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Creatine phosphokinase: in vitro activity modification and proteolysis with calpain Arthur, Gavin D. 1994

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CREATINE PHOSPHOKINASE: IN VITRO ACTIVITY MODIFICATIONAND PROTEOLYSIS WITH CALPAIN.ByGAVIN. D. ARTHURA THESIS SUBMITTED TN PARTIALFULFILLMENT OF THEREQUIREMENTSFOR THE DEGREE OF MASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESSchool ofHuman KineticsWe accept this thesis as conforming to the required standardTHE UNIVERSITY OF BRITISH COLUMBIAApril 1994Gavin D. Arthur 1994In presenting this thesis inpartial fulfilment of therequirements for an advanceddegree at the Universityof British Columbia,I agree that theLibrary shall make itfreely available for referenceand study. I furtheragree that permissionfor extensivecopying of this thesisfor scholarly purposes may begranted by the head ofmydepartment or by his orher representatives,It is understoodthat copying orpublication of thisthesis for financial gainshall not be allowedwithout my writtenpermission.Department of flEr.’c-SThe University of British ColumbiaVancouver, CanadaDate 5/41 ‘74-DE-6 (2/88)ABSTRACTThepurpose ofthisstudy was to determineifthe muscle isoformofthe energytransmitting kinase;Creatine Phosphokinase (CPK- EC N° MM-isozyme,is asubstrate for calciumactivated neutral protease (CANP- ECN03.4.22.17).CPKactivity was measuredunder three different conditions:(1) control assay; (2)with 5mMCa2+,(5x103ca2j;(3) with 5mMCa2+and a range ofCANPamounts from 10 tolOOug. 5mM Ca2+consistently caused significantinhibition ofthe CPK activityto36% ofcontrol (p<0.05).In the presence of5mMCa2+and lOug ofCANP,CPKactivity was not significantlydifferent from the control activity.With 27ug CANPthere was a slightbut significant activationofCPK to 123.18 + 12.9%above thecontrol activity (p<0.05).As the amount ofCANP presentwas increased to 54, 67,84 and lOOug, theCPK activity was reduced to56.96 + 0,31%, 50.46+ 2.65%,36.06 + 0.5%,and 2.08 + 2.56% respectively.SDS-PAGE showedthat significant proteolysis ofCPK occurred with a rangeof CANPfrom 10 to 3Oug. Densitometricscanning ofthe CPKband and the 28kDa CANPsubunit showed that proteolysisof CPK was dependent onthe amount of CANPpresent. The proteolysisofCPK resulted in the formationof two large fragments.Themolecular weight ofthese proteolyticfragments were estimated tobe 38 and 35kDa.The results ofthis studyshow that CPK is a substratefor CANP in vitro and thatminorproteolysis results in activationof CPK, while increasedproteolysis results in lossofactivity.TABLEOFCONTENTSABSTRACTTABLE OF CONTENTS:fltLIST OF FIGURESACKNOWLEDGMENTSINTRODUCTION1Statement of the problem1Hypotheses2Physiological significance3Definiton of terms4Assumptions and limitations52. REVIEW OF THE LITERATURE6CANP: Discovery6Structure7Activation8Regulation9Substrate chatacteristics13Myofibrillar substrates14Enzyme substrates14PEST sequences15Examples of CANP activation in skeletal muscle17CPK: Structure18Function19Distribution and physiological significance20Muscle dysfunction22Examples of CPK disturbances23A possible mechanism243. PROCEDURES26Objective 126Measuring CPK activity26Measuring CANP activity27Measuring protein concentration280Statisticalanalysis.28Objective 228Objective 329SDS-PAGE29Densitometry29Statistical analysis294. RESULTS31Objective 131CPK activity with Ca2and CANP31Objective 335CPK proteolysis and CANP35Fragment analysis375. DISCUSSION 406. CONCLUSIONS46Suggested further research467. REFERENCES478. APPENDICES55LISTOFFIGURESFIGUREPAGE1. Schematic of the large and small CANP subunits72. Pest regions and primary sequence of CPK193. Enzymatic function of CPK 204. CPK activity with 10 & lOOug CANP325. CPK activity with increasing amounts of CANP336. CPK activity across all CANP amounts 347. SDS-PAGE of CPK with 10, 27 & lOOug CANP358. SDS-PAGE of CPK with 10-3Oug CANP369(A). CPK proetolysis with CANP389(B). CPK & 28kDa CANP with 0-3Oug CANP3810. Fragment production and 28kDa CANP with 0-3Oug CANP39ACKNOWLEDGMENTSI would like to thank my committee members Dr Don McKenzie andDrWadeParkhouse for theirvaluable input during this researchproject, their expertise, help andsupport was much appreciated. The process ofproposing carrying outand presentingthis final thesis was made almost a fun experience thanks to my supervisor DrAngeloBelcastro. I feel privileged to have worked with someone soknowledgeable,experienced and supportive (financially & academically). Thanksalso to my lab matesand friends whose support is much appreciated.1INTRODUCTIONStatement ofthe problemCalcium activated neutral protease, also called calpain or CANP, and its specific inhibitorcalpastatin is a non lysosomal proteolytic system found in mammalian cells. CANP is acysteine protease which is present in a wide variety of tissues including mammalian skeletalmuscle and has a pH optimum from 7 to 8. CANP is known to exist as two isozymes, bothisoforms having a large subunit (80-82kDa) which contains four distinct domains. Domainfour at the COOH terminal has a calmodulin like binding domain and this region appears todefine the Ca2+dependance of CANP. Each of the isoforms has an absolute requirementfor Ca2,uCANP requires uM Ca2+and mCANP requires mMCa2+concentrations tobecome active. Both have identical small subunits of 28kDa which are believed to haveregulatory roles. CANP undergoes autolytic activation to become fully active and hasattracted a great deal of attention recently due to its likely role as a regulatory or processingprotease in response to increased intracellular Ca2+concentrations. CANP seems ideallysuited for this role because CANP substrates are not degraded but are modified by limitedproteolysis. Included among the substrates known to date for CANP are:Muscle proteins like desmin and C-protein. Cytoskeletal proteins such as fodrin, viementinand neurofilaments. Enzymes, including protein kinase C, phosphorylase b kinase andtyrosine hydroxylase.CANP’s restricted proteolytic activity on its enzyme substrates has been suggested to increasetheir activity, indeed CANP was once known as “kinase activating factor”. CANP mediatedproteolysis may be facilitated by binding of the CANP to specific amino acids on itssubstrates, (Wang, Roufogalis, & Villalabo, 1989b). These sequences are called “PEST”sequences and many of CANP’s substrates carry these sequences and CANP cleavage seemsto occur at locations at or near these sequences.Situations where CANP is found to be active within the cell include: Duchennemusculardystrophy, where CANP activity has been shown to be increased ten fold, (Nagy & Samaha,21986). In the same condition a 5-6 fold and 3-5 fold increase wasmeasured for mCANP anduCANP respectively, (Reddy, Anandavalli, & Anandaraj, 1986). Afterprolonged runningexercise the activity ofCANP is elevated in rat skeletal muscle, (Belcastro,1993).As CANP is aCa2+activated protease then it is not surprising that common totheconditions where CANP is active within the cell are elevated intracellularCa2+levels.Muscles affected by Duchenne muscular dystrophy show increased intracellular Ca2+levelsand prolonged exercise leading to fatigue results in a Ca2+imbalance and a Ca2+overloadwithin the cell, (Croall & DeMartino, 1991). During many typesof muscle dysfunctionthere are disturbances in the enzyme creatinephosphokinase, CPK (E.C.No Againas an example is Duchenne musculardystrophy where CPK disturbances are present and therelease of CPK into the plasma is actually used to screen for the diseasein newborns. Inprolonged exercise the CPK system is disturbed andCPK release in this case is used as amarker of muscle damage. In addition to being disturbed duringperiods when intracellularCa2+levels are elevated and CANP activity is increased, theMM-CPK enzyme contains“PEST” like sequences that are thought to be recognition sites forCANP. Considering allthis, the hypothesis that CPK will be a substrate foractive CANP in vitro and proteolysis ofCPK will be concurrent with increasedCPK activity, seems warranted.To test this hypothesis the following objectives wereproposed.1: Incubation ofCPK with CANP to measure anychanges in the activity of CPK,2: Incubation ofCPK with CANP to measureany changes in the enzyme kinetics ofCPK.3: Visualization ofany proteolysisof CPK by CANP on SDS-PAGE gels.3Physiological SignificanceAny role for CANP mediatedmodification ofCPK could wellbe significantbecause CPKplays a major role in energytransmission within the musclecell. It has been shownto bepresent at specific sitesofenergy consumption withinthe cell such as; the M-lineofskeletalmuscle, (Turner, Walliman, &Eppenberger, 1973); at theCa2+ATP’ase on thesarcoplasmic reticulum and Na/KATP’ase on plasma membranes,(Baskin & Deamer, 1970).CPK is thought therefore to beable to supply the energy requiredfor muscle contraction,Ca2+pumps and Na/K pumps.CPK is also localized at sites ofenergy production withinthe cell. CPK is found atthe outer side ofthe innermitochondrial membrane and alsowiththe complex ofglycolyticenzymes at the I-band, (Walliman &Eppenberger, 1985). It doesseem that the energy producingand energy consuming sitesin the cell rely on these pools ofCPK for proper cellularenergy transmission, (Walliman& Eppenberger, 1985).Yet thereaie many conditionswhere the CPK systemis disturbed and if CPK withinthe cell is asubstrate for CANP, thenits proteolysis by CANP couldbe ofsignificance in understandingthe events during manytypes ofmuscle dysfunction.Therefore the aims ofthisstudy were to determineif CPKwas a substrate for CANP invitroand to determinethe effect ofCANP on bothCPK activity and structure.4Definition oftermsCPK = Creatine PhosphokinaseCANP = Calcium Activated Neutral ProteaseCa2+= CalciumPCr = PhosphocreatineSDS-PAGE = Sodium Dodecyl Sulphate Polyacrylamide ElectrophoresiskDa = kilo Daltonsug = micro grams(10-6grams)uM = micro molar(10-6molar)mM = milli molar(l0- molar)PEST sequence = Amino acid sequence with: Proline (P); Glutamic acid (E); Serine (S);andThreonine (T). PEST scores are determined using the PEST-Find computer programme,(details in Rogers, Wells, & Rechsteiner, 1986).CPK activity is expressed as Sigma units. One Sigma unit ofCPK activity willphosphorylate one millimicromole (nanomole10-9M)ofcreatineper minute at 25°C. In thisassay the reaction proceeds in the direction ofcreatineproduction. For practical purposesempirical conversion in this assay seems warranted. To convert Sigma units to internationalunits ofactivity (IU = u/mol/min/L) the following regression equation can be used:IU = 8.3344 X + 0.9. Where X = Sigma units ofactivity.5Assumptions and LimitationsResults ofthis study are limited to in vitro conditions.Ratios ofCANP to CPK may not be strictly comparable to those found within the cell.The pH ofthe CPK activity assay was constant at 7.5, this is optimum for CANP but abovethe optimum for CPK, pH is not held constant within living muscleThe temperature was held constant at 37°C during all ofthe assays. Temperature is notconstant within living muscle.Observations ofthis study are limited to the mM requiring form of CANP, (mCANP) only.To date no difference has been found between u &mCANP substrates.Only