by NO₃⁻ while the other contained a H⁺-ATPase inhibited by vanadate. We have previously proposed that these H⁺-ATPases are associated with tonoplast (NO₃⁻-sensitive) and plasma membrane (vanadate-sensitive), respectively. Both ATPase were examined to determine to what extent their activity was influenced by variations in the concentration of ATPase substrates and products. The substrate for both ATPase was MgATP²⁻, and Mg²⁺ concentrations in excess of ATP had only a slight inhibitory effect on either ATPase. Both ATPases were inhibited by free ATP (i.e. ATP concentrations in excess of Mg²⁺) and ADP but not by AMP. The plasma membrane ATPase was more sensitive than the tonoplast ATPase to free ATP and the tonoplast ATPase was more sensitive than the plasma membrane ATPase to ADP.

Inhibition of both ATPases by free ATP was complex. Inhibition of the plasma membrane ATPase by ADP was competitive whereas the tonoplast ATPase demonstrated a sigmoidal dependence on MgATP²⁻ in the presence of ADP. Inorganic phosphate moderately inhibited both ATPases in a noncompetitive manner.

Calcium inhibited the plasma membrane but not the tonoplast ATPase, apparently by a direct interaction with the ATPase rather than by disrupting the MgATP²⁻ complex.

The sensitivity of both ATPases to ADP suggests that under conditions of restricted energy supply H⁺-ATPase activity may be reduced by increases in ADP levels rather than by decreases in ATP levels per se. The sensitivity of both ATPases to ADP and free ATP suggests that modulation of cytoplasmic Mg²⁺ could modulate ATPase activity at both the tonoplast and plasma membrane.

     

The kinetics and divalent cation inhibition of plasma membrane ATPase in the yeast Candida albicans

The kinetics of ATP hydrolysis and cation effects on ATPase activity in plasma membrane from Candida albicans ATCC 10261 yeast cells were investigated. The ATPase showed classical Michaelis-Menten kinetics for the hydrolysis of Mg X ATP, with Km = 4.8 mM Mg X ATP. Na+ and K+ stimulated the ATPase slightly (9% at 20 mM). Divalent cations in combination with ATP gave lower ATPase activity than Mg X ATP (Mg greater than Mn greater than Co greater than Zn greater than Ni greater than Ca). Divalent cations inhibited the Mg X ATPase (Zn greater than Ni greater than Co greater than Ca greater than Mn). Free Mg2+ inhibited Mg X ATPase weakly (20% inhibition at 10 mM). Computed analyses of substrate concentrations showed that free Zn2+ inhibited Zn X ATPase, mixed (Zn2+ + Mg2+) X ATPase, and Mg X ATPase activities. Zn X ATP showed high affinity for ATPase (Km = 1.0 mM Zn X ATP) but lower turnover (52%) relative to Mg X ATP. Inhibition of Mg X ATPase by (free) Zn2+ was noncompetitive, Ki = 90 microM Zn2+. The existence of a divalent cation inhibitory site on the plasma membrane Mg X ATPase is proposed.     

The effects of caldesmon on the ATPase activities of rabbit skeletal-muscle myosin.

