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CALCIUM-DEPENDENT ATPASE.
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Analytic techniques for understanding processes involving common eukaryotic enzyme.... More...
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Paper Abstract:
Analytic techniques for understanding processes involving common eukaryotic enzyme.
Paper Introduction:
Calcium-Dependent ATPase: Analytic Techniques
A common eukaryotic enzyme, calcium-dependent ATPase has been extensively investigated. The ion-transport enzyme uses energy derived from the hydrolysis of adenosine triphosphate (ATP) to move Ca2+ against a concentration gradient. Innumerable techniques have been applied to Ca2+-ATPase analyses. These have included proteolytic, genetic, immunologic, and molecular approaches.
Calcium-dependent ATPase was first isolated in 1970 (3:696-700). This heterogenous family of enzymes can be broadly subdivided into two separate groups. The plasma membrane Ca2+-ATPase occurs in most eukaryotic tissues. This 140-kDa enzyme binds calmodulin and is stimulated by calcium ion (10:285-297). Although it may be derived from plants, yeasts, or, for example,
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For example, raisingthe temperature from 1 ?C to 38?C has been found to increase the normalizedenergy transfer between AEDANS and FITC by between 13 and 2 percent. By monitoring such phenomena, researchers can obtain importantstructural information (7:217-218). Second, Matsushita and Pette (1992) usedchronic low-frequency stimulation to induce SR Ca2+-ATPase inactivation inrabbit fast-twitch muscle. R.; Inesi, G. Tricine-sodium dodecyl sulfate- polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 1 kDa. In concurrence with the fluorescence resonanceenergy transfer data, the distance between these two sites is approximately6 Å. Inactivation of sarcoplasmic-reticulum Ca2+-ATPase in low-frequency-stimulated muscle results from a modification of the active site. These various contacts can provideresearchers with important information with regard to the ATPase molecules(4:365-37 ). In addition, scientificinvestigators have analyzed the effects of antibodies on ATPase activityand Ca2+ transport. 316:696-7 ; 1985, August 22. It has been estimated that thedistance between the Ca2+-ATPase's ATP-binding domain and high affinityCa2+ binding sites is between 2 Å and 4 Å. For example, the ion-transport enzyme's full sequence hadbeen elucidated using complementary genetic clones. Amino- acid sequence of a Ca2+ + Mg2+-dependent ATPase from rabbit muscle sarcoplasmic reticulum, deduced from its complementary DNA sequence. Two means of locating Ca2+-ATPase functional sites include bothantibody and fluorescent probe analyses. 9. Thus, it can distinguish proteinswhich range in size from 1 to 1 kDa (8:368-379). Another important fluorescent probe which has been used toinvestigate SR Ca2+-ATPase is N-cyclohexyl-N'-(4-dimethylamino-1-naphthyl)carbodiimide (NCD4). The mechanism by which Ca2+-ATPases couple ATP hydrolysis to Ca2+transport has yet to be fully elucidated. Febs Letters. Structural information may also come from site-directed mutagenesisstudies. Researchersessentially probed complimentary deoxyribonucleic acid (cDNA) librarieswith radiolabelled synthetic oligomeric nucleotides. (199 ) tested 19 anti-ATPase antibodies. Calcium-dependent ATPase was first isolated in 197 (3:696-7 ).This heterogenous family of enzymes can be broadly subdivided into twoseparate groups. While the stimulation preserved the enzyme'scalcium binding ability, it did affect phosphorylation by both ATP and Pi.This led the researchers to conclude that the stimulation caused some"essential structural change" at the enzyme's active site (5:3 3-3 9). It thus introduces a structuralperturbation into the ATPase. By separately labelling ATPase moleculeswith either IAEDANS or IAF, intermolecular Förster type energy transfersmay be demonstrated in reconstituted artificial phospholipid bilayers. 