The Biochemistry of the Muscle Contraction Process An Undergraduate Laboratory Experiment Using Viscosity to Follow the Progress of a Reaction J a m e s F. Belliveau, Gerard P. O'Leary, Jr., Harry Hajian' Providence College, Providence, RI 02918
is now commonly presented in undergraduate hiochemistry texthooks (. I . 2). A numher of undereraduate ex~eriments have heen developed to illustrate the contractile p;ocess, hut they seem to suffer either from a very sensitive technique yielding questionahle reproducihility in undergraduate hands or they require specialized equipment with time-consuming procedures. Detailed below is a simple, short experiment which uses common laboratory equipment and which has a high probability of yielding the desired results. This experiment illustrates the action of ATP (adenosine triphosphate) as the source of energy in the contractile process and the catalytic effect of Ca2+as a control in the energy producing process. Suggestions also are given to expand this experiment to an extended project. Theory In muu.lt,, lhr maill stnlc lur31 1 ~~nll,ontlll~nrt.tu,t~ prt>ttili~; ~ , I ~m I Id my
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of the control mechanism of contraction and relaxation in muscle (4). Two minor protein components, troponin and tropomyosin, are associated with the polymerized actin and this troponin-tropomyosin system exhibits an inhibition effect on the ATPase activity of muscle actin and myosin. However, calcium ions act on the troponin-tropomyosin moiety, overcoming the inhibition effect and catalyzing the ATPase activity. Thus, the release or withdrawal of calcium from the surrounding membranes allows the contraction-relaxation process to he controlled by external electrical signals from the nervous system ( 5 ) . The components of the muscle system can he solubilized in buffered solutions approximating physiological conditions. In the absence of ATP, actin and myosin form an extended polymerized network called actomyosin (6). The formation of this polymeric complex is accompanied by a rise in the viscosity of the solution. These actomyosin solutions exhibit similar ATPase activitv and similar calcium ion catalvsis as the actin-myosin moiety in muscle. Therefore, these solutions are suitable analorues for studvine " .and illustratine- the contractile mechanism biochemically. The overall energy producing reaction can he represented as:
TIME
(MIN)
Figure 1. Viscosity study on crude preparations of
rabbit muscle actomyosin. A: (a) addition of one drop 01 buffer showing dilution effect.(b) addition of one drop of ATP solution causing dispersal of actomyosin complex, and (c)addition of onedrop of Cazi solution causing only dilution effect. B: (a') addition of one drop af EDTA solution complexing the Ca2+. (b') addition of one drop of ATP Solution Showing only the dilution effect without the presence of CaZ+, and (c') addition of one drop of Ca2+ solution swamping the EDTA, thus causing the
dispersal of actomyosin complex. Solutions of actomvosin show a dramatic decrease in viscosity when a small amount of huffer solution containing ATP is added. This effect is much areater than the dilution effect of addition of an equal amount of buffer only (Fig. 1A). Because of the less structured nature of solution actomyosin compared to the orientation of actin and myosin in muscle, the ATPase activity in solution results in the dispersal of the comnlex into actin and mvosin monomers. The viscositv of the mixture drops to the sum of the viscosities of actin and myosin taken separately. The reaction of ATP at the attachment sites of actin and myosin is cyclic Once one ATP molecule has been hydrolyzed to ADP, another ATP molecule will displace this ADP and undergo hydrolysis. Hence, the actin and myosin will not reassociate in the presence of an ATP ~ o o lAs . the ATP pool is used up, the complex gradually reforms with the ac-
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actin,myasin, t ATP + HzO + Ca2+
rn actin
+ n myosin + ADP + HPO2-
'Rhode Island Junior College, Warwiek, RI 02886.
442
Journal of Chemical Education
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decrease in viscosity on the order of the dilution effect is seen upon addition of ATP. Addition of excess Ca2+ to this solution causes the dispersal of the actomyosin to take place as evidenced by the dramatic decrease in viscosity (Fig. 1B). Experimental Solutions: Prepare 100 ml of a KCl-Tris buffercontaining 0.5 M KC1 and 0.05 M ?'risma2base adjusted t o pH 7.8 with concentrated
Figure 3. Ca2+ Effect on the ATP-actomyosin viscosity response. (a) Addition of one drop of 8 mMATP solution, causing dispersal of the actomyosin campien. (bl addition of one drop of 5 m M EDTA solution partially removing the Cast. (c)addition of one drop of ATP solution showing abbreviated dispersal of actomyosin complex pmialiy using the ATP pool, (dl addition of one drop of 30 m M Ca2+ enabling remainder of ATP pool to be utilized.