the MM CPK isozyme was studied.The BB, MB, and MiMi isozymes ofCPK may respond differently to mCANP.6Chapter twoLITERATURE REVIEWCANP: DiscoveryThe Calcium activated neutral protease, (CANP), is one ofthe few well characterized nonlysosomal proteolytic systems in mammalian cells. This neutral protease, which shows anabsolute requirement forCa2+for its activity, was discovered by Guroffin 1964 in thesoluble fraction ofrat brain homogenate, (Guroff, 1964). The second demonstration ofCANP was when aprotein factor was found in rabbit skeletal muscle that would activatephosphorylase b kinase in thepresence ofCa2+,(Meyer, Fisher, & Krebs, 1964). It wasnamed “kinase activating factor”. Subsequently, this kinase activating factor was shown tobe the same intracellularCa2+dependent protease that Gurrofhad documented, (Huston &Krebs, 1968). A specific inhibitor ofcalpain was discovered in the late seventies, (Nishiura,Tanaka, Yamoto, & Murachi, 1978). The existence ofthis specificCa2+protease inhibitorwas also shown by Waxman & Krebs in 1978. This endogenous specific inhibitorprotein,now called “Calpastatin”, indicated the CANP system to be specifically regulated andcontrolled within the cell. The discovery ofa lowCa2+requiring form of CANP in dogcardiac muscle by Mellgren in 1980 also increased the likelihood that the CANP system wascomplex and closely regulated, (Meligren, 1980) Further workby Murachi and co-workersin 1981 also showed that the two CANP’s had distinctly differentCa2+requirements andthat calpastatin was inhibitory toward both “the micromolarCa2+requiring CANP, (uCANPor CANP1) and the millimolarCa2+requiring CANP (mCANP or CANP2)” , (Murachi,Tanaka, Hatanaka, & Murakami, 1981). It seemed that the two forms of CANP and thespecific inhibitor protein constituted an intracellular regulatory system which was regulatedbyCa2+levels. The two different calcium sensitive forms of CANT’ and their specificinhibitor differ from tissue to tissue, both in relative and absolute amounts. It was thoughtlikely that these different amounts could well reflect a different physiological role for CANP7in each cell or tissue containing it. The increasing interest in the CAMP system resulted inthe analysis of CAMP structure in the 1980’s.StructureThe two isoforms ofCAMP both have a large (80-82kDa) subunit and a small (29kDa)subunit. The larger subunit ofthe CANP’s are genetically different proteins while the smallsubunits are from the same gene, (Suzuki et al, 1988). The large uCANP & mCANPsubunits have the same fundamental structures and share 50% sequence identity. Suzuki andco-workers elucidated the primary structure ofthe 80 kDa protein and showed it to have fourmain domains, (Ohno, Emori, Kawasalci, Kisaragi, & Suzuki, 1984). The 29kDa subunit hastwo main domains, see figure 1.III IIIIVCalmodulin80 kDaPropeptideProteaseBindingProtein“Calmodulin” likeCa2+ binding domainsV IV’30 kDaGlycine richdomainFigure 1: Schematic diagrammeofthe large and small CAMP subunits8Domain two shows a high sequence homology with papain, cathepsinsB, H & L. Domainfour ofeach subunit has calmodulin like calcium binding domains,ofthese four EF handstructures, the first and fourth are active calcium binding sites. A possible reasonthat CANPrequires Ca2+for activity is that domain four interacts with domain two and inhibits itsproteolytic ability. WhenCa2+binds to the enzyme, domain four undergoes aconformational change which removes this interference with domain two.Participation ofcalmodulin binding domains or proteins can usually beseen in Ca2+andcaimodulin activation ofenzymes. Domain three oftheCANP molecule is thought to act asa calmodulin binding region and to interactwith theCa2+bound domain four to renderdomain two free and active. The u and mCANP 8OkDa subunits appear tohave essentiallythe same domain two protease, however they do differ in domainfour, which is the domainthat defines theCa2+sensitivity ofthe protease. Domain one can also inhibit the intrinsicprotease activity but it is removed during autolytic activation. Twokinds ofcalpastatin havebeen found, the most prominent is a 73kDa protein.A 43kDa calpastatin has been found inerythrocytes. The large calpastatin molecule contains four repeatingslightly homologousdomains ofabout 140 amino acids. Each ofthe four repeatingdomains has inhibitory ability,therefore theoretically one large calpastatinmolecule can inhibit up to four CANP molecules,(Maid, Hatanaka, Takano, & Murachi,1990).ActivationWithCa2+ions playing a major role in mediating intracellular signalling andregulationthrough various calcium binding proteins etc, then thequestion was, “how is thisCa2+dependant intracellular protease activated and its activityregulated within the cell”?Incubation ofCANP in the presence ofCa2+and a caesin substrate initially results inmodifications ofboth the CANP subunits. This modificationofthe subunits occurs beforeany proteolysis ofthe substrate occurs. CANP is therefore aninactive pro-enzyme, (Saido etal, 1992). During the activation process, a 76kDafragment is formed from the large 8OkDa9sub-unit and an l8kDa fragment is formed from the small 30 kDa sub-unit. TheN-terminalregions ofboth the sub-units have been shown to change duringthis period ofautolysis. Thelarge sub-unit is modified first and modification ofthe small sub-unit followsjustprior to theappearance ofproteolytic activity. The N-terminal region ofthe8OkDa sub-unit must bemodified before proteolytic activity occurs. Modification ofthe small sub-unitapparentlyhas no effect on the proteolytic ability ofthe CANP but seemsessential for its interactionwith phospholipids, (Suzuki et al, 1988). Because the activationrate ofCANP has beenshown to be independent ofthe CANP concentration studied,it is suggested that theautocatalytic method of CANP activation is most likely anintramolecular event rather than anintermolecular one, (Inomota, Kasai, Nakamura, &Kawashima, 1988).RegulationThe calcium sensitivity ofnative or “pro-canp” in vitro increases significantlywhenpreincubated with Ca2•This Ca2+sensitivity is generally recognized to be the Ca2+required for autolysis and not theCa2+level required for proteolytic activity. The autolysedCANP requires lower Ca2+for proteolytic activity and therefore may be active at nearphysiologicalCa2+levels. The question is then: how does CANP become active in vivo ifit requires higher than physiological Ca2+levels for activation in vitro? Coolican andHathaway in 1984 first reported that phospholipids could significantly lowertheCa2+concentration required for autolytic activation ofCANP. This possiblemechanism forreducing theCa2+required for CANP activation was further investigated, (Suzuki, et al,1988 also, Saido, Mizuno, & Suzuki, 1991).Biological disuiphides were reported to reducetheCa2+required by CANP for its activation, this was suggested as a possible physiologicalmechanism that could play a role in the regulation ofCANPactivation, (Sacchetta,Santarone, & Dicola, 1990). Acidic phosholipids were found tolower theCa2+concentration required for autocatalytic activation,theCa2+level required was lowered withincreased phosphate groups on the phospholipid. Withphospatidylinositol-4-phosphate (PIP)10orphosphotidylinositol-4,5-bisphosphate (PIP2), then autocatalyticactivation occurred at 106to io-MCa2+,a plausible physiologicalCa2+concentration. Thephospholipids PIP3or PIP4, recognized as precursors or storage forms of2nd messenger, may be more effectivein assisting the activation ofCANP. The amount ofPIP2 shown toreduce theCa2+levelsrequired were within the levels normally found at typical biological membranes.Pro-CANPhas been shown to be able to translate to the plasma membrane fromthe cytoplasm in thepresence ofuM Ca2+and autocatalytic activation could then occur at the membrane in vivoin rat liver. This possible activation of CANP at biologicalmembranes would suggest thatCANP substrates could be membrane associatedproteins. Howeverit does not preclude therelease ofthe activated CANP from the membrane to acton cytosolic protein substrates, orthat it could be activated at sites other than biological membranes,(Meligren, 1987). Thereis evidence to suggest that the specific CANP inhibitorprotein calpastatin could be associatedwith the sarcolemma and possibly the sarcoplasmicreticulum, (Lane, Meligren, & Mericle,1985 also Meigren, Lane, & Kakar,1987). Considering the suggested method ofCANPactivation at the membrane, then a membrane associatedcalpastatin is an attractive idea. Thecalpastatin would be ideally placed to modulate the autoactivation ofthe CANP and sopossibly regulate its action on membrane bound substrates. It wasproposed that when theCANP to calpastatin molar ratio is greater than 1:1 thenthe calpastatin could be removedfrom its membrane sites, leaving the membranebound protein substrates open to proteolyticmodification by the CANP. The role ofthe membrane bound calpastatincould thereforebeto act as aprotective buffer againstany briefexposure to elevated Ca2 Intuitively itmakes sense to have this intracellularproteolyticsystem under some kind oftight spatialcontrol to ensure no unwanted cleavages of substrateswithin the cell. Examples of somemembrane associated events include: The fact that uCANP can be translocated toboth theplasma and granule membranes as the inactive“proCANP” and then be activated at themembrane during thrombin stimulatedplatelet activation, (Kuboki,Ishii, & Kazama, 1990).Another example is the long term potentiation producedfrom repetitive stimulation of11hippocampal neurons resulting from post synaptic activation ofuCANP. A suggestion as tohow this occurs is that the pre-synaptic release ofglutamine, opens thepost synapticCa2+channels and the resulting Ca2+influx causes activation ofuCANP and then hydrolysis ofthe membrane cytoskeletal attachment protein fodrin. It is thought that this post synapticremodelling makes latent glutamatereceptors expose themselves to subsequent release ofneurotransmitters, (Lynch & Baudryn, 1984). Further evidence for a CANP involvementinthis process is that the cysteine protease inhibitor Leupeptin can prevent the increase inglutamate receptors.Another example ofa membrane location for CANP activity is the CANP dependentactivation ofProtein Kinase C (PKC). CANP has been shown to be able to proteolyzePKC,the rate ofcleavage is enhanced by the addition ofphospholipid and diacyiglycerol. Theproteolysis ofthe 8OkDa PKC produces an active 5OkDa enzyme fragment requiring neitherCa2+or phospholipid for activity, (Murray, Fournier, & Hardy, 1987 also Melloni etal,1986). A model ofCANP activation and regulation that explains all ofthe data to date hasnot been agreed upon.The membrane activation hypothesis is an attractiveone and has been given generalwidespread acceptance. However it does not accommodate some other experimentalfindings. Except for theCa2+required for theproteolytic activity of CANP after autolysisthen the Ca2+levels required by CANP for autolysis and proteolysis are higher than thel0O-800nM freeCa2+found in vivo (0.1 - 0.8 uM). It may be possible to suggestmechanisms that allow physiological Ca2+levels to initiate autolysis ofCANP whichrequiresCa2+levels of 1-5uM in the presence ofphospholipid. An