Purification and characterization of DNA-dependent ATP phosphohydrolases from KB cells

DNA-dependent ATPases have been purified from logarithmically growing KB cells by chromatography on single-stranded DNA cellulose and phosphocellulose. Phosphocellulose resolved the DNA-dependent ATPases into three activities designated ATPase I, II and III, respectively. From gel filtration and sedimentation analysis ATPases II and III were found to be very similar, both with calculated molecular weights of 78,000. Due to the extreme lability these enzymes were not purified further. The molecular weight of ATPase I determined by gel filtration and sedimentation analysis was calculated to be 140,000. ATPase I was further purified by gradient elution on ATP-agarose, revealing two peaks of activity (IA and IB), and by sucrose gradient sedimentation. Analysis of the fractions from the sucrose gradient by sodium dodecylsulphate gel electrophoresis revealed only one broad polypeptide band co-sedimenting with both ATPase IA and ATPase IB. This band was composed of four closely spaced polypeptides with apparent molecular weights of 66,000, 68,000, 70,000 and 71,000. Comparison of the native molecule weight (140,000) with these results suggests that ATPase I is a dimer. ATPase IA and IB were indistinguishable in their structural and enzymatic properties and presumably represent the same enzyme. The purified enzyme has an apparent Km of 0.5 mM for ATP producing ADP + Pi. A maximum activity of 2,100 molecules of ATP hydrolyzed per enzyme molecular per minute was found. Hydrolysis of ATP requires the presence of divalent cations (Mg2+ greater than Ca2+ greater than Mn2+ greater than Co2+). A broad pH optimum (pH 6--8) was observed. The enzyme uses ATP or dATP preferentially as a substrate, while other deoxyribonucleoside or ribonucleoside triphosphates were inactive. ATPase I prefers denatured DNA as cofactor. The activity with native DNA is 40% of that with denatured DNA.     

Properties of detergent-dispersed adenylate cyclase from cerebral cortex. Presence of an inhibitor protein

Adenylate cyclase was solubilized from washed particulate fraction of rabbit cerebral cortex with the nonionic detergent Lubrol 12A9 and subjected to either gel filtration on Ultrogel AcA 34 or chromatography on DEAE Bio-Gel A. By both procedures the enzyme was resolved into two components, one insensitive to guanyl 5'-yl imidodiphosphate [Gpp(NH)p] and NaF but stimulated by Ca2+ and calmodulin, and another that was sensitive to Gpp(NH)p and NaF but relatively insensitive to Ca2+ and calmodulin. The data support the possibility that two independent forms of adenylate cyclase exist in cerebral cortex, one regulated by guanine nucleotide regulatory protein and another by Ca2+-calmodulin. Fractions containing the guanylnucleotide-sensitive activity were found to contain a factor that inhibited basal and Ca2+-stimulated adenylate cyclase in the Ca2+-sensitive fraction. The inhibitor was inactivated by heating at 60 degrees C and by incubation with trypsin. Inhibition was not time-dependent, and it was not due to destruction of cAMP by phosphodiesterase or of ATP by ATPase. Inhibitory action was not reversed by calmodulin and therefore it does not appear to be a calmodulin binding protein. Sucrose density gradient sedimentation indicated a sedimentation coefficient of 4S for the inhibitor; by this technique it co-sedimented with the adenylate cyclase sensitive to Gpp(NH)p and NaF.     

Interactions of the Complex Secretory Vesicle Binding Protein Chromobindin A with Nucleotides

Chromobindin A is a large, multisubunit protein that binds to chromaffin granule membranes in a Ca2+- and ATP-regulated manner. Ca2+ stimulates binding to the membrane, whereas ATP, in the the absence of Ca2+, is required for release of the protein from the membrane. We now report that spectral and HPLC data indicate that nucleotides are associated with the native chromobindin A complex and that the protein can bind two molecules of [3H]ATP in vitro. Chromobindin A also appears to be a novel nucleotide triphosphatase. ATPase activity was detected in fractions containing chromobindin A isolated by affinity chromatography, gel filtration, or ion exchange chromatography. Kinetic studies indicated that the Vmax is 44 nmol of Pi/mg/min and the Km is 0.115 mM, whereas the nonhydrolyzable ATP analog 5'-adenylylimidodiphosphate acts as a competitive inhibitor of this reaction with a Ki of 0.08 mM. The activity was found to be sensitive to protease treatment or to preincubation at 65 degrees C and was inhibited by Ca2+ or low pH. The ATPase activity was not inhibited by N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide, vanadate, oligomycin, or azide.     