2. Under optimal conditions, about half of the ATPase molecules arelabeled by EITC and about half by FITC. W.; Jona, I.; Martonosi, A. Sarcoplasmic reticulum vesicles werefirst labeled with FITC. For example, various researchershave examined the enzyme by using proteolytic techniques on intact membranesystems and purified erythrocyte enzyme. The hydrophobic transmembrane sequences, however, are moredifficult to obtain (3:696-7 ). Such methods have been used to identify the individual ATPaseamino acid residues involved in calcium binding. 5. Wuytack, F.; Raeymaekers, L. There are many ways toapproach Ca2+-ATPase structural analysis. The SERCA 2a Ca2+-ATPase isoform is found in skeletal muscle; whereas, the SERCA 2b isoformoccurs in smooth muscle. Perhaps the mostwell-known of these enzymes comes from the SERCA 1 gene. Innumerable techniques have been applied to Ca2+-ATPase analyses. The binding of monoclonal and polyclonal antibodies to the Ca2+-ATPase of sarcoplasmic reticulum: Effects on interactions between ATPase molecules. Labeledenzyme maintains its characteristic response to such things as calcium ionconcentration or the presence of vanadate. Thisessentially removes the enzyme's hydrophilic portions located after thelast putative transmembrane helix (11:8 7 -8 76). Then, they were diluted, washed, resuspended, andlabeled again with NCD4. 7. Fluorescence energy transfer as an indicator of Ca2+-ATPase interactions in sarcoplasmic reticulum. Characterization of the plasma-membrane calcium pump from Trypanosoma cruzi. (199 ) assessed the effects of 14 monoclonal and 5 polyclonalantibodies on Ca2+-ATPase activity and interactions. Therefore, it must attach close to the tryptophan residueseither within or near the enzyme's transmembrane region. The plasma membrane Ca2+-ATPase occurs in most eukaryotictissues. Thisextramembranal head accommodates both the ATP binding site and an aspartylresidue phosphorylation site (Asp-351). The epitope of this monoclonal antibody occurred atresidues 659-668, between the head and lobe regions of the ATPase molecule. W.; Buchet, R.; Varga, S. 24:285- 3 ; 1992, June.11. Structural perturbation of the transmembrane region interferes with calcium binding by the Ca2+ transport ATPase. A "P-type" ionic pump, the Ca2+-ATPase forms a phosphorylated intermediate during its catalytic cycle(1:299). For example, specific inhibitors of SR ATPaseinclude thapsigargin, cyclopiazonic acid, and 2,5-di(tert-butyl)-1,4-benzohydroquinone (2:293-3 4). 3. According to MacLennan and associates (1985), calcium-dependent ATPases generally contain three major domains. Thesarcoplasmic reticulum (SR) is also a very good source of Ca2+-ATPase. The Journal of Biological Chemistry. In general though, most polyclonal and monoclonal anti-Ca2+-ATPase antibodies have "either no effect on ATPase activity ofsarcoplasmic reticulum or produce only partial inhibition, even atsaturating concentrations (6:147-166)." Molnar et al. The SR Ca2+ pump differs from the plasma membrane Ca2+ pump by thefact that it does not have a calmodulin binding site. In yet another study, Martonosiet al. Although it may be derived from plants, yeasts, or, forexample, pig stomach smooth muscle, perhaps the most thoroughly studiedplasma membrane Ca2+-ATPase is found in erythrocytes (1:3 3). In addition to their different regulatory mechanisms, plasma membraneand SR Ca2+-ATPases also have distinct functions. In fact, EITC and FITC causesteric hindrance of both nucleotide binding and enzyme phosphorylation(5:3 3-3 9). Variousresearchers, for example, have employed antibodies with epitopes located ondifferent regions of the Ca2+-ATPase molecule to perform isoenzyme cross-reactivity and cellular localization studies. In fact, the NCD4 label has beenassociated with the intramembranous portion of Ca2+-ATPase's A1 proteolyticfragment. M. Literature Cited 1. Other tools used to investigate Ca2+-ATPases include the variouscalcium pump inhibitors. Hence, this region of the molecule is thought to behighly accessible. Biochimica