example ofthis could betransient large increases in Ca2+locally at sites such as Ca2+channels. Immunolocalizationstudies show CANP and calpastatin are distributed throughout the interior ofcells withnopreferential location near theplasma membrane, (Kumamoto, et al 1992). Several studieshave shown that CANP binds to proteins and not the phospholipids in membrane vesicles,(Kuboki et at, 1990). It has been shown thatCa2+levels required for either u or mCANP12autolysis were not changed by the presence ofinsideout erythrocyte membrane vesicles,(Zalewska, Thompson, & Goll, 1991). No changesin the ratio ofcytosolic to bound CANPwas found despite increased overall CANP activity inexercised rat skeletal muscle,(Belcastro, 1993).Immunolocalization studies show that in addition toplasma membranes, CANP will bind to awhole variety ofsubcellular structures eg; myofibrillsand Z-disks. It could be that somefeature ofthe binding ofCANP to its substrate is requiredto lower theCa2+required forautolysis, (Melloni & Pontremoli, 1989). However workby Barret, 0011, and Thomson(1991) suggests that in general the substrate has no effect on theCa2+required by theCANP. The specific role ofcalpastatin is also unclear in the membrane activationhypothesis. Calpastatin completely inhibits both autolyzedand unautolyzed u and mCANPand does so at Ca2+concentrations below those required for either autolysis orproteolyticactivity, (Kapprell & Goll, 1989). Also puzzling is the factthat most cells contain enoughcalpastatin to inhibit all the CANP that immunolocalization studies showit to be co-localizedwith. Therefore cells must have a means for regulation ofthe inhibition ofCANP bycalpastatinifCANP is to have any role in the cell.A simple and direct explanation for the various contradictory pieces ofevidence regarding theactual and requiredCa2+levels could be that there is some other piece to the CANPpuzzle.This “piece” could take the form ofan “activator” that couldenhance the affinity oftheCANP-proteolysis binding sites so that Ca2+can bind at physiological concentrations. Thiscould involve a kinase or phosphatase that is activated by a physiologicalCa2+influx, theactivator may then phosphorylate or de-phosphorylate the CANP, thereby increasingtheCa2+affinity. Such a factor may be analogous to Calmodulin eg; can bindcalcium atphysiological Ca2+then bind to CANP and change its Ca2+affinity. A 40 kDa protein hasbeen reported in neutrophils and muscle extracts which lowers the Ca2+required bymCANP 50 fold but does not affect theCa2+required by uCANP. This protein alsoprevented the inhibition ofCANP by calpastatin, (Pontremoli et al, 1990). Anotherprotein13factor has been reported which seems to modify CANPactivity, (Shiba et al, 1992). This socalled “CANP activator” was purified from humanplatelets and could increase CANPactivity two fold although it did not alter theCa2+sensitivity ofthe CANP.Since the process ofCANP activation and regulation cannot be explained in a way toencompass all thepieces ofevidence to date, it may bethat CANP activation and regulationdiffers depending upon the location and characteristicsofthe substrate being targeted.Substrate characteristicsBy knowing which proteins are substrates for CANP,then the physiological role(s) oftheCANP system should become clearer. Proteolysis ofa particularsubstrate may be dependenton the metabolic state ofthe tissue and thereforethe proteins within the cell. For example,ATP depletion has been shown to stimulate CANPdependent protein breakdown in chickskeletal muscle, (Fagan, Wajnberg, Culbert, & Waxman,1992). Certain conditions couldalso make CANP substrates resistant to proteolysis.Covalent modifications of substrates andoxidation reduction status ofproteins are suggested toallow targeting by CANP in skeletalmuscle, (Belcastro, 1993). Phosphorylation ofactinbinding protein by cAMP dependentprotein kinase increases its resistance toproteolysis by CANP, (Zhang, Lawrence, &Stracher, 1988). Another example is themicrotubule associated protein tau, whenphosphorylated by cAMP-dependent protein kinaseits proteolysis by CANP is significantlyreduced compared to un-unphosphorylated tau, (Litersky& Johnson, 1992). These authorssuggest a role for phosphorylationin regulating the degradation oftau, and that abnormalphosphorylation may result in a resistant populationoftau which could lead to the formationofpaired helical filaments found inpeople with Alzheimer’s disease.A wide range of substrates for CANP have been demonstratedin vitro and despite theadvances in knowledge about thestructure and the enzymaticproperties ofpurified CANP,its physiological roles are still poorly understood.It is clear that CANP does not actjust as ageneral protease, CANP has specific substrates whichit cleaves in a specific and usually14limited fashion. The two differentCa2+requiring isozymes will proteolyze the sameproteins in vitro, (Wang et al, 1989b).Myofibrillar substratesInitially the CANPs were suggested to be involved in the turnover of myofibrillarproteins,(Revile, Goll, Stromer, Robson & Dayton, 1976). Contractileproteins are known to becolocalized with the CANPs and remodelling or metabolic turnover ofthese contractileproteinsis thought to be one ofCANPs main physiological functions. Initial studies on the effectofCANP on muscle myofibrillar proteins showed that mCANP was able to cleave desmin andtroponin T rapidly. Troponin I, tropomyosin and C-protein were cleaved more slowly, whileactin, myosin and troponin-C were resistant to modification by CANP, (Reddy, Anandavalli,& Anandaraj, 1986). Activation ofCANP under physiological conditions during prolongedrunning exercise in rat skeletal muscle was shown by Belcastro in 1993. The myofibrils fromthe exercised animals showed increased rates ofCANP induced degradation ofdesmin,vimentin, C-protein, and the removal ofaipha-actinin. This study showed thatphysiologically induced modifications ofthese CANP substrates due to exercise madethemmore succseptable to CANP cleavage, (Belcastro, 1993).The CANPs have also been attributed with the role ofdisassembly ofcell cytoskeleton,especially at sites where the cytoskeletal proteins are attached to the plasma membrane, (Ek& Helden, 1986 also Kay, 1983). This ability ofthe CANPs to influence the cytoskeletalproteins has been well studied in platelets. The activation ofplatelets is accomplished by anincrease in intracellularCa2+concentration and this is followed by the specific cleavage offilamen, talin and spectrin, (Barret et al, 1991).Enzyme substratesSpecific cleavage by CANP also activates or alters a number ofdifferent enzymes. Anunknown protein factor, later shown to be CANP, was found to activate phosphorylase-b15kinase in thepresence ofCa2+,(Myer et al, 1964). This unknown factor was giventhename “kinase activating factor”. Usuallywhen a kinase or phoshaptase is proteolysed byCANP the remaining large fragments retaintheir activity, but they are usually now free fromthe controls that regulated the intact enzyme.An example ofthis is protein kinase C, (PKC),which is cleaved from an 8OkDaprotein to a5OkDa protein which no longer needs thecofactors that the 8OkDa protein requiresfor activity, (Murray et al, 1987). Phosphorylasekinase is also activated by mCANPproteolysis, this occurs independently from its normalmode ofactivation by cyclic AMP andphosphorylation. Another substrate that CANPconverts to an irreversibly active formis myosin light chain kinase, which usually requirescalmodulin and Ca2+for activation. Some other enzymes which are activated byCANPactivity are; Protein activated kinase II, which is convertedto an active form by uCANP(Perrisic & Traugh, 1988); as is the D form ofglycogensynthase, (Hiraga & Tsuiki, 1986).Calcineurin, a calmodulin dependentprotein phosphatase was also found to be a substrateforCANP and was converted to an active,calmodulin independent form. Its activation wasconcurrent with the formation ofa 45kDaprotein after cleavage ofthe original 6OkDa proteinby uCANP, (Wang et al,1989a). A specific cleavage by CANP on tyrosine hydroxylasealso results in the formation ofa large,(56kDa), fragment from the 6OkDa original.Thisremoval ofthe N-terminal end ofthe tyrosinehydroxylase resulted in a slight but significantactivation ofthe enzyme. Although thelist ofenzymes that are substrates for CANP isgrowing, only selected enzymes are substrates forCANP. Obviously these enzymes havesome common feature to set themapart for recognition by CANP.PEST sequencesWhat makes certain proteins recognizable substratesfor CANP? Rogers and co-workersexamined the amino acid sequencesoften proteins with intracellular halflives ofless than2hours. Common to each protein was atleast one region rich in proline (P), glutamic acid(E), serine (S) and thereonine (T), now known as“PEST” regions. The analysis ofanother16group of35 longer lived proteins showed that only 15 ofthese contained these so calledPEST regions. Two types ofPEST regions have been defmed,determined on how closelyrelated they are to the above amino acid sequence. These scores can be greaterthan zero or“strong pest regions”, or they can be between -5 and zero, or“weakpest regions”. Ofthegroup of35 longer lived proteins, only 3 ofthem containedstrong PEST regions. Rogerssuggested that as well as conferring the propertyofrapid degradation to these proteins, thenegatively charged and phosphorylatable PESTregions could bind Ca2 This localizedincrease inCa2+concentration could possibly activate CANP, (Rogers et al, 1986).PESTregions are very hydrophilic and are thought to form surfaceloops on the protein, thesewould be easily accessible to CANP, which would thencleave the protein nearby,(Rechsteiner, Rogers, & Rote, 1987). The fact thatmany calmodulin binding proteins areknown to be substrates for CANP prompted a searchfor PEST like regions in these proteins,(Wang et al, 1989b). The substrates for CANP generallyshowed the presence ofPEST likeregions located at or near the site ofcleavage by the CANP.These included; Insulinreceptor substrate-i which contains PESTlike sequences and is degraded by CANP froml7OkDa to 90 & 79kDa fragments, (Smith, Bradshaw,Croall, & Garner, 1993). PlasmamembraneCa2+ATP’ase, where the C-terminal end ofthe protein showedPEST likeregions near the sites ofCANP cleavage, (Wang,Roufogalis, & Villalabo, 1988). u&mCANP also cleave alpha-fodrin (24OkDa)selectively to produce a specific fragment(i5OkDa). It as been shown that locationofthe CANP cleavage on the fodrin is in the samearea as the only PEST like region in theprotein. The CANP cleavage site on protein kinaseC has also been found to be inthe same area as the region containing thepest like sequence.Another enzyme substrate for CANPproteolysis is HMG-CoA reductase, the rate limitingenzyme in cholesterol biosynthesis. It containstwo PEST like sequences at approximately 40and 47kDa from the N-terminal,(Chin et al, 1984). The CANP cleavage sites are found atapproximately 35 and 49kDa from the N-terminus,(Liscum, Finer-Moore, Stroud, Luskey,Brown, & Goldstein, 1985).17The degradation rates ofproteins may also depend on their association withcellularstructures, membranes, filaments etc. This binding to intracellular sites could maskthe