Reactive sulfhydryl groups of sarcoplasmic reticulum ATPase. III. Identification of cysteine residues whose modification with N-ethylmaleimide leads to loss of the Ca2+-transporting activity

The reactive sulfhydryl group (SHD) (Kawakita et al. (1980) J. Biochem. 87, 609-617) which is essential for the decomposition of the E-P intermediate of Ca2+-transporting ATPase of the rabbit skeletal muscle sarcoplasmic reticulum has been identified. One sample of sarcoplasmic reticulum membranes was reacted for 3 min with 0.4 mM N-[3H]ethylmaleimide at pH 7.0 at 30 degrees C to a labeling density of 1 mol/mol ATPase without loss of the Ca2+-transporting activity. Another sample of the membranes was treated similarly with non-radioactive N-ethylmaleimide and then labeled with 0.4 mM N-ethyl[14C]maleimide for 17 min. An extensive loss of the Ca2+-transporting activity occurred during the period of this radio-labeling, thus substantiating the 14C-labeling of SHD. The labeled membranes were digested by thermolysin, and the labeled peptides were fractionated by gel filtration and reversed-phase HPLC. Two major radioactive peptides were present in both 3H- and 14C-labeled thermolytic digests, and each of the major components of 14C-labeled peptides had a counterpart in the major components of 3H-labeled peptides which behaved identically on HPLC. The major 14C-labeled peptides were purified and found to be identical with the two SHN peptides, TL-I and TL-II (Saito-Nakatsuka et al. (1987) J. Biochem. 101, 365-376), and 0.5 mol/mol ATPase each of Cys344 and Cys364 was assigned as SHD. It seems that the Ca2+-transport system retains its activity while either of the two Cys residues is unoccupied, but loses it when both of them are modified with N-ethylmaleimide.     

Ca2+ sensitization and potentiation of the maximum level of myofibrillar ATPase activity caused by mutations of troponin T found in familial hypertrophic cardiomyopathy

Human wild-type cardiac troponin T, I, C and five troponin T mutants (I79N, R92Q, F110I, E244D, and R278C) causing familial hypertrophic cardiomyopathy were expressed in Escherichia coli, and then were purified and incorporated into rabbit cardiac myofibrils using a troponin exchange technique. The Ca2+-sensitive ATPase activity of these myofibrillar preparations was measured in order to examine the functional consequences of these troponin mutations. An I79N troponin T mutation was found to cause a definite increase in Ca2+ sensitivity of the myofibrillar ATPase activity without inducing any significant change in the maximum level of ATPase activity. A detailed analysis indicated the inhibitory action of troponin I to be impaired by the I79N troponin T mutation. Two more troponin T mutations (R92Q and R278C) were also found to have a Ca2+-sensitizing effect without inducing any change in maximum ATPase activity. Two other troponin T mutations (F110I and E244D) had no Ca2+-sensitizing effects on the ATPase activity, but remarkably potentiated the maximum level of ATPase activity. These findings indicate that hypertrophic cardiomyopathy-linked troponin T mutations have at least two different effects on the Ca2+-sensitive ATPase activity, Ca2+-sensitization and potentiation of the maximum level of the ATPase activity.     

Ca2+- and Sr2+-sensitivity of the ATPase activity of rabbit skeletal myofibrils: effect of the complete substitution of troponin C with cardiac troponin C, calmodulin, and parvalbumins

The Ca2+-sensitive ATPase activity of rabbit skeletal myofibrils disappeared completely after treatment with a solution containing CDTA, a strong divalent cation chelator, at a low ionic strength. A gel electrophoretic study revealed that all troponin C and about half of myosin light chain 2 were removed from the myofibrils by the CDTA treatment. The CDTA-treated myofibrils, when reconstituted with skeletal troponin C, showed almost exactly the same Ca2+- or Sr2+-sensitive ATPase activity as that of intact myofibrils. The CDTA-treated myofibrils reconstituted with porcine cardiac troponin C showed the same Ca2+- or Sr2+-sensitivity of the ATPase as that of porcine cardiac myofibrils; Sr2+-sensitivity relative to Ca2+-sensitivity was about ten times higher than, and the maximal slope of the activation curve was about half that of skeletal myofibrils. These findings indicate that these characteristic features of divalent cation regulation in the contraction of skeletal and cardiac muscles are determined solely by the species of troponin C. Bovine brain calmodulin hardly activated the ATPase activity of the CDTA-treated myofibrils even in the presence of Ca2+. Excess calmodulin, however, was found to give Ca2+- or Sr2+-sensitivity to the ATPase activity of the CDTA-treated myofibrils. Frog skeletal parvalbumins 1 and 2, even in excess, did not affect the ATPase activity of the CDTA-treated myofibrils.     