et Biophysica Acta. High calcium ion concentrationscause a shift to the E1 state; whereas, vanadate induces the enzyme to formE2 type crystals. Workingtogether, the pumps provide cells with free cytosolic Ca2+ concentrationsthat are 1, - to 1 , -fold less than those of the extracellular space(1 :285-297). Biological Journal. As cytosolic concentrations ofcalcium increase, however, the ions inevitably bind to membrane ATPases.Once the enzymes are activated, they are able to restore the cytosol'sambient calcium levels. Hence, considerable uncertainty stillsurrounds this particular issue (4:365-37 ). It was found thatmost of the antibodies with epitopes on the B tryptic fragment reacted withthe native enzyme. This is accomplished by measuring energytransfer between fluorophores bound at specified locations within theATPase molecules (4:365-37 ). Unfortunately though, the changes associated withtransition between E1 and E2 appear to involve little alteration of theenzyme's secondary structure. Second, the enzymepossesses a large pear-shaped, cytosolic head (5:3 3-3 9). In addition, the alteration of membranepotential will also influence both the rate of formation and stability ofCa2+-ATPase crystals (4:365-37 ). The researchers hypothesized that disruption of the Ca2+-ATPase crystalsby A52 might be caused by its interference with B-type interactions (4:365-37 ). Muscle contraction, for example, is marked by a sudden and rapidincrease in the concentration of cytosolic Ca2+ concentration. TheSR Ca2+-ATPase is derived from the SERCA gene family. Sumbilla, C.; Cantilina, T.; Collins, J. Obviously, thisis a relatively large distance from the ATPase's extramembranous catalyticsite (9:12682-12688). Various studies have shown that trypsin proteolysis progressivelyreduces Ca2+-ATPase to 9 -kDa, 85-kDa, and 81-kDa fragments. Biological Signals. Over the years, researchers haveused many different approaches to analyze the enzyme. Moreover, these states differ in their affinities and vectorialspecificities for ATP, Pi, and Ca2+. 266:12682-12689; 1991.1 . Fluorescenceresonance energy transfer measurements have calculated that the distancebetween the AEDANS site and the FITC site is 56 Å. For example, the 9 -kDa fragment obtained from plasma membrane ATPase"functions as a fully competent, calmodulin- and phospholipid-stimulatedCa2+-ATPase (11:8 7 -8 76)." Detailed information with regard to Ca2+-ATPase's structure andtopography can also be obtained by sequencing. 2:293-3 4; 1993, September-October. Biochemical Journal. kilobar causes reversible inhibition of Ca2+-ATPase activity. 8. Furthermore,this inhibition has been associated with conformationally specific changesin the structure of the enzyme (4:365-37 ). This resultsin two fragments of equal length: A and B. For one, artificially imposed membrane potentials of about 1 millivolts reversibly alter Ca2+-ATPase structure. 29:8 7 -8 76; 199 .----------------------- 1 The Ca2+-transport ATPases from the plasma membrane. Analytical Biochemistry. Eosin-5'-isothiocyanate (EITC) and fluorescein-5'-isothiocyanate(FITC), for example, selectively label a lysine residue. In 1977, researchers demonstrated thatcalmodulin binding activates the plasma membrane ATPase (11:8 7 -8 76).This generally results in an increased affinity for Ca2+, and an enhancedthe maximal rate of Ca2+ pumping. Certain of these fluorescent probes have also be used to estimateintermolecular distances. These have included proteolytic, genetic, immunologic,and molecular approaches. For example, reaction rate analyses indicate that theprocess preferentially involves "hydrophobic carbodiimide derivatives andoccurs with four orders of magnitude faster rates than the correspondingreaction in aqueous media (9:12682-12688)." Hence, the carbodiimidederivatization which inhibits calcium binding probably occurs within ahydrophobic region of the enzyme. 