sitesrecognized by intracellular proteolytic agents. It is likely that CANP localizationcan givesome clues to its role.Examples ofCANP activation in skeletal muscleCANP has been shown to be activated in a number of “physiological” situations.Anexample where CANP has been shown to be active is in dystrophic muscle, where there areincreased rates of protein degradation and increased levels of CANPactivity. Increasedmeasurable CANP activity of 3-5 fold for uCANP and 5-6 fold for mCANP inmuscle fromDuchenne muscular dystrophy patients has been reported, (Reddy et al, 1986). A tenfoldincrease in CANP activity was found in the same condition by Nagy & Samaliain 1986.Skeletal muscle CANP activity was increased and the calcium required for 50%of maximalactivity (pCa5),was reduced after prolonged running exercise, (Belcastro,1993). CardiacCANP activity was increased by 60 minutes of level runningin rats, (Arthur & Belcastro,1994) abstract.?. In skeletal muscle of mice with diabetic amyotrophy, intracellularCa2+levels and CANP activity were both elevated, (Kobayashi,Fujihara, Hoshino, Kimura &Kimura, 1989). Common to many situations whereCANP activity is increased aredisturbances in the localization of the enzyme creatine phosphokinase (CPK),In Duchennemuscular dystrophy the plasma levels of CPK are often twoorders of magnitude greater thannormal and elevated levels of CPK in theblood are used as a marker for the disease innewborns. During episodes of malignant hyperthermia,(MH), that occur in susceptibleindividuals after exposure to depolarizing muscle relaxants or anaesthesia,there are musclecontractures, rigidity and disturbances of CPK in the muscle, (Lang,1981). Elevation ofCPK levels in the serum were once used to screen forMH, (Gronert, 1980). Disturbancesin CPK from cardiac muscle after coronary damagehas been extensively studied and theplasma levels ofCPK are used as an indicator ofinfarct size, (Roe, 1977 also Jones, Jackson,18& Edwards, 1983). CPK disturbances are found with prolonged running exercise,whichalso increases CANP activity. Denervation or fasting and refeeding increases theconcentration of CANP and calpastatin in rat soleus muscle, and serum CPK levels are alsoelevated, (Kumamoto et al, 1992). The disturbance and release of CPK has long been usedas a marker of muscle damage, (Linjen et al, 1988 also Amelink, Bar, Van DerKalen, &Wokke, 1990). CPK has also been shown to become unbound from its intracellular locationsin isolated muscles stimulated to fatigue, (Guderley, Jean, & Blovin, 1989), Total CPKactivity is reduced in the myocardium of cardiomyopathic hamsters, (Khuchua, VenturaClapier, Kusnetsov, Grishin & Saks, 1989). Parathyroid hormone induces increases in CPKactivity which can be mimicked by synthetic diacyl glycerol or the Ca2+ionophore A23187.It is likely that CANP will be active at these times, (Somjen, Zor, Kaye, Harell & Itzhak,1987). However CPK activity was unchanged in diabetic amyotrophy whileCa2+levels andCANP activity were elevated, (Kobayashi et al, 1989).Situations where CPK disturbances are found are usually associated with elevatedintracellular Ca2+concentrations which increase the possibility that CANP is active withinthe cell.Thus a relationship may exist between the increased CANP activities and disturbancesin theCPK system. However no data exists for CPK being a substrate for CANP mediatedmodification.Initial study of the amino acid sequence of CPK has shown that CPK does contain someweakpest regions, see figure 2.CPK: StructureThe enzyme ATP:Creatine N-Phosphotransferase (EC No 2.7.32), commonlyknown ascreatine phosphokinase (CPK), is a dimeric enzyme composed of two 360/380 amino acidmonomers and contains two catalytic centers. The molecular weight of the enzyme as19determined in a non denaturingelectrophoresis system is approximately82,600 daltons, madeup oftwo subunits of41,300 each. Theprimarysequence ofMM-CPK is shown in figure2.The PEST sequences ofthe MM-CPKare:POSITIONFrom To14 2446 6567 85PEST SCORECOMMENT-0.21 weak-0.4 weak-12.48 weakpSEOUENCEFSAEEEFPDLSETPSGFTLDDVIOTGVDNPGPFIMTVGCVAGDEESYEVF1 MPFSSTHNKHKLKFSAEEEFPDLSKHNNHMp p31 AKVLTPELYKRLRDKETPSGFTLDDVIOTG61 VDNPGHPFIMTVGCVAGDEESYEVFKDLFDp91 PVIQDRHGGYKPTDKHRTDLNHENLKGGDDp pp121 LDPKYVLSSRVRTGRSIKGYSLPPHCSRGEp151 RRAVEKLSVEALNSLEGEFKGRYYPLKAMT181 EQEQQQLIDDHFLFDKPVSPLLLASGMARD211 w P 0 A R G I W H N 0 N K T FL V W V N E E D H L R V IS M241 EKGGNMKEVFRRFCVGLKKIEEIFKKAGHP271 FMWTEHLGY I LTCPSNLGTGLRGGVHVKLPpP301 K L SQH P K F E E I L H R L R LQK R G T G G V 0 T A A Vp p331 GAVFDISNADRLGFSEVEQVQMVVDGVKLM361 VEMEKKLEQNQP I DDMIPAQKFigure 2: The primary sequence of MM-CPK.Sequences recognized as possiblePESTsequences are underlined. The active site is in boldand underlined (residues 283 to 289).Phosphate groups are represented witha 11p’ above the appropriate amino acid.FunctionThe first event in the activation of the myofibrillaradenosine triphosphatase by Ca2+ishydrolysis of ATP, leading to a local accumulationof ADP andH+,both of which actuallyserve as substrates for the Lohman reaction by CPK.20CPK catalyses the reversibletransfer of a phosphate residue inhigh energy bondingbetweenadenosine triphosphate (ATP) andCreatine (Cr) as follows:Cl-f3 NH2CU, NH,O2C—CH,—NC+ MTP’MgADP+ O,CH,—N<2 + WNI-f,“NHPO,CreatinePhosphocreanc)(11)Figure 3: Enzymatic reactioncatalyzed by CPK.Increased availability ofADP, phosphocreatine (PCr),and the lowering of intramyofibrilarpH will all activate CPK inthe direction of ATP regeneration.The Km values for PCr andcreatine for MM-CPK are 1.7mMand 16mM respectively, (Saks,Chemousova, Gukovsky,Smirnov, & Chazov, 1976). Thehigh affinity of MM-CPKfor Mg-ADP (Km=O.08 mM)causes the latter to be trappedand rephosphorylated intoATP by CPK as long as creatinephosphate is present atconcentrations of 3-4 mM or higher.The rephosphorylation of ADPto ATP also acts to buffer hydrogenions and maintain pH levels withinthe cell. The pHoptimum ofthe CK reactionis between pH 6.5 and 6.6, (Walliman& Eppenberger, 1985).Distribution & PhysiologicalsignificanceCPK is the key energy transmittingkinase in the muscle celland is known to exist in fourdifferent isozyme forms.Three of these isozymes are formedby combination of either twohomologous subunits, MM-CPKfor muscle, BB-CPK forbrain or hetrologous subunits, MBCPK. In skeletal musclethe predominant form of CPK is theMM isoform. CPK wasoriginally thought to be strictlycytoplasmic and thereforesoluble. However it becameapparent that CPK is also specificallybound at strategicallyimportant locations as well.From ten to thirty percentof total CPK activity, dependingon muscle type is located on theinner mitochondrial membraneas the fourth isozyme of CPK, MiMi-CPKa specificmitochondrial adenylatetranslocase isoenzyme. CPK is foundat the sites of energyproduction within the cell,such as oxidative phosphorylation, ATP/ADPtranslocase and21glycolysis, (Walliman & Eppenberger, 1985). These sites ofenergy consumption within thecell where CPK is bound include the M-line of skeletal muscle, (Turner et al, 1973),sarcoplasmic reticulum and ribosomes, (Sharov, Saks, Smirnov, & Shazov, 1977), andplasma membranes, (Baskin & Deamer, 1970). The small strategically located sites of CPKare thought to be able to respond quickly to changes in their local environment such as ATPhydrolysis and rephosphorylation of ADP. CrP is thought to move from the sites of energyproduction to the sites ofconsumption and Cr moves from the sites ofenergy consumption tothe sites ofenergy production.The functional significance of these “pools” of CPK within the muscle cells have beendemonstrated by numerous experiments, CPK bound to skeletal muscle sarcoplasmicreticulum maintains a much higher rate of Ca2+ion uptake in the presence of CrP ratherthan an added ATP regenerating system at the same adenine concentration,(Levitsky,Levchenko, Saks, Sharov, & Smirnov, 1977). Ion transport across the cell membranecanalso get its energy supply from CrP via local CPK.The adequacy of the CPK system hasbeen demonstrated using the rate of hydrolysis of ATP, proportion ofM-line bound CK, andits ATP regeneration potential. That is, the intra myofibrillar regeneration ofATP by theMline bound CPK can account for the entire energy required for contraction, (Walliman &Eppenberger, 1985). When ATP containing radio labelled gammaphosphate (5mM) andnormal CrP (10mM) was supplied to the myofibrillar ATP’ase, there wasvery little of thelabeled phosphate produced via the muscle contraction. This shows that the phosphate groupused to rephosphorylate the ADP formed during the contraction wasfrom the CrP,(Bessman, Geiger, Erickson-Vitanen, & Yang, 1980). The rate of amino acid uptakebyisolated polysomes was found to be much higher in the presence of CrP and low ATP(0.05mM), than with an equimolar or even higher ATP concentration or an added ATPregenerating system, (Savabi, Carpenter, Mohan, & Bessman, 1988).CPK both bound and free at the sites of energy production and consumption couldplay thekey role in energy transmission within the cell.This system is known as the creatine22phosphate shuttle, (Bessman et al, 1980 also Walliman & Eppenberger, 1985 and Savabi etal, 1988). With CPK playing such a key role in energy transmission within the cell, it wouldbe expected that alterations in CPK activity by inhibition, proteolysis or removal, may lead tofunctional changes within the muscle.Muscle DysfunctionDisturbances in the normal functioning of muscle often occurs when theCPK system isperturbed or modified. In several muscle diseases in both humans and experimental animals,including muscular dystrophy and nutritional myopathy, there is a distinct inability of themuscle to retain creatine. It has been demonstrated that CPK changes are a causative factorof muscle dysfunction. Chicks fed with B-guanidobutyric acid which inhibits creatinetransport into the muscle show ultrastructural changes within the muscle. These changesinclude disruption of normal filament organization, Z band streaming, scattering of thesarcoplasmic reticulum and t-tubules, (Laskowski, Chevli, & Fitch, 1981). These changeswere not attributed to any loss of a direct structural role of creatine. Removal of the CPKlocalized at the M-line in skeletal muscle using a low ionic strength buffer, (Walliman &Eppenberger, 1985), resulted in a loss ofability to regenerate ATP and an obvious functionallimitation in that the remaining CPK was unable to maintain the supply ofATP and also wasunable to buffer theH+ion production. The deterioration in CPK activity found inmyocardial cells in rats has been proposed to affect the transfer of high energy phosphateswithin the myocardial cells and the consequent defect in energy utilization may explain thealtered myocardial function present in diabetes mellitus, (Mokhtar, Rouseau, Migneron,Tancrede, & Nadeau, 1992). Prevention ofthe oxidative deactivation of cardiac CPK duringischemia with myristic acid results in improved CPK activity in rats. The enhanced CPKactivity after treatment correlated well with functional benefits to the reperfusedmyocardium, (Kaplan, Blum, Banerjee, & Whitman, 1993). It is clear that abnormalities inthe CPK system ofenergy transmission are associated with many