Figure 2.Viscometric determination of myokinase in crude preparatian of rabbit muscle actomyosin. A: (a) addition of one drop of 8 mMATP solution, (bladdition of one drop of 0.7 mMAPsA solution. ( c )addition of one drop of ATP soiution. (d) addition of one drop of APsA solution. (e)addition of one drop of ATP solution, (f) addition of one drop of ATP solution. S (a') addition of one drop of 8 m M ADP solution. (b') addition of one drop of 0.7 mMAPsA solution. (c') addition of one drop of ADP solution. id') addition of one drop of AP6A solution, (e')additian of one drop of ADP solution, if') addition of one drop of ATP solution.
HCI. This buffer is used to prepare 10 ml each of the following three solutions: 20 m M EDTA (disodium), 30 m M CaClz and 8 m M ATP (sodium salt). ATP solutions should be made fresh daily. The buffer, EDTA and calcium solutions may be stored for a month a t 4°C. Use metal free distilled water from glass vessels because small amounts of certain metal ion impurities can denature the proteins. Prpparation oiSoluhilized Actomyosin: Glycerinated psom muscle From rabbits is eommerically available.3 Do not allow the muscle to come in contact with metal. Use glass or teflon-coated forceps. T h e glycerol is washed off these muscle bundles (weighing between 0.k0.6 g) by dipping them in ice cold KC1-Tris buffer solution. Quickly weigh the muscle bundle and put it into a tissue grinder? Five milliliters of cold KCl~Trisbuffer solution is added for every 0.5 g of muscle. Mince the muscle carefully avoiding excess foaming which indicates denaturation of the proteins. I t is important that the sample he kept cold during this procedure. After mincing, store this solution overnight in a refrigerator to allow the actin and myosin to dissolve in this KCI-Tris medium. Centrifuge this solution at approximately 100-500 kg for 15 min to pack the course detritus and carefully decant the supernatant which contains the actomyosin complex. (This centrifugation step can he critical. T h e preparation must be centrifuged a t enoughg force to pellet out the cell debris, yet t h e g force must not be high enough to pellet uut the actomyosin). A solution that has been suitahly centrifuged is slightly opaque, not clear. I t should be noted that all solutions (except the ATP solution) and the mincing of the rahhit muscle should be done on the day preceding the viscosity measurements. Viscosity Detwrninations: Viscosity runs were carried out a t 20°C using a l-ml Cannon-Manning semimero ~ i s e o m e t e r Data . ~ is represented as relative outflow time (outflow time for solution/outilow time for same volume of buffer) versus time. The viscometer is filled with a charge of 0.8 ml of the actomyosin preparation. T h e initial viscometer outflow time is measured a few times to check the stability of this preparation. I t should be a t least 50% higher than the outflow time of the KCI-Tris huffer, indicating that a suitahle amount of actomyosin has been soluhilized. The outflow times of different muscle preparations will vary greatly. This variance is a function of mincing and centrifueation technioues. (A few triais at these techniaues mav
side of the viscometer. T h e viscometer is shaken immediately for a few seconds before further outflow times are taken. Students should work in pairs. One student should investigate the ATP viscosity re^ sponse on one sample by buffer, A T P and Ca2+ additions (Fig. 1A). T h e other student should investigate the Ca2+ catalysis effect on another samole bv" EDTA. ATP. and CaZ+additions (Fie. . ,, 1B). Suitahle viacnsitv rims imuallv one ....~., ~ .~~ .~ ~ take ~ ~hetween ~ ~ , ~and -a half , to three hours depending on the length of time needed to use up the ATP pool. Student Reports: Students shuuld graph their results and submit a report which illustrates that they understand the hiochemical interpretation of the various viscosity effects. ~~