Inhibition of basal and calmodulin-activated Ca2+-pump ATPase by fractionated compound 48/80

Compound 48/80 (48/80), a mixture of polycationic compounds was fractionated using affinity chromatography on calmodulin-Sepharose. Unfractionated 48/80 and various fractions were tested for their potential inhibitory effects on ATPase activities of isolated human red blood cell membranes. ATPase activities tested included: Mg2+-ATPase, the Na+/K+-pump ATPase, and the Ca2+-pump ATPase in both its basal (calmodulin-independent) and calmodulin-activated state. Neither 48/80 nor its various fractions were very potent or efficacious inhibitors of the Mg2+-ATPase or the Na+/K+-pump ATPase. In agreement with previous reports, 48/80 was found to be an inhibitor of the calmodulin-activated Ca2+-pump ATPase. By contrast, we found that unfractionated, as well as some fractionated, material inhibited both the basal (calmodulin-independent) and calmodulin-activated Ca2+-pump ATPase activity. A fraction designated as Fraction III bound to calmodulin-Sepharose in the presence of Ca2+ and low salt and was eluted in the absence of Ca2+ and 0.15 M NaCl. By gel filtration, Fraction III had an apparent average molecular weight of 2064 (1320 for unfractionated material). Fraction III was the most potent inhibitor of the Ca2+-pump ATPase with IC50 values for the basal and calmodulin-activated forms of the enzyme of 0.6 and 1.2 micrograms/ml, respectively. Inhibition by Fraction III was cooperative with n apparent values of 2.4 and 5.7, respectively, for the basal and calmodulin-activated forms of the enzyme. Thus, binding of 48/80 constituents to calmodulin can not fully account for the observed data. Direct interaction of 48/80 constituent(s) with the enzyme and/or the lipid portion of the membrane is suggested.     

Properties of a series of tegumental membrane-bound phosphohydrolase activities of Schistosoma mansoni.

1. Incubation of Schistosoma mansoni for 5 min in a phosphate-buffered medium, pH 7.4, released tegumental material containing the following phosphohydrolase activities: alkaline phosphatase, 5'-nucleotidase, glycerol-2-phosphatase, glucose 6-phosphatase, phosphodiesterase and ATPase. 2. Maximum activity of these enzymes was measured at pH 9.5; however, the phosphodiesterase and ATPase activities were also appreciable at pH 7.0. 3. Solubilization of the released tegumental material in 1% Triton X-100 followed by gel filtration distinguished three peaks of enzyme activity: an ATPase (mol.wt. greater than 1000 000), a phosphodiesterase (mol.wt. 1 000 000) and an alkaline phosphomonoesterase with broad specificity (mol.wt. 232 000). 4. The ATPase activity was highly activated by 10 mM-Mg2+ or 1 mM-Ca2+ and was inhibited by chelating agents. Ouabain, Na+ and K+ had little effect on enzyme activity, whereas activity was increased by 50% in the presence of calmodulin. The phosphodiesterase activity was highest in the presence of 100 mM-Na+ or -K+, and 10 mM-Mg2+ or -Ca2+. Alkaline phosphatase activity was also stimulated by 100 mM-Na+ or -K+, and 10 mM-Mg2+; however Ca2+ inhibited at greater than 1 mM. 5. Surface iodination of parasites followed by detergent solubilization and gel filtration of the released tegumental membranes indicated that these enzymes were not accessible. A major surface component, apparent mol.wt. 80 000, was iodinated. 6. Rabbit anti-(mouse liver 5'-nucleotidase) antibodies did not inhibit the phosphohydrolase activities. However, an immunoglobulin G fraction from sera of mice chronically infected with S. mansoni partially inhibited alkaline phosphatase activity, but was without effect on the phosphodiesterase and ATPase activities. 7. The location of the enzymes in the double membrane of the tegument and their significance in host-parasite interactions is discussed.     