We studied the effects of caldesmon, a major actin- and calmodulin-binding protein found in a variety of muscle and non-muscle tissues, on the various ATPase activities of skeletal-muscle myosin. Caldesmon inhibited the actin-activated myosin Mg2+-ATPase, and this inhibition was enhanced by tropomyosin. In the presence of the troponin complex and tropomyosin, caldesmon inhibited the Ca2+-dependent actomyosin Mg2+-ATPase; this inhibition could be partly overcome by Ca2+/calmodulin. Caldesmon, phosphorylated to the extent of approximately 4 mol of Pi/mol of caldesmon, inhibited the actin-activated myosin Mg2+-ATPase to the same extent as did non-phosphorylated caldesmon. Both inhibitions could be overcome by Ca2+/calmodulin. Caldesmon also inhibited the Mg2+-ATPase activity of skeletal-muscle myosin in the absence of actin; this inhibition also could be overcome by Ca2+/calmodulin. Caldesmon inhibited the Ca2+-ATPase activity of skeletal-muscle myosin in the presence or absence of actin, at both low (0.1 M-KCl) and high (0.3 M-KCl) ionic strength. Finally, caldesmon inhibited the skeletal-muscle myosin K+/EDTA-ATPase at 0.1 M-KCl, but not at 0.3 M-KCl. Addition of actin resulted in no inhibition of this ATPase by caldesmon at either 0.1 M- or 0.3 M-KCl. These observations suggest that caldesmon may function in the regulation of actin-myosin interactions in striated muscle and thereby modulate the contractile state of the muscle. The demonstration that caldesmon inhibits a variety of myosin ATPase activities in the absence of actin indicates a direct effect of caldesmon on myosin. The inhibition of the actin-activated Mg2+-ATPase activity of myosin (the physiological activity) may not be due therefore simply to the binding of caldesmon to the actin filament causing blockage of myosin-cross-bridge-actin interaction.     

Biphasic steady-state kinetics of myosin adenosine triphosphatase. Evidence for a substrate effector site

The steady-state kinetics of the K+, Ca2+, and Mg2+-activated adenosine triphosphatase (ATPase) activities of rabbit skeletal myosin were investigated in the substrate concentration range from 0.05 microM to 5 mM and found not to follow Michaelis-Menten kinetics but rather to display biphasic behavior. The Ca2+-ATPase activity of myosin chymotryptic subfragment-1 (S-1), which has only one active site, also exhibits biphasic kinetics, thus excluding the possibility that the biphasic behavior is caused by negative cooperativity between the two active sites of myosin. Myosin K+ and Mg2+-ATPase are both activated by 5'-adenyl methylenediphosphonate (AdoPP[CH2]P) in a competitive manner at high substrate concentrations; i.e. the maximal velocity observed at high substrate concentrations is independent of the AdoPP[CH2]P concentration. This result provides evidence for substrate activation via binding to a regulatory site. Pyrophosphate inhibits myosin ATPase in a competitive manner at low substrate concentrations and in an uncompetitive manner at high substrate concentrations, with the uncompetitive Ki being smaller than the competitive Ki; i.e. pyrophosphate binds more tightly to the effector site than to the active site.     

Localization of endogenous ATPases at the nerve terminal

We have investigated the localization of a set of intrinsic ATPase activities associated with purified synaptic plasma membranes and consisting of (a) a Mg²⁺-ATPase; (b) an ATPase active at high concentrations of Ca²⁺ in the absence of Mg²⁺ (Ca_H-ATPase); (c) a Ca²⁺ requiring Mg²⁺-dependent ATPase (Ca + Mg)-ATPase, stimulated by calmodulin (Ca-CaM-ATPase); (d) a Ca²⁺-dependent ATPase stimulated by dopamine (DA-ATPase); and (e) the ouabain-sensitive (Na + K)-ATPase. The following results were obtained: (1) All ATPases are largely confined to the presynaptic membrane; (2) the DA-, (Ca + Mg)-, (Ca-CaM)-, and (Na + K)-ATPases are oriented with their ATP hydrolysis sites facing the synaptoplasm; (3) the Mg- and Ca_H-ATPases are oriented with their ATP hydrolysis sites on the junctional side of the presynaptic membrane and are therefore classified as ecto-ATPases of as yet unknown function.     