51:2 5-22 ; 1987. Furthermore, in the absenceof Ca2+, ATPase molecules may also be crystallized by the addition ofeither lanthanides, vanadate, or inorganic phosphate. It has been found that incubation of SR vesicles with increasingconcentrations of NCD4 causes a progressive inhibition of ATPase activity.The NCD4 probe is a rather large moiety. This region contains two highaffinity Ca2+ binding sites and a Ca2+ pathway. Schägger, H.; Von Jagow, G. Biochemical Journal. For the most part,membrane-bound Ca2+-ATPase is cleaved instantly at site T1. Various analyses have been employed in an attempt to locate the NCD4binding site. The delineation of Ca2+-ATPase's mechanism of action requiresaccurate and comprehensive structural information. In the plasmamembrane pump, this binding site is found near the enzyme's C terminus onthe membrane's cytosolic side. During this transition from the E1 state to the E2state, the probes undergo a large change in fluorescence intensity (6:147-166). Mapping of functional domains in the plasma membrane Ca2+ pump using trypsin proteolysis. Hence, about half the enzymemolecules are labeled by the donor fluorophore and about half are labeledby the acceptor fluorophore: Double labeling of the same ATPase moleculeby acceptor and donor fluorophores is for the most part avoided. In recent years, a wide selectionof monoclonal and polyclonal ATPase antibodies have been created. Eventually, two cross-hybridizing clones were found thatencoded for two different forms of the enzyme. Calcium-dependent ATPase is a rather ubiquitous ion-transport enzymethat has received considerable attention. Through both steady state and intensity decayfluorescence characterization measurements, the researchers demonstratedonly negligible energy transfer between the NCD4 label and FITC-labelledLys-515. (1991) used double labeling proceduresto locate the NCD4 binding site. Benaim, G.; Moreno, S. In order for coupling tooccur, it has been hypothesized that this distance could be altered by someconformational change. Once Ca2+-ATPases have undergone proteolysis, the resulting fragmentsmay be separated by electrophoresis. Use of calcium pump inhibitors in the study of calcium regulation in smooth muscle. Brief exposure to pressures of between .5 and 1. This 14 -kDa enzyme binds calmodulin and is stimulated by calciumion (1 :285-297). Although theresearchers found that 12 of these interacted with membrane Ca2+-ATPase,only 6 of these bonded so well that they were able to interfere withvanadate-induced ATPase crystallization. This system--which wasdeveloped to separate the beef heart bc1 complex' 11 subunits--uses Tricineas the trailing ion and tailors the stacking limits as narrowly as possiblewithin the low-molecular-mass range. (199 )'s antibody binding data, researchers have determined that theIAEDANS and A52 binding sites (residues 67 and 674, and residues 659-668,respectively) are both located near the tip of the lobe region. the "L-type,receptor-operated Ca2+ channels") and the SR (i.e., the "inositol 1,4,5,-triphosphate (IP3)-sensitive channel and/or the ryanodine-sensitive Ca2+-induced Ca2+ release channel") (2:293-3 4). Inaddition to structural information, this approach to Ca2+-ATPase analysisalso provides researchers with insights on protein-protein interactions(7:2 5-2 6). Biochemistry. On freeze fracture the transmembrane helices showup as intramembranous particles. The Ca2+-ATPase molecule extends about 6 Å above the surface of anygiven plasma bilayer. N.; Hutchinson, G.; Cervino, V.; Hermoso, T.; Romero, P. 6. Inaddition, excimer fluorescence of N-(1-pyrene)maleimide covalently bound toCa2+-ATPase molecules may also provide data on enzyme interactions (7:2 5-2 6). Moreover,these different fragments can be correlated with specific functional sites. This signal permits thevisualization of labeled protein in electrophoretic gels. This occurswith the opening of channels on both the plasma membrane (i.e. J.; Korczak, B.; Green, N. Such observations indicate that the NCD4 label does not occurwithin the ATPase's extramembranous region (9:12682-12688). Five cytoplasmic segmentsof the enzyme which are readily