Purification of (Ca2+-Mg2+)-ATPase from rat liver plasma membranes

The Ca2+-stimulated, Mg2+-dependent ATPase from rat liver plasma membranes was solubilized using the detergent polyoxyethylene 9 lauryl ether and purified by column chromatography using Polybuffer Exchanger 94, concanavalin A-Sepharose 4B, and Sephadex G-200. The molecular weight of the enzyme, estimated by gel filtration in the presence of the detergent on a Sephadex G-200 column, was 200,000 +/- 15,000. The enzyme was purified at least 300-fold from rat liver plasma membranes and had a specific activity of 19.7 mumol/mg/min. Polyacrylamide gel electrophoresis under nondenaturing conditions of the purified enzyme indicated that the enzymatic activity correlated with the major protein band. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one major band in the molecular weight range of 70,000 +/- 5,000 was seen. The isoelectric point of the purified enzyme was 6.9 +/- 0.2 as determined by analytical isoelectric focusing. The enzyme was activated by Ca2+ with an apparent half-saturation constant of 87 +/- 2 nM for Ca2+. Calmodulin and trifluoperazine at the concentration of 1 microgram/ml and 100 microM, respectively, had no effect on the enzymatic activity.     

Adenosine triphosphatases associated with bovine brain microtubules. I. Presence of two distinct ATPases and their partial purification

Brain microtubules purified by cycles of assembly and disassembly contained an ATPase activity in the fraction of microtubule-associated proteins (MAPs). This ATPase activity was found to be stimulated by 6S tubulin in the presence of Ca2+ ions, suggesting its functional association with brain microtubules (Ihara et al. (1979) J. Biochem. 86, 587-590). On further purification by DEAE-cellulose column chromatography, two peaks of ATPase activity were separated; one, eluted at 0.2 M KCl (ATPase I), was dependent on added 6S tubulin but the other, eluted at 0.5 M KCl (ATPase II), was not. ATPase I was highly unstable but could be stabilized by the addition of 0.1 mM ADP, 50% (v/v) glycerol or 0.3 mg/ml tubulin. ATPase I was further purified by CM-cellulose column chromatography, and by gel filtration on Sephacryl S-300. Its molecular weight, estimated by gel filtration, was 33,000. ATPase II had a high molecular weight and appeared to be associated with membrane vesicles. It sedimented on glycerol density gradient centrifugation with an s value of 27S. It was purified by high speed sedimentation and hydrophobic chromatography, and was observed under an electron microscope to consist of membrane vesicles of about 70 nm in diameter containing knob-like structures similar to those of H+-pump ATPase.     

Purification and characterization of phosphatidylinositol-specific phospholipase C from bovine spermatozoa

1. The distribution of phosphatidylinositol3, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate hydrolysis or phosphatidylinositol-specific phospholipase C (PI-PLC), activity in the bull reproductive system showed the highest specific activity in the isolated spermatozoa (SZ) followed by testis and different epididymal segments. Both the head and tail fractions of SZ were active. 2. The optimal solubilization of the enzyme from SZ was obtained with 0.2% Triton X-100 or at 0.05% detergent concentration when combined with a 60 sec sonication. The sucrose gradient centrifugation showed that PI-PLC was enriched in membrane fraction distinct from mitochondria and acrosomes. 3. The enzyme was purified by ammonium sulphate precipitation and fractionations by hydrophobic interaction chromatography, gel filtration, Con A-Sepharose affinity and chromatofocusing columns. The purified enzyme was able to hydrolyse all phosphatidylinositol substrates with optimum at pH 7.0 and activation by Ca2+, Cd2+ and Mn2+ but not phospholipids lacking the inositol residue. 4. In PAGE (8-25% gradient) the purified (aggregated) enzyme did not enter the gel. In SDS-PAGE two closely located bands were found with Mr-values of 15,000 and 18,000. Isoelectric focusing showed a wide band at pl 4.5-5.1. 5. Gel filtration resulted in a broad elution peak indicating multiple molecular forms (aggregates); the basic form had an apparent molecular weight of 100,000. The binding of the enzyme to Con A-Sepharose indicated that the enzyme is a glycoprotein.     