types of muscle dysfunction23and that the energy consuming sites within the cell rely on the proper functioning of theirlocalized CPK pool. Therefore alteration of CPK activity by proteolysis, and or removalwould likely cause functional changes to energy transmission within the cell. Theserelationships between disturbances in CPK activity and function have been used successfullyin numerous clinical studies to assess the presence or absence oftissue damage (see below).Examples of CPK disturbancesThe release ofintracellular CPK from diseased muscle is a well known phenomena and wellcharacterized in a number ofconditions, (Pennington, 1981). There are many such examplesof disturbances in the distribution of CPK and the majority of these examplesinvolve somesort of muscle dysfunction. These include Duchenne muscular dystrophy, As mentionedbefore, CANP activity is also elevated in muscle from patients with Duchenne musculardystrophy. MH causes contractures, rigidity, and disturbances of CPK in the muscle.Disturbances in CPK from cardiac muscle after coronary damage has been extensivelystudied and theplasma levels of CPK are used as an indicator ofinfarct size, (Roe, 1977 alsoJones et al, 1983). CPK disturbance and release has long been used as a marker of muscledamage, (Linjen et al, 1988 also Amelink et al, 1990). CPK has also been shown to becomeunbound from its intracellular locations in isolated muscles stimulated to fatigue, (Guderleyet al, 1989). Although experimentally induced CPK disturbances can lead toloss of musclefunction and the clinical evidence is extensive for these disturbances. Little is known abouteither the chronic changes in disease states or the more transitory alterations in the CPKsystem as with acute exercise. A number of factors have been suggested to give rise to CPKdisturbances and its release from skeletal muscle. Increased membrane permeability due todetergents initiates a quicker release of CPK than is found with contractile activity,suggesting against a simple release of cytoplasmic CPK due to increased membranepermeability in the exercise condition. The time course of CPK release was found to besimilar to that of lactate dehydrogenase which has a molecular weight of 140kDa as24compared to the 8lkDa of CPK, also suggesting against only an increased membranepermeability. Acidosis has also been suggested as a cause of CPK release, however musclesstimulated under conditions considered normoxic will release CPK, as do muscles that havebeen poisoned with iodoacetatic acid and therefore cannot produce lactate. Some of thesuggested causes of the CPK release are, influx of extracellular Ca2+and consequentdamage to mitochondria, activation of proteases and lipases. Despite much research in thisarea, the mechanism or mechanisms underlying CPK disturbance and release or reduced CPKactivity is not well understood. The modification of CPK activity at its sites within theskeletal muscle cell may cause significant changes in the energy transmission throughout thecell. It is during these times ofhigh energy requirements that the CPK system is used most.These conditions are also where the greatest alterations in the CPK system seem to occur.Therefore any agents that could modify CPK activity may be important during these periods.A possible mechanismElevated intracellular Ca2+concentrations are commonly used to stimulate CPK releasefrom muscle in studies attempting to determine the underlying cause and/or mechanism ofCPK release. These include experiments with the calcium ionophore A23187 and caffeine,(Duncan, 1978 also Jones et al, 1983). Incubation of rat skeletal muscle with the calciumionophore A23187 or with the mitochondrial poison 2.4. Dinitrophenol increasesintracellularcalcium levels and stimulates CPK disturbances and release from the cell, (Brazeau & Fung,1990). The same group also found that release of CPK from skeletal muscle exposed topropylene glycol or ethanol was increased when muscles were incubated in a mediumcontaining calcium chloride compared to one containing sodium chloride. The addition ofdibucaine, a non specific phospholipase A2inhibitor, only modestly reducedthe CPK releasefrom the muscle, (Amelink et al, 1990). It was concluded that the cosolvent induceddisturbances of the CPK system in skeletal muscle may be caused by anintracellularmechanism rather than by a direct solubilization of the sarcolemma and that thisintracellular25mechanism may involve the mobilization ofCa2•Further support of the role ofCa2+ininducing CPK disturbances comes from exercise or disease studies. The exercise inducedrelease of CPK from rat skeletal muscle is reduced after treatment with dantroline sodium,(Amelink et al, 1990). Dantroline sodium inhibitsCa2+release from the sarcoplasmicreticulum and has been used as an effective treatment for MH. Common to thesedysfunctions of skeletal muscle are disturbances in the Ca2+homeostasis within the musclecell, an example of this is found in prolonged exercise leading to fatigue where aCa2+imbalance develops. As mentioned previously, CANP is also activated during prolongedrunning exercise where Ca2+levels have also been reported to be increased with fatigue,(Sembrowitch, Johnson, Wang & Hutchison, 1983 also Belcastro, 1993). This CANPactivation may promote a disrupted state within the muscle during running, (Belcastro et al,1985). Similarly muscles affected by Duchenne muscular dystrophy appear to have increasedintracellularCa2+levels, (Croall & De Martino, 1991). AlteredCa2+levels have beenhypothesized to cause MH as a result of a sudden increase in intracellular Ca2+within themuscle. MH muscle has been shown to have a so called “leaky sarcoplasmic reticulumCa2+release channel” and MH susceptible individuals do demonstrate significantlyincreased levels ofintracellularCa2+It seems clear that elevated Ca2+levels are consistently associated with myopathies andCPK release from the muscle. How this process works is completely unknown. Howeverelevated Ca2+levels may be linked to CPK activity through the Ca2+dependent proteolyticsystem, CANP/calpastatin.CANP activity may be elevated during muscle dysfunctions where Ca2+levels are elevatedand the CPK system is disturbed. Thus a relationship may exist between increased activity ofCANP and these disturbances and or modifications to the CPK system. However no dataexists for CPK being a substrate for CANP or ofany effect ofCANP on CPK activity.26Chapter 3PROCEDURESObjective 1: To determine ifCPK activity is changedby active CANP invitro.CPK activity was measured in vitro, as a standard control and then with 5mM Ca2,anddifferent amounts and specific activities ofactive CANP present during the assay.The MM-CPK purified from rabbit skeletal muscle was obtained from Sigma and itspuritywas assessed by scanning the control CPK lanes in the SDS-PAGE gels. Purityof thesamples ranged from 83% to 98% of the total protein being the42kDa MM-CPK band, seeappendix D (1).Measuring CPK activityCPK activity was measured using a colorometric assay basedon the method of Hughes,(1962). The assay relies on the rephosphorylation of ADP at the expense of PCrand theliberation ofcreatine as shown below:Napthol+Phosphocreatine CreatineDiacetyl+ ADP+‘1Coloured ComplexThe basic assay solution contained 6.48mM PCr, 10mM Dii’, low saltTris buffer, pH 7.5,(1M KC12, 1mM MgC12, and 1mM Dii’), & 25u1 of CPK (200ugIul). The assaycontentswere incubated at 37°C in a water bath and the CPK reaction was started by adding 50ul ofADP-Glutathione (final concentration = 13.3uM).The reaction was then stopped after thirtyminutes by the addition of 50u1 of p-Hydroxymercuribenzoatesolution (0.05 mol/L). Then250u1 of alpha-napthol, 250 ul of diacetyl and l.75m1 of waterwere added to the assay andmixed. The creatine that was liberated during the reaction forms acomplex with the alpha-27napthol and diacetyl. This complex is coloured and its formation takes 10 to 15 minutes.The assay contents were then centrifuged in a Hermle Z360K centrifuge with a four placeswing out rotor at 3000rpm for five minutes. The amount of coloured complex formed isproportional to the CPK activity and was measured by spectrophotometry using a ShimadzuUV-160 spectrophotometer. The activity of the CPK was found by comparing the absorptionat 520 nm to a standard curve using known amounts of creatine treated in the same way asthe samples. This was the standard or “control” CPK assay condition. This standard assaywas reliable from day to day within each batch of CPK, the descriptive statistics for fourassays on different days are shown in Appendices E(1) & E(2). The activity from these fourstandard assays had a standard deviation of6.4% ofthe mean activity.Because it is necessary to have sufficientCa2+present to activate the CANP, (1mM freeCa2j.ACa2+containing assay was used as aIICa2+control”. When modifications aremade to the control assay, the results for each set were normalized to the control value forthat particular series of experiments and the appropriate blank used to correct for backround.This is because each batch of CPK could vary slightly in activity depending on purity of theCPK. The activity of CPK during the thirty minute incubation was recorded with differentamounts and specific activities of CANP present in the assay, (details are provided in theresults section, tables and figures).Measuring CANP activity:Partially purified (anion exchange chromotography), m-CANP (Sigma), was used in all theassays. Calpain was stored in a buffer containing 50u1 of 200mM DTT, 4.9m1 of 250mMTris pH 7.4, and 50 ul of 100mM EGTA. The EGTA is required to chelate any freeCa2+ions and therefore prevent autolytic activation of the calpain during storage. Activity of 0,10, and SOul CANP was standardized to the proteolysis ofcasein substrate assayed at 37°Cin the presence of2 mgml4casein, 25u1 of200 mM DTT, 75ul of50mMCa2+(this gives1mM freeCa2+due to EGTA) and the total volume for each condition made up to 500ul28with 250mM Tris (pH 7.4).After 30 minutes, caseinolysis wasquenched by the addition of500u1 of ice cold 5% TCA.A unit of calpain activityis defmed as the amount of TCAsoluble product resulting in an increaseof 1 unit at 280nm after the calpain digestof caseinsubstrate. Estimates ofcalpain activitywere performed in duplicates.Measuring Protein ConcentrationProtein concentration ofthe CPK and CANP was measuredusing the method of Lowry etal., (1951). See appendixA.Statistical AnalysisTo detect any statisticallysignificant alteration in the activityof the CPK with differentamounts (0-lOOug)of CANP, a repeated measuresANOVA was used. Scheffes posthocanalysis allowed the studyofindividual differences across conditions,(alpha 0.05)Objective 2: To study theenzyme kinetics ofthe CPK.To study the enzyme kineticsof CPK it was necessary to studythe activity of the enzymeusing an end point that lay on theinitial linear portion of theplot of enzyme activity againsttime. Pilot work had shownthat with the control assay condition,an end point of 1.5minutes would ensure thatthe enzyme kinetics are beingstudied under a steady state.However when a seriesof assays with ranges of substrate concentrationswere measuredunder (a) control conditions, (b)control + Ca2,(c) control + Ca2++ CANP. Thesmall amount of CrP liberatedin 90 seconds meant that the variabilityof the resultingactivities were high andmade any meaningful enzyme kinetic dataanalysis unfeasible. SeeAppendix B; 1 & 229Objective 3: To determine ifCPK would be proteolyzed byactive CANP in vitro.CPK was incubated with different amounts ofCANP and proteolytic products visualized.SDS-PAGE.The following methods were used.For the CPK control solution = 8mMCa2+,17mM DTT’, 14.75mM Tris (pH 7.4) and lOulofCPK stock.In the trials where CANP was present, the volume of CANP added was accommodatedbyreducing the volume of the Tris buffer to keep the total volumeconstant. The CANP wasadded to the solution last and the contents incubated at 37°Cfor 30 minutes and eachcondition was duplicated. The samples were then mixed with20u1 of digestion buffer(consisiting of l2ul of 200mM DTT, imi of 3 X sample buffer and200u1 of 2% bromophenyl blue). The samples were then incubated for 10 minutes at 47°C and loadedonto 10%gels. A solution containing known molecular weight markerswas loaded into one of thewells on the gel. The gels were then run overnight for approximately16 hours, then stainedin Coomassie brilliant blue stain for 2 hours and destained in graded methanolsolutions.After destaining, the gels were dried down in 70% methanol between cellophane sheets.DensitometryQuantification of protein bands was carried out using anLKB 2202 Ultrascan LaserDensitometer and an LKB Integrator. The percent total proteinof both the CPK, the smallsubunit ofCANP, and any proteolytic products were recorded from these scans.The molecular weights ofthe proteolytic fragments were estimated fromthe gels.Statistical Analysis: The % total protein of each band visualizedon the SDS-PAGE withincreasing CANP was compared using a repeated measures ANOVA.The effects of each30increasing amounts of CANP were compared to each other using aScheff&s post hoc test,(alpha = 0.05)31Chapter 4RESULTSObjective 1: Is CPK activity affected by active CANP in vitro?CPK activity with Ca2+and CANPThe values for CPK activity of the control assay were typically from 45 to 70Sigma units(375 to 585 U), the protein concentrations of the CPK used was 200ugIul. The activity ofthe control CPK was stated as 100-200 U ofactivity/mg. The control assay contained 25u1 x200ug = 5mg of CPK. Therefore I could have expected CPK activity of500 to 1000 units.The assay results were reliable within each CPK batch, see appendix B (1&2).Howeverwhen new samples of CPK were reconstituted, the activity from one batch to another rangedfrom 43.7 to 70.3. This was reflected in the SDS-PAGE scans where the CPK batcheswithhigher activity showed higher enzyme purity on the gel, with CPKranging from 83 to 98%of total protein. The CPK activity data has been represented as percentages ofthe controlassay used for each trial or condition toallow a clearer comparison ofthe data from differentCPK batches.A preliminary comparison of control CPK activity and CPK with 200u1(200ug) CANPpresent but no Ca2+allowed any possible effect of the inactive CANP on the activitymeasured. There was no effect of the inactive CANP on the CPK activity withcontrolactivity = 51.36 + 1.83 and with CPK plus inactive CANP = 52.67 + 2.48.Initially theeffect ofonly two different volumes ofCANP were measured.CPK activity was measured as, (1) = standard or “control” assayor, (2) = as control butwith 5mMCa2+added, and also (3) = as in (2), but with lOul CANP present in the assay,and (4) = as in (2) but with lOOul CANP present inthe assay. These two different volumesof CANP resulted in very different CPK activities, see figure 4. In the samples with 5mMCa2+present, the CPK activity was significantly reduced to 37.64% + 2.6% ofthe controlassay, (p<0.05). When 10 ul of CANP waspresent, the CPK activity was recovered to97.11 + 1.7% of the control value. The addition of lOOul of CAN? tothe assay caused32tz.(-)0I-.0C-)0”very different results, therewas a reduction of CPK activityto 2.08 + 2.56%of controlwhich was significantly differentto all three other conditions.See appendix C (5).140120100806040200Figure 4: Comparisonof two diferent amountsof CANP on CPK activity.1 Controlassay, 2 = Controllus 5mM Ca‘ (*= significantly differentfrom 1,3 & 4). 3 = As in2 plus lOul CANP(= Not significantlydifferent from 1). 4 = As in 2 pluslOOul CANP(# = significantlydifferent from 1,2 & 3).These results suggestedthat different amountsof CANP will have different effectson CPKactivity.The results ofincubatingCPK with a range ofincreasing CANP amounts is shown infigure5. In this case afurther “control” was addedfor each condition. In addition to the“calciumcontrol”, each CANP conditionhad a duplicate containingan equal volume of the buffer that1 2 3 433the CANP is stored in. This was done to measure any effects of the EGTAin the storagebuffer on the inhibition of CPK by Ca2•The approximately 36% inhibition of CPKactivity by the addition of 5mMCa2+is consistent and this inhibition was not significantlydifferent across conditions, the amount ofEGTA added in comparison to theCa2+ present isso small that there is no significant removal of the Ca2+ inhibition of the CPK, Seeappendix C (3). This inhibitory effect is overcome by the initial stimulation of CPK activitywith 27u1 CANP and then CPK activity is lost with increasing amounts ofCANP. See figure5.1401201o08004-.0:‘0Fig 5: CPK activity with increasing amounts ofCANP.1 = Control assay with CANP buffer.2 = As in 1 plus 5mMCa2+also CANP storagebuffer. 3 = As in 2 plus CANP. A = 27ul ofCANP buffer or CANP. B = 54u1 ofCANPbuffer or CANP. C = 67u1 of CANP buffer or CANP. D =84u1 of CANP buffer orCANP. CANP buffer is the buffer that the CANP is reconstitutedin.*= Significantly different from control activity (p<0.05).= Significantly different from controlactivity (p<0.05).Each of the conditions from A toD has increasing amounts of CANP in sample number 3.With the presence of27u1 ofCANP, (A3),CPK activity is increased slightly but significantly123 123 123 123A B C D34to 123.18 + 12.9% ofthe control value, (p<0.05). As the amountof CANP present in theassay is increased to 54u1(B:3),then the CPK activity is recovered to 56.96 + 0.31% ofcontrol. With 67u1 of CANP (C3)in the assay the CPK activity was again higher than the“calcium control” but is now only 50.46 + 2.65% of the control activity. When 84ulofCANP (D3)was present then the CPK activity was 33.06 + 0.5% ofthe control whichis thesame as with the 5mM Ca2+control.When these results are combined with those from Figure 4, we can see the inhibitionof CPKactivity with5mMCa2+is removed by the initial stimulation of CPK activity and then CPKactivity is lost with increasing amounts of CANP. See figure 6.140120100-0- 5rnMCa2+ &CANP80-Li-5mMCa2+600Q0CANP ugFig 6: CPK activity as a percentage of controlwith increasing amounts of CANP. # =significantly different from control (p<0.05).*sigiifcantly different from control(p<0.05), but notsignificantly different from each otherCaLrntrial.0 20 40 60 80 10035Objective 3: CPK proteolysis by CANPinvitroCPK proteolysis and CANPSDS-PAGE of CPK with active CANP shows that CPK is proteolyzed by CANP.Proteolysis of CPK is shown in figure 7 on a 5-15% gelwhich has been overloaded withCPK, (1412 ug per lane). CPK proteolysis increases with increasing amounts of CANP.When lanes 1, 2 and 4 are compared with the CPK activity in figure 4, then the degree ofproteolysis reflects the resulting CPK activity. The resultsof the densitometric scansshowing significantproteolysis ofthe CPK, (p<0.05) are shown in appendix D (1).SM1234abCdefCPKF1F2Figure: 7. A 5-15% SDS-PAGE with 1416ug of CPK per lane. Lane 1 = Control CPK, 2= CPK + lOul CANP, 3 = CPK + 27u1 CANP, 4 = CPK + lOOul CANP. a = 200kDa,b = 97kDa, c = 66kDa, d = 45kDa, e = 3lkDa. Position ofCPK band is indicated by thesolid arrow. F1 = fragment 1, F2 = fragment 2.36When l2Oug ofCPK was loadedin each lane and increasingamounts ofCAMP werepresent,the degree ofCPKproteolysis wasdependent on the amountofCANP present, see figure 8.S 1 2 3 45 6..ab —C—dI—CPKE—F1•—e- 28kDaCANPFigure 8: Gel showing proteolysis ofl2Oug CPK with increasing amountsof CAMP. Lane 1= Control CPK, 2 = CPK +lOul CAMP, 3 = CPK+ 15u1 CAMP, 4 = CPK + 20u1CAMP, 5 = CPK + 25u1CAMP, and 6 = CPK+ 30u1 CAMP. S Molecular weightstandards. a = 200kDa, b= 97kDa, c = 66kDa,d = 45kDa, e = 3lkDa. The CPKandsmall CAMP subunit bands arelabelled. F1 = fragment1, F2 = fragment 237The data from densitometricscans of each lane from five SDS-PAGEtrials are plotted infigure 9(A). Data was reportedfor each protein band as a percentageof total protein on thelane being scanned, See appendixD (1).The increased CPK proteolysisis shown in figure 8. The correspondingdata from this gel isshown in figure 9(B). In thecontrol lane the CPK represents 83.2%of the total proteinloaded in that lane. Lane 1has lOul of CANP added and this constitutes2.79% of the totalprotein, the small 28kDa subunitof CANP is visible on the gel. Lane 2has 15u1 of CANPwhich was 4.93% of total protein,the resulting decrease of CPK to 61.23%total protein forCPK shows the effect of the increasingCANP. The loss of the CPK bandcontinues withincreasing CANP, 20u1 CANPleft 59.95% as CPK, 25u1 CANP left43.79%, and 30u1CANP left 36.38%. These resultsdemonstrate the dose dependency ofthe CPK proteolysison the amount ofCANP actuallypresent.Fragment analysisThe proteolysis of the CPK, asshown by the loss of the CPKband in figures 7 & 8, resultsin the formation of two mainfragment products. Thesefragments are a few kDa smallerthan the original CPK.The significant formation ofthesefragments with increasing amountsofCANP is shown infigure 10.381009590850c 8007065605550Figure 9(A): CPK proteolysis with CANP. Data= significantly different from control (p<0.05).is the mean for five SDS-PAGE trials.*Figure 9(B): Data from densitomethc scans of 10% SDS-PAGE from figure 8. The % totalprotein of CPK and the 28 kDa CANP subunit are shown. Each lane was loaded with l2OugCPK.