accessible to sequencing techniques includethe amino-terminus, the carboxy-terminus, and three long, internalsegments. The plasma membrane pumptransports calcium ions from the cytosol to the extracellular space;whereas, the SR enzyme transfers calcium ion into the SR lumen. Within such crystals different types ofinteractions may be observed. This perturbation is sufficiently large toinhibit the binding of calcium ion at both the enzyme's activating andtransporting sites (9:12682-12688). Nature. Zvaritch, E.; James, P.; Vorherr, T.; Falchetto, R.; Modyanov, N.; Carafoli, E. In the presence of Ca2+, the ATPasemolecules may be crystallized in the E1 state. In contrast, the FITC binding site (Lys-515) islocated at the opposite end of the molecule. The ion-transport enzyme uses energy derivedfrom the hydrolysis of adenosine triphosphate (ATP) to move Ca2+ against aconcentration gradient. In addition to revealingthe ATPase's primary structure, characterization of the cDNA clones alsoprovided a base from which researchers could predict the enzyme's secondaryand tertiary structure (3:696-7 ). 4. Once the Ca2+ subunitsare separated, they can be blotted onto membranes; the resulting bands willthen correspond to the enzyme's tryptic fragments (11:8 7 -8 76). The first ofthese consists of a transmembrane domain. In contrast, with the SR pump,regulation occurs through an associated SR membrane protein known asphospholamban (2:293-3 4). It has been found that Ca2+-ATPase molecules canoccur in at least two structurally distinct conformational states (E1 andE2). One particularly usefulelectrophoretic technique consists of discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Journal of Bioenergetics and Biomembranes. J.; Ruiz, F.; De Souza, W.; Docampo, R. N. E. H.; Malak, H.; Lakowicz, J. Lys-515 occursnear the Ca2+-ATPase's primary tryptic cleavage site within the molecule'sputative ATP binding domain (7:2 5-2 6). Matsushita, S.; Pette, D. Papp, S.; Pikula, S.; Martonosi, A. Suchobservations have led scientific investigators to postulate that theenzyme's thermal expansion results in increased structural fluctuation(4:365-37 ). (199 ) observed that one antibody in particular,A52, was quite capable of interfering with vanadate-induced Ca2+-ATPasecrystallization. In addition, a distanceof 68 Å has been obtained between the AEDANS site and the ATP binding site. While these data maybe used to produce a fairly complete representation of the Ca2+-ATPase,much about the molecule remains unknown. Thermal studies may additionally be employed to elucidate thestructure of Ca2+-ATPase. This 56 Å to 68 Å distance roughly corresponds to the maximum dimension ofthe ATPase molecule's cytoplasmic domain (6:147-166). MacLennan, D. It occurs near the bridge onthe enzyme's head region. The distributionof the label may then be evaluated by electrophoretic separation of the SRcomponents (9:12682-12688). Temperature-dependent changes in the energytransfer efficiency between site-specific donor and acceptor fluorophoreshave been used to reveal ATPase structural changes. 3 6:299-3 3; 1995, February 15. These effects areevidenced by changes in the steady state fluorescence of covalently boundFITC and intrinsic tryptophan. Researchers have suggested that "threeextramembranous globular domains form a headpiece" which "lies on top of astalk comprised of five helices which are, in turn, adjoined to fivetransmembrane helices (3:696-7 )." These transmembrane helices thencombine with five additional helices at the molecule's carboxy-terminalend; together, the ten helices form the ATPase's basepiece. In contrast, of 6 antibodies directed against the A1region, 5 would only react with solubilized or denatured enzyme.Furthermore, the apparent scarcity of inhibitory antibodies could indicatethat Ca2+-ATPase's active site is either inaccessible or poorly antigenic;in addition, the molecule could also possess some "unique secondarystructure (6:147-166)." Martonosi et al. Martonosi, A. Carbodiimide derivatization of SRATPase, however, does not interfere with the phosphorylation of theenzyme's active site by Pi in the absence of Ca2+. For example, Clark et al.