The rat liver plasma membrane high affinity (Ca2+-Mg2+)-ATPase is not a calcium pump. Comparison with ATP-dependent calcium transporter

The high affinity (Ca2+-Mg2+)-ATPase purified from rat liver plasma membrane (Lin, S.-H., and Fain, J. N. (1984) J. Biol. Chem. 259, 3016-3020) has been further characterized. This enzyme also possesses Mg2+-stimulated ATPase activity with K0.5 of 0.16 microM free Mg2+. However, the Vm of the Mg2+-stimulated activity is only half that of the Ca2+-stimulated ATPase activity. The effects of Ca2+ and Mg2+ on this enzyme are not additive. Both the Ca2+-stimulated ATPase and Mg2+-stimulated ATPase activities have similar affinities for ATP (0.21 mM and 0.13 mM, respectively) and similar substrate specificities (they are able to utilize ATP, GTP, UTP, CTP, ADP, and GDP as substrates); both activities are not inhibited by vanadate, p-chloromercuribenzoate, ouabain, dicyclohexylcarbodiimide, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, oligomycin, F-, N-ethylmaleimide, La3+, and oxidized glutathione. These properties of the Mg2+- and Ca2+-ATPases indicate that both activities reside on the same protein. A comparison of the properties of this high affinity (Ca2+-Mg2+)-ATPase with those of the liver plasma membrane ATP-dependent Ca2+ transport activity reconstituted into artificial liposomes (Lin, S.-H. (1985) J. Biol. Chem. 260, 7850-7856) suggests that this high affinity (Ca2+-Mg2+)-ATPase is not the biochemical expression of the liver plasma membrane Ca2+ pump. The function of this high affinity (Ca2+-Mg2+)-ATPase remains unknown.     

The sulfhydryl groups involved in the active site of myosin B adenosinetriphosphatase. I. Relantionship of the sulfhydryl group responsible for Mg2+-ATPase activation to the S1 and S2 groups.

The reactivity of the sulfhydryl groups in myosin B to N-ethylmaleimide (NEM) was investigated under various conditions. Under the conditions where actin and myosin associate, i.e. at low ionic strength, only Mg2+-ATPase [EC 3.6.1.3] activity was markedly activated by NEM treatment, whereas coupling of EDTA-ATPase inhibition with Ca2+-ATPase activation, which was seen on blocking S1 of myosin A with NEM, was observed under conditions at which the dissociation of actomyosin occurs, i.e. at high ionic strength, suggesting the covering with actin of the S1 region of myosin. Nevertheless, APT accelerated the reactivity of S1 and S2 much more in the myosin B system than in myosin alone. NEM-modified myosin B ATPase exhibited a shift of the KCL dependence curve to high concentration, a shift of the maximum activation of ATPase activity to high Mg ion concentration and a suppression of substrate inhibition at high substrate concentrations. These all indicate that the blocking by NEM of Sa, the sulfhydryl group related to the activation of Mg2+-ATPase of myosin B, brings about an increase in the association of myosin and actin in the myosin B system, resulting in an activation of Mg2+-ATPase activity. In addition, the relationship between Sa and a sulfhydryl group(s) essential for Ca2+ sensitivity was discussed.     

Modulation of monomer-polymer equilibrium of phosphorylated smooth muscle myosin: effects on actin activation

Actin activation of the adenosinetriphosphatase (ATPase) of phosphorylated gizzard myosin at low (2 mM) free Mg2+ concentration and 50 mM total ionic strength continues to increase on raising the free Ca2+ concentration near pCa 3. Similar levels of activity can be obtained by increasing the free Mg2+ concentration to a higher (in excess of 4 mM free) concentration. In the presence of micromolar concentrations of free Ca2+ and low free Mg2+ concentration, the actin-activated adenosine 5'-triphosphate (ATP) hydrolysis exhibits an initial rapid rate which progressively slows to a final, lower but more linear rate. In the presence of high divalent cation concentrations, the fast rate of ATP hydrolysis is maintained during the entire ATPase assay. The ionic conditions which favor the slow rate of ATP hydrolysis are correlated with increased proportions of folded myosin monomers while higher rates of ATP hydrolysis are correlated with increased levels of aggregated myosin. Elevating the thin filament proteins to saturating concentrations does not abolish the change in ATPase rate or the final distribution of myosin aggregates and monomers; however, the stability of the myosin aggregates is enhanced by the presence of thin filament proteins in low divalent cation conditions. The nonlinear profile of the actin-activated ATP hydrolysis in low divalent cation concentrations is eliminated by utilizing nonfilamentous, phosphorylated heavy meromyosin. The data presented indicate that Ca2+ and Mg2+ alter monomer-polymer equilibrium of stably phosphorylated myosin. The alteration of monomer-polymer equilibrium by Ca2+ at low Mg2+ concentration modulates ATPase rates.     