*•- CPK0 5 10 15 2025CANT’ ug0C-)80706050403020100-0- 28kDaCANT’ subunit-0- CPK10C8 ‘.64200 510 1520 2530CANPug39Figure 10: Data from densitometric scansof 10% SDS-PAGE with l20ug CPKper laneincubated with increasing volumes ofCANP.An approximation of the molecular weightsof these proteolytic productswas made bycreating a linear regression equation using thelog Molecular weight of the knownstandardsplotted against their relative mobilities from the gel.30252015105aa.aG)10800.64000-A-2SkDa CANPsubunit—- smaller fragment(F2)-0- largerfragment (Fl)0 5 10 15 20 25 30CANP ug40Chapter 5DISCUSSIONFrom the results ofthis study, two ofthe three proposed hypotheses can be accepted.The increase in CPK activity and then its loss as the ratio ofCANP to CPK is increasedconfirms the first hypothesis. That is: “CANP will change CPK activity in vitro”.The data from the SDS-PAGE ofCANP and CPK showing significant yet restrictedproteolysis ofCPK by CANP also leads to the confirmation ofthe third hypothesis, which is“CANP will cause proteolysis ofCPK in vitro”.The inhibition ofCPK with 5mMCa2+was consistent across trials and 10 to 20% CPKinhibition with 4mM ATP and Ca2+has been reported before (Walliman & Eppenberger,1985). FreeCa2+resulting from the 5mMCa2+added to the control assay will likely bereduced upon the addition ofthe CANP buffer which contains 0.1mM EGTA. Thereforeitmay have been expected that the addition ofincreasing volumes of CANPstorage buffer mayhavejust increased the chelationofCa2+and would have caused removal ofthe inhibitionon the CPK activity. The fact that the effecton CPK activity of the Ca2+conditions withincreasing volumes ofCANP buffer were not significantly different from each otherdemonstrates that this effect did not occur. Any possible recovery of CPK activity withremoval ofCa2+by the presence ofthe CANP alone is unlikely because ifthis was the caseit is unlikely that the CPK activity could have been increased to a level greater than thecontrol value. Indeed any removal of this inhibitory effect would have caused recovery ofCPK activity with increasing amounts ofCANP and not the opposite, as observed.This is the first demonstration that MM-CPK is a substrate for CANP in vitro. The activityofCPK was increased above control values when 27ug ofprotease was incubatedwith1412ug of substrate, this is a protease to substrate ratio of 1:185. Other examples in theliterature demonstrating CANP mediated proteolysis ofenzyme substrates have usedproteaseto substrate ratios ranging from 1:500 with tyrosinehydroxylase, (Kirsch, Kirsch, Titani,Fugita, Suzuki & Nayatsu, 1991),; 1:3, 1:16.67 or 1:33.33 with PKC, (Kishimoto etal,41l989),and 1:25 with calcineurin, (Wang et al, 1989a). The CANP content of skeletal muscleis approximately 2mg/g wet weight and the CPK content is around 5mg/g wet weight,(Walliman & Eppenberger, 1985). This gives a ratio of 1:2.5 which is obviously a grossestimate and has many confounding factors, including the various different locations ofbothCANP and CPK. Conclusions and assumptions made regarding the ratio ofprotease tosubstrate at this time is at best a rough indicator ofthe actual intracellular ratios.The modification ofCPK activity can be loosely correlated to the changes in CPK structurevisualized on the SDS-PAGE. The SDS-PAGE gel in figure 7 shows the effect of lOulCANP. There is definite proteolysis ofthe CPK at this ratio ofprotease to substrate and inthe activity assay this is reflected as a recovery ofthe CPK activity back to control values.The 27ug of CANP shows increased proteolysis ofthe CPK and activity is increased abovethe control level. In these cases the ratio ofprotease to substrate on the gel is 1:148 and inthe assay is 1:185. Similar activation ofsubstrate enzymes after proteolysis by CANP hasbeen documented with, for example: tyrosine hydroxylase, (Kirsch et al, 1991),; PKC,(Kishimoto et al, 1989), and phosphorylase-b-kinase, (Meyer et al, 1964). The loss ofCPKactivity with lOOug ofCANP correlates to the almost complete loss ofthe original 4lkDaCPK band on the SDS-PAGE.The fact that two large fragments are formed rather than an absolute degradation oftheenzyme suggests some kind ofregulatory role ofCANP with CPK and the other enzymesubstrates that are not degraded but are slightly modified, such as PKC, TN, and calcineurinas mentioned previously. The sites of CANP cleavagein these enzymes have been shown tobe near to the PEST regions on their amino acid sequences. The fact that CPK contains twoweak PEST sequences of-0.21 and -0.4 adds to the evidence that these sequences are likelyto be involved in some kind of substrate recognition process before CANP proteolysisoccurs. The PEST sequences on CPK were found to be at the amino end ofthe protein andifthe CANP cleavages were to occur close to these sequences, then the approximatemolecular weight ofthe fragments produced would be 39 and 35 kDa. The approximate42molecular weights ofthe fragments as determined under the conditions in this study were 38and 35kDa. Production oflarge fragments after cleavage of substrates close to their PESTsequences has been shown previously, (Chin et al, 1984 also Liscum et al, 1985). PESTlocalized CANP cleavage was shown by Liscum et al with the sites ofCANP proteolysis onHMG-CoA reductase being 5 and 2kDa from the two PEST sequences on the enzyme. Itseems that on CPK, like the other enzyme substrates, there are sites that CANP canrecognize and proteolysis then occurs at or near these PEST sequences, (Wang et al, 1989b).Proteolysis ofCPK has been shown before with proteinase K, in this case the fragmentproduced was 37kDa this proteolysis resulted in a loss ofCPK activity.The inactivation wasthought to be due to a small change in the tertiary structure ofthe enzymewhich preventedthe formation ofa transition state analogue, (Price, Murray, & Mimer-White,1981). CANPis known to activate some ofits kinase substrates, just how the proteolysis ofthese enzymesby CANP results in their activation is unclear.An explanation ofthe bi-phasic response ofCPK activity to increasing CANPthat isdemonstrated in this work could be explained in terms ofquaternary structure changes withspecific CPK cleavage. The quaternary structure ofCPK is as yet unknown.Research todate has shown that there are certain amino acid residues that are close to Cys-278 attheactive site. These residues are; Cys-69, Lys-191 and Asp-335, (James, Wyss, Lutsenk,Walliman, & Carafoli, 1990). The close proximity ofthese residues makes it likely thattheCPK molecule is complex and folded. The presence ofCys-69 near the active site meansthat CPK’s two pest sequences could be quite close to the active site where thecatalyticcomplex is formed. An initial cleavage at or near the first and stronger pest sequence couldremove the first 30 or so amino acids. This could act to remove Ca2+ions that could bindto the pest sequence and possibly interfere withthe formation of the active complex. Withincreasing amounts of CANP present then the second and weaker pest sequencewouldbecome a target for CANP proteolysis. Cleavage at this site on the enzyme may act toremove essential residues involved in active site complex formation.These explanations are43at best speculative but do fitthe available data. The fact that the fragments formedwere thesize that cleavage at or near the PESTsequences would predict, makes it likely that somekind oflimited proteolysis is occurring. Then with increasedproteolysis ofthe CPK residuesessential for activity could be lost. How the activity is related toproteolysis in vivo could beaffected by a range ofconditions not found in the invitro experiments. These includebinding ofthe protein to the M-line or to plasmamembranes which may mask sites ofproteolysis etc.Studies ofCPK’s reactive cysteine side chain suggeststhat the environment ofthis side chain,which is believed to be close to the active site is somewhatmodified in theproteolyzedenzyme. It has been proposed on the basis ofstructureprediction methods that this sidechain is at the beginning ofa B turn separatingtwo portions ofa B sheet, (Maggio, Kenyon,Markham, & Reed, 1977). Ifthis is the case then this side chainmay play a role inmediating conformational changes associated with the formationof the catalytically activecomplex. In support ofthis is the fact that small modificationsofthis side chain led to onlysmall decreases in activity but larger modifications resultedin more significant activitylosses.The effect of CANP on this side chain could be to alloweasier formation ofthe catalyticcomplex. More severe proteolysis ofthe CPK may then act toprohibit the formation oftheactive complex. An example ofincreased CPKactivity has been shown during naturaldevelopment, myofibrillar CPK Vmax is increasedwith no change in the Km ofthe enzyme.It is suggested that the alteration ofCPK Vmaxoccurs via an interacting subunit domaineffect on the conformation ofthe enzyme which in turninfluences enzyme activity, (Dowell& Fu, 1992). However the detailed structureofthe enzyme is not known and therefore theexact role that this reactive cysteine side chain may playin inhibition or activation is unclear.Comparison ofthe MB-CPK andMM-CPK isozymes suggested that the succeptability ofthedifferent CPK isozymes to proteolysis was not thesame but that the MM (muscle) isozymewas a more compact, less flexible proteinthan the brain (BB) isozyme which is thought to be44a “looser” protein, (Grossman,Akinade, & Garcia-Rubio, 1990). The conformationalflexibility ofthe CPK may be linked to its catalyticcooperative & regulation. Therefore itwould be interesting to compare the CANP to CPK ratio requiredfor activation and/orinactivation for both BB & MM CPK isozymes.The degradation rate ofa protein might depend on itsintracellular location and its associationwith intracellular structures such as membranesor myofilaments. The localization ofCPKwithin the cell may increase its chances ofproteolysis byCANP. CPK is commonly locatedat cellular membranes close to sitesofenergy consumption. These include theCa2+ATP’ase on the sarcoplasmic reticulum and at theplasma membrane Na/K ATP’asedependent pump, (Levitsky et al, 1977 and Sharov etal, 1977). Considering the supportgiven to the membrane activation hypothesis ofCANP, it is likely that this position on themembrane could locate CPK close to the membranelipids that can reduce the Ca2+requiredfor CANP activation. CPK has alsobeen shown to be localized with the enzymes ofglycolysis at the I band in rat skeletal muscle, (Walliman& Eppenberger, 1985).Immunogold labelling and EM also showed uCANP tobe located predominantly at the Iband, (Kumamoto et al, 1992). The location ofaprotein can also confer added resistance toproteolytic agents in the cell, for example;neuron specific enolase and CPK both associatewith structural proteins during axonal transport andare remarkably stable, (Rechsteiner et al,1987). MM CPK is localized withinthe M line of skeletal muscle and has both an enzymaticand structural role. These different locationsofCPK pools within the cell may help todetermine their succeptability to CANP proteolysis.Therefore the susceptibility ofthedifferent CPK isozymes to CANPproteolysis within a cell could vary due to their locationand conformation ofthe actualisozymes.CANP may act on CPK at its specific intracellularlocations within the cell depending on thefactors previously mentioned, forexample; protein to protein; or protein to membraneinteractions; and local environmental factors such ashydrophobicity, phosphorylation ofCPK45etc. CPK is subject to phosphorylation anddephosphorylation, (Hemmer, Skarli, Perriard &Walliman, 1993).Conclusions regarding the role ofCANP proteolysis ofits in vitro substrates must thereforeconsider the many possible regulatory factors involved such as: substrate availability;intracellular location of substrate; the substrates local environment; endogenous inhibitors ofthe protease eg: calpain to calpastatin ratio. The physiological role ofthe CANP/calpastatinsystem is not clear despite various suggestions for its role in many different cell types.Whether CANP proteolysis ofCPK plays a physiological role within the muscle cell remainsto be determined. A protease that can cause limited but specific proteolysis ofCPK would bewell suited to influenceprocesses such as energy transmission, ion transport,Ca2+transport, and protein synthesis in the muscle cell. As it stands, the conclusions from thisstudy should provide a basis from which to investigate the CANP/calpastatin systemand itseffects on CPK during periods ofacute and chronic muscle dysfunction.46Chapter 6CONCLUSIONSThe muscle isoform ofCPK is a substrate for CANP in vitro.Proteolysis ofthe CPK resultsin a stimulation ofCPK activity with lower amounts ofCANP andactivity ofCPK is lost asthe ratio ofprotease to CPK is increased.CANP induced activation ofCPK can overcome the inhibitory effect ofCa2•CANP proteolysis ofCPK results in the production oftwo large fragments.The size ofthesefragments suggest that the CPK is cleaved at or near one ofthe two “weak”PEST regionsnear the N-terminal end ofits primary structure.Suggested further researchThe effect ofCANP on muscle myofibrillsshown to rely on CPK for the re-synthesis ofATPat the M-line would be a suitable place to start workon a more physiological model.The succeptability ofthe different isozymesofCPK to CANP proteolysis would giveinformation on the specificity ofCANP towards CPK.Development of specific antibodies towards the fragments produced by CANPproteolysis ofCPK would allow these fragments to be visualized in muscletissue. 