(1989) found that "conservative mutations of any of 6 amino acid residuesin the transmembrane domain of the ATPase interfere with activation of thisenzyme by Ca2+ (9:12682)." Researchers have even employed genetic approaches to the analysis ofCa2+-ATPase. Combining both the fluorescence energy transfer data and Molnar etal. Abouttwo-thirds of the enzyme's mass is exposed on the cytoplasmic surface ofthe plasma bilayer; most of the remaining one-third is contained within thelipid phase (6:147-167). Calcium-stimulated hydrolysis of ATP is also altered by highhydrostatic pressure. Once it becomes covalently attached to Ca2+-ATPase, the FITC probeacts as a sensitive determinant of the molecule's conformation. Eventually, fragment A iscleaved at site T2 into two unequal subfragments, A2 and A1 (3:696-7 ).Although these tryptic fragments are typically held together by multipleweek interactions, they can be dissociated by solubilization with strongdetergents (9:12682-12688). 166:368-379; 1987. Further, the enzyme's total length is approximately1 Å (4:365-37 ). The primary structureof these nucleotide probes was based on previously determined ATPase aminoacid sequences. Hence bothsites occur on the lower surface of the cytoplasmic mass close to thephospholipid headgroups. Attack by trypsin generallycleaves the ATPase in the region of the calmodulin binding domain. Darby, P.J.; Kwan, C.Y.; Daniel, E. All three compounds have been extensivelyemployed to study the mechanisms behind the enzymatic entry of Ca2+ ion.In addition, the inhibitors may also be used to define the relativeimportance of plasma membrane or SR ATPases in either cellular orsubcellular contexts (1 :285-297). N.; Jona, I.; Molnar, E.; Seidler, N. Ultimately, their action causes muscles to relax(9:12682-12688). 285:3 3-3 9; 1991, December 9. Information may also be obtained about Ca2+-ATPase through analysisof the enzyme's crystalline structure. Calcium-Dependent ATPase: Analytic Techniques A common eukaryotic enzyme, calcium-dependent ATPase has beenextensively investigated. Molnar, E.; Seidler, N. Thus, ofthe different alternative coupling mechanisms that have been proposed,perhaps the most accurate consists of the "conformational coupling"hypothesis (4:365-37 ). 1 23:147-167; 199 April 13. Researchers generally believethat the phenomenon involves the spatial relationship of the enzyme'sphosphorylation and ATP-binding domains to their Ca2+ channel. Finally, electrical studies have been used to evaluate the Ca2+-ATPase. H.; Brandl, C. The resultingenzymatic states may be characterized as follows: (1) E-La3+; (2) E-V; and(3) E-Pi, respectively. Other fluorescence techniques used to measure ATPase-ATPaseinteractions employ the following probes: (1) N-iodoacetyl-N' (5-sulfo-1-naphthyl)-ethlyenediamine (IAEDANS); (2) iodoacetamidofluorescein (IAF);and (3) N-(1-pyrene)maleimide. Emerging views on the structure and dynamics of the Ca2+- ATPase in sarcoplasmic reticulum. For example, Ca2+-ATPase can be selectivelylabeled with IAEDANS at Cys-67 and Cys-674 (6:147-166). Finally, a narrow stalk region links the membranous region to thecytosolic head. Various fluorescent probes have been used to characterize Ca2+-ATPase. The NCD4 moiety gives off a fluorescent signalfollowing the formation of bound N-acylurea. In addition, Sumbilla et al. Electron micrographs of Ca2+-ATPase typically show an outline of aglobule structure attached to the plasma bilayer by a narrow stalk. 268:365-37 ; 199 , August 1. Furthermore, the FITC site's position on the enzyme's head regiongives a distance of 47 Å between the FITC site and the Ca2+ binding site.Finally, the distance between the IAEDANS site and the Ca2+ binding sitehas been determined to be 16-18 Å (6:147-166). By blocking the binding site, this hindrance essentiallyinhibits ATPase activity and Ca2+ transport.
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