Vanadate inhibition of brain (Ca+Mg)-ATPase

Vanadate was a potent inhibitor of the membrane-bound (Ca+Mg)-ATPase from rat brain, the concentration required for 50% inhibition under conditions optimal for enzymatic activity being 3 M. Vanadate inhibition increased with the MgCl₂ concentration, half-maximal inhibition occurring at 2 mM MgCl₂, near the MgCl₂ concentration required for half-maximal activation of the ATPase activity. MnCl₂ could substitute for MgCl₂, and at concentrations of 1 mM (Ca+Mn)-ATPase activity was greater than (Ca+Mg)-ATPase activity, although sensitivity to vanadate was less. Vanadate inhibition increased also with the KCl concentration, half-maximal inhibition occurring at 8 mM, again near the concentration required for half-maximal activation of ATPase activity. By contrast, NaCl stimulated (Ca+Mg)-ATPase activity without potentiating vanadate inhibition. These effects of cations on ATPase activity and vanadate inhibition resemble properties of certain transport ATPases and thus suggest mechanistic and functional similarities.     

A comparative study of the rat heart sarcolemmal Ca2+-dependent ATPase and myosin ATPase

In order to gain some information regarding Ca²⁺-dependent ATPase, the enzyme was purified from cardiac sarcolemma and its properties were compared with Ca²⁺-ATPase activity of myosin purified from rat heart. Both Ca²⁺-dependent ATPase and myosin ATPase were stimulated by Ca²⁺ but the maximal activation of Ca²⁺-dependent ATPase required 4 mM Ca²⁺ whereas that of myosin ATPase required 10 mM Ca²⁺. These ATPases were also activated by other divalent cations in the order of Ca²⁺ > Mn²⁺ > Sr²⁺ > Br²⁺ > Mg²⁺; however, there was a marked difference in the pattern of their activation by these cations. Unlike the myosin ATPase, the ATP hydrolysis by Ca²⁺-dependent ATPase was not activated by actin. The pH optima of Ca²⁺-dependent ATPase and myosin ATPase were 9.5 and 6.5 respectively. Na⁺ markedly inhibited Ca²⁺-dependent ATPase but had no effect on the myosin ATPase activity. N-ethylmaleimide inhibited Ca²⁺-dependent ATPase more than myosin ATPase whereas the inhibitory effect of vanadate was more on myosin ATPase than Ca²⁺-dependent ATPase. Both Ca²⁺-dependent ATPase and myosin ATPase were stimulated by K-EDTA and NH₄-EDTA. When myofibrils were treated with trypsin and passed through columns similar to those used for purifying Ca²⁺-ATPase from sarcolemma, an enzyme with ATPase activity was obtained. This myofibrillar ATPase was maximally activated at 3–4 mM Ca²⁺ and 3 to 4 mM ATP like sarcolemmal Ca²⁺-dependent ATPase. K⁺ stimulated both ATPase activities in the absence of Ca²⁺ and inhibited in the presence of Ca²⁺. Both enzymes were inhibited by Na⁺, Mg²⁺, La³⁺, and azide similarly. However, Ca²⁺ ATPase from myofibrils showed three peptide bands in SDS polyacrylamide gel electrophoresis whereas Ca²⁺ ATPase from sarcolemma contained only two bands. Sarcolemmal Ca²⁺-ATPase had two affinity sites for ATP (0.012 mM and 0.23 mM) while myofibrillar Ca²⁺-ATPase had only one affinity site (0.34 mM). Myofibrillar Ca²⁺-ATPase was more sensitive to maleic anhydride and iodoacetamide than sarcolemmal Ca²⁺-ATPase. These observations suggest that Ca²⁺-dependent ATPase may be a myosin like protein in the heart sarcolemma and is unlikely to be a tryptic fragment of myosin present in the myofibrils.     