Partial purification and properties of cGMP-dependent protein kinase from prawn tissue

Using ion-exchange chromatography and gel filtration, cGMP-dependent protein kinase was purified from prawn tissues 220-fold with a yield of activity of 12%. The apparent Ka values for cGMP, cAMP and 8-Br-cGMP are 1 . 10(-7), 5 . 10(-6) and 5 . 10(-8) M, respectively; the apparent Km values for ATP in the presence of cGMP is 9 . 10(-6) M. The cGMP-stimulated protein kinase activity was observed only in the presence of SH-compounds and high Mg2+ concentrations (500-100 mM). The protein kinase demonstrated a broad pH optimum wih a maximum at pH 6.8-7.2. The elution volume of the enzyme during gel filtration corresponded to a globular protein with molecular weight of 140,000.     

Large-scale isolation of human erythrocyte Ca2+-transport ATPase

A rapid procedure for preparing large quantities of purified erythrocyte Ca2+-transport ATPase is presented. The method involves: (1) fast preparation of calmodulin-deficient, essentially haemoglobin-free, erythrocyte membranes by molecular filtration using Pellicon filters; (2) solubilization of membrane proteins by deoxycholate; and (3) a batch procedure using calmodulin-Sepharose 4B gel for purification of Ca2+-transport ATPase.     

Fluorescence quenching in model membranes. 3. Relationship between calcium adenosinetriphosphatase enzyme activity and the affinity of the protein for phosphatidylcholines with different acyl chain characteristics

The dependence of function and lipid binding affinity of an integral transport protein on the fatty acyl chain characteristics of a membrane-forming phospholipid have been determined. When a newly developed fluorescence quenching technique [London, E., & Feigenson, G. W. (1981) Biochemistry (first paper of three in this issue); London, E., & Feigenson, G. W. (1981) Biochemistry (preceding paper in this issue)] is used for examining lipid-protein interactions in membranes, the Ca2+ ATPase from rabbit sarcoplasmic reticulum is found to bind with equal affinity a large variety of phosphatidylcholines used to reconstitute the protein into enzymatically active vesicles, regardless of fatty acyl chain length or details of unsaturation. In parallel with the lipid binding studies, we have measured the sensitivity of the catalytic activity of the Ca2+ ATPase to the fatty acyl chain characteristics of the phosphatidylcholine membranes in which the enzyme was reconstituted. The enzyme appears to be sensitive only to the effective fatty acyl chain length, which determines the thickness of the bilayer in which the protein is inserted and displays little sensitivity to such details of unsaturation as degree, position, and isomeric type. Both ATP hydrolyzing and Ca2+ transporting activities of the enzyme were similarly affected by bilayer thickness, and maximum activity was observed in membranes of intermediate thickness. These observations are reconciled in a number of possible models for the manner in which this integral protein interacts with membranes of varying thickness. A freeze-thaw method was used to reconstitute the Ca2+ ATPase, and the vesicles so obtained have been characterized by gel permeation chromatography, density gradient centrifugation, and electron microscopy, (thin section). Convenient methods are described for (a) rapidly separating reconstituted Ca2+ ATPase from unincorporated protein simultaneously in a large number of small samples, giving good recovery of fractionated vesicles without significant dilution, and (b) measuring leakiness to Ca2+ of reconstituted vesicles. Additionally, the gel and liquid-crystal phase transition temperature and bilayer thickness have been determined respectively by differential thermal analysis and low-angle X-ray diffraction for some of the synthetic phosphatidylcholines, which range in chain length from 12 to 24 carbon atoms.     

Separation of Ca2(+)-ATPase from Mg2(+)-ATPase in plasma membrane-rich fraction of bovine parotid gland

Ca2(+)-ATPase, which does not require Mg2+ for its activation, was separated from Mg2(+)-ATPase by papain treatment of a membrane-rich fraction of bovine parotid gland. The enzyme was partially purified 48-fold by subsequent chromatography on DEAE-cellulose, gel filtration on HPLC, and ion-exchange HPLC. The enzyme showed a molecular weight of 100,000, as estimated by gel filtration on HPLC. The Ca2(+)-ATPase was activated by Ca2+ but not by Mg2+, and this enzyme did not require Mg2+ for its activation by Ca2+. In fact, Mg2+ was inhibitory. p-Nitrophenyl phosphate was not hydrolyzed in the presence of Ca2+ or Mg2+, and this enzyme had no activities of other phosphatases tested. These results suggest that the Ca2(+)-ATPase is a separate enzyme from Mg2(+)-ATPase, Ca2(+)-stimulated Mg2(+)-dependent ATPase, and alkaline phosphatase, all of which are well known to be present in other tissues.     