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Further Extension of the Experiment
I) The actamyosin preparations given above is only a solubiliaation
.~ . . systems. often, the myokinase is also active along with the actomyosin
nrocedure. and it is ouite oassihle to soluhilize associated chemical ~~~
system. This enzyme system ( 7 , s )can be illustrated as follows: myoliinare
2 ADP LATP
+ AMP
Thus, addition of ADP to actomyosin solutions with active myokinase systems will cause the same viscosity response due to the formation of ATP. I t can he shown that ADP does not directly react with actomyosin by poisoning the myokinase system with APsA idiadenosine pentaphosphate). In solutions where APsA has been added, only ATP will cause the viscosity response peculiar to the actomyosin dispersal (Figs. 2A & 2R). 11) Calcium is a catalyst in the actomyosin system, yet with these crude preparations, there seems to be an unknown chemistry which uses calcium as a reactant. On occasion, addition of EDTA has not totally removed the ATP-actomyosin viscosity response but has decreased the length of time that the actin and myosin stay dispersed (Fig. 3). The addition of more Ca2+ shows a second ATP-actomyosin effect which would seem to indicate that Ca2+ is somehow acting as a reactant and enabling the remainder of the ATP p o d to be utilized. No detailed explanation of this effect is available currently, and this problem can make the basis of a good undergraduate research project. Persons interested in performing ext.ended projects can gain additional experimental details by contacting one of the authors personally."
Summary T h e use of t h e s i m p l e O s t w a l d viscometer t o s t u d y t h e chemistry of t h e c o n t r a c t i o n process gives a n o t h e r o p t i o n i n 'Sigma Chemical Co., St. Louis, MO 63178. Tarolina Biological, Burlington, NC 27215. "Tissue grinder (homogenizer), Arthur H. Thomas, Inc. 114288-G. Cannon Instrument Co., P.O. Box 16, State College, PA 16801 (size 150, 7 to 35 centistokes).
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Number 5
May 1981
443
the physical chemistry laboratory. Most viscosity experiments are divided into two categories: (i) the determination of viscosity of liquids as a function of temperature to obtain various thermodynamic parameters of activation for viscous flow ( 9 ) and (ii) the determination of intrinsic viscosity of polymeric solutions to ohtain the molecular weights of polymers (10). Thus, this experiment expands this list by using viscosity as a physical measurement to follow the course of a chemical reaction. This experiment also lends itself to the physiology and biochemistry laboratories. Literature Cited Ill Lehningei. AiberlL.. "Biochemiitry."2nd
444
Ed.. Wirith Pablishera. l n c , IY77.p 749.
Journal of Chemical Education
121 white, A . st ul, "Principles uf ~ i i a h e m i i t r y , "6th ~ d . ~. c ~ r a w -BOO* ~ i ~CO.. i 1978, i'.10%.
(:I) H U X I B ~ .H E . , s ~ ~ 164. ~ ~ ,356 c ~ .(1969). 14) ~ i r r i e yH , E., ( d d ~ , w ~ ~,symp. i p qunnt ~ ~ ~ ~i i 0 ~ i ~. , ~~ 7 11!972). ,m I51 MecLennan, D. H., Holland, P. C.,AnnHru. Htunhys Rineng .4,277 (19751. 16) kent~Gyorgyi.A,, T h e m i s t r y o i Muscular Contraction? Academic Press. Inc.. 1947. p. 24.
171 Szasr,c.. ~ ~ r h a r dw., t , cruber, W.a n d ~ e r n tE.. . c l i n c h m . 2 2 , m x (19761. 181 l.lmhard,G.E.,andSecemski.I...J.Ri~il. Them ,248, 1121 (19731. 191 i k n i d s . F., et a ~ .", B x p e r i m c n ~ lphysical chemistry? 6th ~ d . M. C G I ~ W - H ~RUOL II r u m m n y , hc., 1 9 6 2 , ~ 147 . (101 Shoemaker, David P , et 81.. "Experiments in P h y r d Chemistry.‘. :3rd Ed.. M C G ~ ~ W - HI ~~ CI .I. I W ~ P. . :)YO.
%damesF. Belliveau, Department of Chemistry, Providence College, Providence, R102918.