The Role of Phospholipids in Plasma Membrane ATPase Activity in Vigna radiata L. (Mung Bean) Roots and Hypocotyls

Root and hypocotyl plasma membrane H⁺-ATPases were partially purified from deoxycholate-solubilized fractions of microsomes in mung bean (Vigna radiata L.) plants in the presence of glycerol. Certain properties of the ATPases and the manner in which phospholipids affect their activity were compared. Root ATPase was similar to hypocotyl ATPase with respect to substrate specificity, salt stimulation, pH dependence, K_m for ATP·Mg²⁺ and inhibitor sensitivity, except for inhibition by vanadate. Both purified ATPases required phospholipids for their activation. Optimum concentrations of exogenously added phospholipid mixture (asolectin) to hypocotyl and root ATPase mixture were 0.03% and 1.0%, respectively. Root ATPase activation did not decrease if more than 1.0% asolectin was added. Qualitatively, phosphatidylserine and phosphatidylcholine brought about greater ATPase activation than other phospholipids. The hypocotyl ATPase was activated by phosphatidylinositol, phosphatidylserine and phosphatidylglycerol to a greater extent than the root ATPase. Root, but not hypocotyl ATPase, was slightly inhibited by the addition of phosphatidylinositol, phosphatidylethanolamine, and phosphatidic acid. The hypocotyl plasma membrane contained phosphatidylinositol + phosphatidylserine, phosphatidylglycerol and phosphatidic acid, and unsaturated fatty acids in greater abundance than the root plasma membrane. The differential activation of the plasma membrane ATPases may arise from these differences.     

Biochemical and structural studies of actomyosin-like proteins from non-muscle cells. Isolation and characterization of myosin from amoebae of Dictyostelium discoideum

A myosin-like protein was purified from amoebae of the cellular slime mold Dictyostelium discoideum. The purification utilized newly discovered solubility properties of actomyosin in sucrose. The amoebae were extracted with a 30% sucrose solution containing 0.1 m-KCl, and actomyosin was selectively precipitated from this crude extract by removal of the sucrose. The myosin and actin were then solubilized in a buffer containing KI and separated by gel filtration.The purified Dictyostelium myosin bears a very close resemblance to muscle myosin. The amoeba protein contains two heavy chains, about 210,000 molecular weight each, and two classes of light chains, 16,000 and 18,000 molecular weight. Dictyostelium myosin is insoluble at low ionic strength and forms bipolar thick filaments. The myosin possesses ATPase activity that is activated by Ca²⁺ but not EDTA, and is inhibited by Mg²⁺; under optimal conditions the specific activity of the enzyme is 0.09 μmol P₁/min per mg myosin.Dictyostelium myosin interacts with Dictyostelium actin or muscle actin, as shown by electron microscopy and by measurements of enzymatic activity. The ATPase activity of Dictyostelium myosin, in the presence of Mg²⁺ at low ionic strength, exhibits an average ninefold activation when actin is added.     

Calcium regulation of the ATPase activity of Physarum and scallop myosins using hybrid smooth muscle myosin: The role of the essential light chain

To examine the role of two light chains (LCs) of the myosin II on Ca²⁺ regulation, we produced hybrid heavy meromyosin (HMM) having LCs from Physarum and/or scallop myosin using the smooth muscle myosin heavy chain. Ca²⁺ inhibited motility and ATPase activity of hybrid HMMs with LCs from Physarum myosin but activated those of hybrid HMM with LCs from scallop myosin, indicating an active role of LCs. ATPase activity of hybrid HMMs with LCs from different species showed the same effect by Ca²⁺ even though they did not support motility. Our results suggest that communication between the original combinations of LC is important for the motor function.