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The JournalofBiological Chemistry, 253, 5888-5891.Zalewska, T., Thompson,V.F., & Goll, D.E. (1991).Effect ofphosphatidylinositolandinside out erythrocytevesicleson autolysis ofu and m-calpain. Submitted.Zhang, Z., Lawrence, J., &Stracher, A. (1988). Phosphorylationofplatelet actin bindingprotein protects againstproteolysisby calcium dependent suithydrylprotease. Biochemicaland Biophysical ResearchCommunications,j5.j, 355-360.55Appendix A:Lowry assay procedure: Determinationwith Folin-Ciocalteau reagent.Stockreagents:A. 0.5% w/v copper sulphate(CuSO4.5H20)B. 1 % w/v sodium potassium tartrateC. 0.1M sodium hydroxide &2% w/v sodium carbonate, pH 12.5.D. Folin-Ciocalteau Reagent.E. 0.3M potassium hydroxide.F. 5mg/ml Bovine serum albumin(BSA), in distilled water.Method:1. Preperation ofstandards.Protein Stock (ml) Buffer (ml)Concentration (Final)0 0.500.1 0.41 mg/ml0.2 0.320.3 0.230.4 0.140.5 052. Dilute 0.1 ml from all standards andsamples with 0.2 ml ofreagent E, mix andletstand at room temperature for 10 minutes.3. To 48m1 ofreagent Cadd imi ofreagents A and B. This copperalkaline solutionwas prepared fresh when required.4. Dilute reagent D (F-C reagentlv:lv with distilled water).5. Take 0.imi ofall standards and samples and add 2.5 ml ofcopperalkaline solution(from step 3), mix and let stand atroom temperature for 10 minutes.6. Add 0.25m1 ofdilutedF-C reagent (from step 4) to eachtube while mixing with thevortex. Let stand for 30 minutes.7. Measure absorbances at 750nmagainst the 0 (blank tube) prepared asthe standardsand samples.8. Prepare a standard curve of0D750vs protein concentration (mg/ml) todetermineunknown concentrations.56Appendix B(1):Time course ofCPK activity showing the creatineproduced by CPK over increasingdurations ofincubation under the control assay conditions, the fitted curve has R =0.987. The smaller inset graph shows the initial linear section ofthe time course andthe arrow at 90 seconds indicates that using this time as the end point will ensure thatthe measurements ofCPK activity are on the linear portion ofthe curve.Lii40-86-20 - —r ‘ —0 30 60 90 120 150Tiaic (ccon)..L._.L._..1.__.I____J___j______LI I I I I1cP0 600 1200 1800 2400 3000Time (seconds)57Appendix B(2):Data from two independent phosphocreatine curves are shown, the small degree ofPCR hydrolysis allowed in 90 seconds resulted in low values for activity and highdegrees ofvariation. Therefore I decided it would not be valid to make anycomparisons from this data.10 50 100150Volume PCR (ul)200 25098765432C)L)—0- Assay#l-. Assay#258Appendix C (1):Dfor CPK activity (Sigmaj,ts), each trial having acontrol assay; assay plus 5mMCa+;and assay plus 5mM Ca plus CANP.MeanStdDevMeanStdDevControl43.743.943.0843.560.43Control46.28243.15149.16546.203.01Control70.30368.59569.44969.450.85Ca2+15.6116.15517.42516.40.93Ca2+19.05913.61416.34716.342.72Ca2+22.72825.25122.48923.491.53lOug CANP42.341.742.342.142.103527ug CANP51.08662.72356.92156.915.8254ug CANP39.34339.7739.5739.560.21lOOug CMP2.8-1.0620.9761.620.91.93CPK activityControl Ca2+ 84ugCANP54.929 20.6620.6653.015 17.56417.564Mean 53.0119.11 19.11Std Dev 1.921.55 1.55MeanStdDevMeanStdDevControl57.91961.2250.55356.565.46Ca2+18.31217.45823.86319.883.4867ug CANP28.66726.95929.94828.521.50N -t.p6 C oDJI60Appendix C (3):Repeted measures ANOVA ofcontrol CPK activity and CPKactivity with 5mMCatmadded. Again the datais normalized and represented as apercentage ofcontrol.GroupControl (100%)lOCa2+27 Ca2+54 Ca2+67 Ca2+84 Ca2÷100 Ca2+Count3333333Mean10037.6435.3733.8235.1636.0537.64StdDev02.145.892.26.152.922.14StdError01. df Sum ofSquares Mean Square F-test P valueBetween Subjects 13.09 6.55 0.01 0.9891Within Subjects 10760.47 597.8Treatment 10583.4 1763.9 119.54 0.0001Residual 177.07 14.76Total 10773.57Comparison MeanDiff ScheffeF-testControl v 10 Ca2+ 62.3665.88*Control v 27 Ca2+ 64.6370.77*Control v 54Ca2+ 66.1874.2*Control v 67 Ca2+ 64.84 7122*Control v 84 Ca2+ 63.9569.28*Control v 100 Ca2+ 62.3665.88*10 Ca2+ v 27 Ca2-i- 2.27 0.0910 Ca2+ v54 Ca2+ 3.82 0.2510 Ca2+ v 67 Ca2+ 2.48 0.110 Ca2+ v 84 Ca2-i- 1.59 0.04lOCa2+vlOOCa2+ 0 027 Ca2+ v 54 Ca2+ 1.55 0.0427 Ca2-f- v 67 Ca2+ 0.21 0.0727 Ca2+ v 84 Ca2+ -0.68 0.01 —27Ca2+ v 100 Ca2+ -2.27 0.0954 Ca2+ v 67 Ca2+ -1.34 0.0354 Ca2+ v 84 Ca2+ -2.23 0.0854 Ca2+ v 100 Ca2÷ -3.82 0.2567 Ca2+ v 84 Ca2+ -0.89 0.0167 Ca2+ v 100 Ca2+ -2.48 0.184 Ca2+ v 100 Ca2+ -1.59 0.04=significant_at95%61Appendix C (4):Data for CPK activityassays with 5mMCa2+and CANP present. Data isnormalizedwith the activity for eachCANP conditionrepresented as a percentage ofthecontrolactivity.Scource df Sum ofSquares MeanSquare F-test P valueBetween Subjects 28.95 4.48 0.00248 0.9975Within subjects 1832458.2 1803.23 0.0001Treatment 632092.07 5348.68 175.31Residual 12366.13 30.51Total 2032467.15Group CountMean StdDev StdErrorControl (100%)3 100 0 01OugCANP3 97.11 1.38 0.827ugCANP3 123.18 12.59 7.2754ugCANP 3 56.960.31 0.1867ugCANP 3 50.462.65 1.5384ug CANP 3 36.060.5 0.29lOOug CANP 3 2.084.44 2.56ComparisonMeanDiff ScheffeF-testControl v lOug CANP2.89 0.07Control v 27ug CANP-23.184•4*Controlv54ugCANP 43.04 15.18*Control v 67ug CANP49.54 20.11*Control v 84ug CANP63.9433•5*Control v lOOugCANP97.9278.57*lOug CANPv 27ugCANP26.085•57*1OugCANPv54ugCANP40.141321*lOng CANI’v 67ugCANP 46.65 17.83*lOug CANPv 84ug CANP61.0530.54*lOug CANPv lOOugCANP95.0374*27ug CANPv 54ugCANP66.223593*27ug CANPv 67ug CANT’72.724333*27ug CANPv 84ug CANT’87.1362.2*27ugCANPv100ugCANP 121.11 120.18*54ug CANPv 67ug CANP6.5 0.3554ug CANPv 84ug CANP20.913.58*54ug CANP v lOOng CANP54.8924.68*67ugCANPv 84ugCANP 14.41.767ugCANPv100ugCANP 48.38 19.17*84ugCANP v lOOugCANP33.989.46**_signjfjct_at95%62Appendix C (5):Repeated meaures ANOVA ofCPK activity (sho’ninfigure 2). Control assay; assaywith 5mM Ca + added;and assay with 5mM Ca added andeither 10 or lOOug ofCANP added. Activity expressed as Sigma units ofactivity.GroupControlCa2+1OugCANPlOOug CANPCount3333Mean43.5616.442.10.9StdDev0.430.930.351.93StdEffor0.250.540.21.12Source df SumofSquares Mean Square F-test P valueBetween subjects 2 1.98 0.99 0.0023 0.977Within subjects 9 3875.74 430.64Treatments 3 3867.91 1289.3 988.42 0.0001Residual 6 7.83 1.3Total 11 3877.72Comparison MeanDiff ScheffeF-testControl v Ca2+ 27.61282.83*Controlv lOug CANP 1.46 0.82‘ Controlv lOOug CANP 42.66697.44*Ca2+v bugCANP -25.7253.24*Ca2+ v lOOug CANP 15.4992*lOug CANP v lOOug CANP 41.2650.52**_sjgnjficjfl_at95%63Appendix D (1):Data from densitometric scans of 10% SDS-PAGE of CPK and increasing amounts ofCANP, as shown in figure 7. Values for each protein band are reported as percentoftotal protein ofeach lane. Fl = fragment 1, F2 = fragment 2.GEL#1 CPK F2 Fl28kDa CANPControl 83.151 1.8873.14301Oug CANP 62.16411.764 11.5062.793l5ug CANP 50.91716.238 14.9074.932Oug CANP 49.85216.929 14.502 4.2125ugCAN? 36.41322.563 18.7977.783Oug CANP 30.24927.518 19.053 8.805GEL#2Control 97.550.4940lOug CAN? 77.4545.178 10.738 2.177l5ug CAN? 70.6318.137 13.355 2.5922Oug CANP 65.049.445 15.463 2.90825ugCANP 62.0711.679 17.43 2.6463OugCANP 62.10512.189 17.46 2.425GEL#3Control 97.9750.504 0lOugCANP 88.6013.265 0.223l5ugCANP 84.8024.931 2.2232Oug CAN? 85.6974.345 0.8925ug CANP 84.8035.995 0.458GEL#4Control 94.8250.969 0lOug CAN?90.781 2.6540.282l5ug CANP87.9544.379 5392Oug CAN? 84.335.313 1.15925ugCANP 84.7524.204 0.6273Oug CAN? 83.123GEL#5Control 96.9110.4680lOugCAN? 87.8813.887 1.18l5ug CAN? 88.4533.923 0.4642Oug CANP84.251 4.4350.93725ug CANPGEL#6Control 95.2590.9 0lOug CAN?86.723 4.7540.173l5ugCAN? 82.8044.876 1.8622Oug CAN? 84.5154.568 0.625ug CAN?91.485 3.4790.41364Appendix D (2):Repeated measures ANOVA ofthe percent total protein ofthe small CANP subunitband. Control and increasing amounts ofCANP added to the gels. Data from gels 2-6were used.Source df SumofSquares Mean Square F-test PvalueBetween Subjects 20.15 0.07 0.150.8664Within Subjects 126.06 0.51Tiatment 44.43 1.11 5.45 0.0204Residual 81.63 0.2Total 146.21Group CountMean StdDev StdErrorControl 30 0 0lOugCANP3 0.23 0.05 0.03lSugCANP3 1.54 0.89 0.512Oug CANP3 0.88 0.28 0.162SugCANP0.5 0.11 0.07Comparison MeanDiff ScheffeF-testControl v lOug CANP-0.23 0.09Control v l5ugCANP-1.5444*Controlv 2Oug CANP-0.88 1.44Controlv 25ugCANP-0.5 0.46bugCANPv l5ugCANT’-1.32 3.21lOug CANP v 2Oug CANT’-0.66 0.8lOug CANT’ v 25ug CANT’-0.27 0.14lSug CANPv 2Oug CANT’0.66 0.8115ugCANPv25ugCANP1.05 2.012Oug CANPv 25ugCANP0.38 0.27I*=sjgp.jficantat95%65Appendix D (3):Repeated measures ANOVA ofthe percent total protein of fragment I produced withincreasing amounts of CANP.Sourcedf SumofSquares MeanSquareF-testP valueBetweenSubjects30.85 Subjects1243.683.64Treatment3 40.6513.5540.330.0001Residual93.020.34Total1544.53GroupCountMeanStdDevStdErrorControl40.710.260.13lOugCANP43.640.9 0.45l5ugCANP44.530.470.242Oug CANP44.670.440.22ComparisonMeanDiffScheffeF-testControlv lOugCANP-2.93 17.03*‘DontrolvlSugCANP-3.82 28.91*Controlv 2OugCANP-3.95 31.04*lOug CANPv l5ug CANP-0.891.561OugCANPv2OugCANP-1.032.09l5ug CANPv 2Oug CANP-1.40.4I*_sjgp.jficantat95%66Appendix E (1):Data from four independent trials ofcontrol CPK activity(Sigma units) from the samebatch ofCPK.Trial 1 Trial 2 Trial 3 Trial 4CPKactivity “‘I40.944 40.411 39.944 40.83841.709 38.279 36.712 38.4339.865 37.01 38.7339.42141.037 40.62 38.63141.65Mean 40.89 39.08 38.50 40.08StdDev 0.76 1.74 1.34 1.4467Appendix E (2):Repeated measures ANOVA ofthe fourindependent trials ofCPK activity(Sigmaunits) shown in appendix B (1).Source df Sum ofSquares Mean SquareF-test P valueBetween Subjects 3 13.443 4.4812.404 0.1183Within Subjects 12 22.364 1.864Treatment 3 12.115 4.0383.546 0.061Residual 9 10.249 1.139Total 15 35.807Group CountMean StdDev StdErrorTrial 1 4 40.5340.456 0.228Trial2 4 38.783 2.11.05Trial 3 438.756 1.254 0.627Trial 4 4 40.485 1.3060.653Comparison MeanDiff Scheffe F-testTrial 1 v Trial 2 1.7521.796Trial 1 v Trial 3 1.7781.85Trial 1 vTrial40.05 0.001Trial 2 v Trial 3 0.0263.96E-04Trial 2 v Trial4-1.702 1.696Trial3vTrial4 -1.728 1.748


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