Purification and characterization of a deoxyribonucleic acid dependent adenosinetriphosphatase from mouse FM3A cells: effects of ribonucleoside triphosphates on the interaction of the enzyme with single-stranded DNA

There are at least four forms of DNA-dependent ATPase in mouse FM3A cells [Tawaragi, Y., Enomoto, T., Watanabe, Y., Hanaoka, F., & Yamada, M. (1984) Biochemistry 23, 529-533]. One of these, ATPase B, has been purified and characterized in detail. During the purification of the enzyme, we encountered the difficulties that the enzyme could not be recovered well from the single-stranded DNA-cellulose column and that the enzyme activity was distributed very broadly. The problems were resolved by the addition of ATP in the elution buffer. The ATPase has a sedimentation coefficient of 5.5 S in both high salt and low salt. The enzyme hydrolyzes rNTPs and dATP, but ATP and dATP are preferred substrates. Adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S), 5'-adenylyl methylenediphosphate (AMP-PCP), and 5'-adenylyl imidodiphosphate (AMP-PNP) inhibit the enzyme activity. The enzyme is insensitive to ouabain, oligomycin, novobiocin, and ethidium bromide. A divalent cation (Mg2+ congruent to Mn2+ greater than Ca2+) as well as a nucleic acid cofactor is required for activity. Poly(dT), single-stranded circular DNA, and heat-denatured DNA were very effective. Native DNA was little effective with an efficiency of 29% of that obtained with heat-denatured DNA. In addition, the enzyme showed almost no activity with poly(dA).poly(dT) although it showed very high activity with the noncomplementary combination of poly(dT) and poly(dC), suggesting that ATPase B requires single-stranded DNA for activity. ATP altered the affinity of ATPase B for single-stranded DNA. The interaction of the enzyme with DNA was studied by Sephadex G-200 gel filtration assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Affinity chromatography of H+-translocating adenosine triphosphatase isolated by chloroform extraction of Rhodospirillum rubrum chromatophores. Modification of binding affinity by divalent cations and activating anions.

1. ATPase isolated from Rhodospirillum rubrum by chloroform extraction and purified by gel filtration or affinity chromatography shows three bands (alpha, beta and gamma) upon electrophoresis in sodium dodecyl sulphate. 2. Ca2+-ATPase activity of the preparation is inhibited by aurovertin and efrapeptin but not by oligomycin. Activity may be inhibited by treatment with 4-chloro-7-nitrobenzofurazan and subsequently restored by dithiothreitol. 3. The enzyme fails to reconstitute photophosphorylation in chromatophores depleted of ATPase by sonic irradiation. 4. Most of the active protein from the crude chloroform extract binds to an affinity chromatography column bearing an immobilised ADP analogue but not to a column bearing immobilised pyrophosphate. 5. In the absence of divalent cations, a component with a very high specific activity for Ca2+-ATPase is eluted from the column by 1.6 mM ATP. This protein migrates asa single band on 5% polyacrylamide gel electrophoresis and only possesses three subunits. At 12 mM ATP an inactive protein is eluted which does not run on acid or alkali polyacrylamide gels and shows a complex subunit structure. 6. ATPase preparations prepared by acetone extraction or by sonic irradiation of chromatophores may also be purified 10-fold by affinity chromatography. 7. The inclusion of 5 mM MgCl2 or CaCl2 during affinity chromatography of chloroform ATPase increases the capacity of the column for the enzyme and demands a higher eluting concentration of ATP. 8. When the enzyme is more than 90% inhibited by efrapeptin or 4-chloro-7-nitrobenzofurazan, the binding characteristics of the enzyme are not affected. 9. 10 mM Na2SO3, which greatly stimulates the Ca2+- and Mg2+-dependent ATPase activity of the enzyme and increases Ki (ADP) for Ca2+-ATPase from 50 to 850 micron, prevents binding to the affinity column. Binding may be restored by the addition of divalent cations. 10. Na2SO3 increases the rate of ATP hydrolysis, ATP-driven H+ translocation and ATP-driven transhydrogenase in chromatophores. 11. It is proposed that anions such as sulphite convert the chromatophore ATPase into a form which is a more efficient energy transducer.     

Cooperative interactions between the contractile proteins of cardiac and skeletal muscle.

The calcium activation of the ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity of cardiac actomyosin reconstituted from bovine cardiac myosin and a complex of actin-tropomyosin-troponin extracted from bovine cardiac muscle at 37 degrees C was studied and compared with similar proteins from rabbit fast skeletal muscle. The proteins of the actin complex were identified by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Half-maximal activation of the cardiac actomyosin was seen at a calcium concentration of 1.2 +/- 0.002 (S.E. of mean) muM. A hybridized reconstituted actomyosin made with cardiac myosin and the actin-tropomyosin-troponin complex extracted from rabbit skeletal muscle was also activated by calcium but the half-maximal value was shifted to 0.65 +/- 0.02 (S.E. of mean) muM Ca2+. Homologous rabbit skeletal actomyosin showed half-maximal activation at 0.90 +/- 0.01 (S.E. of mean) muM Ca2+ and the value for a hybridized actomyosin made with rabbit skeletal myosin and the actin-complex from cardiac muscle was found at 1.4 +/- 0.03 (S.E. of mean) muM Ca2+ concentration. Kinetic analysis of the Ca2+ activated ATPase activity of reconstituted bovine cardiac actomyosin indicated some degree of cooperativity with respect to calcium. Double reciprocal plots of reconstituted actomyosins made with bovine cardiac actin complex were curvilinear and significantly different than those of reconstituted actomyosins made with the rabbit fast skeletal actin complex. The Ca2+-dependent cooperativity was of a mixed type as determined from Hill plots for homologous reconstituted bovine cardiac and rabbit fast skeletal actomyosin. The results show that cooperative interactions in reconstituted actomyosins were greater when the actin-tropomyosin-troponin complex was derived from cardiac than skeletal muscle.     

Troponin from Akazara scallop striated adductor muscles

Troponin was isolated from striated adductor muscles of the "Akazara" scallop (Chlamys nipponensis akazara), and purified in an active form by DEAE-cellulose (Whatman DE52) column chromatography and subsequent gel filtration on Sephacryl S-300. According to sodium dodecyl sulfate-gel electrophoresis and densitometry, Akazara troponin is composed of three components having molecular weights of 52,000, 40,000, and 20,000 in a molar ratio of 1:1:1. The three components were separated from each other by column chromatography in the presence of 6 M urea and 1 mM EDTA on SP-Sephadex C-50 and DEAE-cellulose. The Mr 20,000 component was regarded as troponin C according to the Ca2+-binding properties, which was found to bind 0.7 mol of Ca2+/mol at 0.1 mM Ca2+. The association constant of Ca2+ to troponin C was estimated to be 5 X 10(5) M-1, and was not affected by the addition of 2 mM MgCl2. The Mr 52,000 component appeared to be troponin I, since it inhibited, together with Akazara tropomyosin, both Mg-ATPase and superprecipitation activities of actomyosin reconstituted from rabbit myosin and actin, and the inhibition of the ATPase activity was diminished by the addition of Akazara troponin C. Finally, the Mr 40,000 component appeared to be troponin T, since it co-precipitated with actin-tropomyosin filament and was indispensable with Akazara troponin C and the Mr 52,000 component (troponin I) for conferring the Ca2+ sensitivity to reconstituted actomyosin.     

Ca2+ binding to skeletal muscle troponin C in skeletal and cardiac myofibrils

Ca2+ binding to skeletal muscle troponin C in skeletal or cardiac myofibrils was measured by the centrifugation method using 45Ca. The specific Ca2+ binding to troponin C was obtained by subtracting the amount of Ca2+ bound to the CDTA-treated myofibrils (troponin C-depleted myofibrils) from that to the myofibrils reconstituted with troponin C. Results of Ca2+ binding measurement at various Ca2+ concentrations showed that skeletal troponin C had two classes of binding sites with different affinity for Ca2+. The Ca2+ binding of low-affinity sites in cardiac myofibrils was about eight times lower than that in skeletal myofibrils, while the high-affinity sites of troponin C in skeletal or cardiac myofibrils showed almost the same affinity for Ca2+. The Ca2+ sensitivity of the ATPase activity of skeletal troponin C-reconstituted cardiac myofibrils was also about eight times lower than that of skeletal myofibrils reconstituted with troponin C. These findings indicated that the difference in the sensitivity to Ca2+ of the ATPase activity between skeletal and cardiac CDTA-treated myofibrils reconstituted with skeletal troponin C was mostly due to the change in the affinity for Ca2+ of the low-affinity sites on the troponin C molecule.