Action of Triton X-100 on the biochemical and functional properties of hake (Merluccius hubbsi) myofibrils

Camp. Biochem. PhysioL Vol. 102B,No. 4, pp. 923-927, 1992 Printed in Great Britain

0305-0491/92$5.00+ 0.00 © 1992Pergamonpress Ltd

ACTION OF TRITON X-100 ON THE BIOCHEMICAL A N D FUNCTIONAL PROPERTIES OF HAKE (MERLUCCIUS HUBBS1) MYOFIBRILS SARA I. ROURA,*'~ADRIANAL. GOLDEMBERG,~RA(.JLE. TRUCCO:~and MARCOSCRUPK1Nt i'Instituto Nacional de Tecnologia Industrial, Centro de Investigacionesde Tecnologia Pesquera, Marcelo T. de Alvear 1168, 7600---Mar del Plata, Argentina; and ~;Dpto de Biologia, Fac. de Ciencias Exactas y Naturales, Funes 3250, 7600---Mar del Plata, Argentina. Tel.: (023)80-2801 (Received 25 November 1991)

Abstraet--l. The effects of Triton X-100 on the kinetic of Mg2+-ATPase and the functional properties of hake myofibrils were investigated. 2. Triton X-100 increased the Hill coefficientto 1.5 in an allosteric type of reaction for the myofibrillar Mg2+-ATpase. 3. Triton X-100 reduced the viscosity and the ATP response of myosin B and changed the contractile response of myofibrils. 4. These changes would be due to the action of Triton X-100 on actin-myosin interacting sites.

INTRODUCTION Non-ionic detergents, such as Triton X-100, are frequently used to dissociate protein from membranes (Wang and Smith, 1974; Dulley and Grieve, 1975). Triton X-100 can effectively remove mitochondrial, sarcolemmal and sarcoplasmatic reticulum membranes from cardiac myofibrils (Solaro et al., 1971). On the basis of an 88% inhibition of myofibrillar ATPase observed in the presence of EGTA, it was reported that the contractile proteins were not inactivated by Triton X-100 (Solaro et al., 1971). Electron microscope observations demonstrated that washing twice with a 1% Triton X-100 solution completely eliminates contaminating sarcoplasmic reticulum membranes without any apparent damage to the myofibrillar apparatus (Gall et al., 1975). It was also reported that changes in the ATPase activity of myofibrils during storage can be successfully studied by purifying skeletal muscle myofibrils with Triton X-100 CYasui et al., 1975). However, in current experiments carried out in our laboratory, actomyosin purified from hake myofibrils treated with Triton X-100 presented non-characteristic physicochemical and biochemical properties. In addition, ATP-sensitivity of 68% in myosin B from sardine myofibrils treated with Triton X-100 was reported (Seki and Narita, 1980). Also, recent evidence suggests that the detergent induces a conformational change of myosin that modifies its function (Highsmith, 1989). These results would indicate that Triton X-100 affects the contractile proteins in hake myofibrils, suggesting that other effects of the detergent on myofibrils should not be excluded. To investigate the effects of Triton X-100, the kinetic properties of the myofibrillar Mg2+-ATPase and the contractile response in hake myofibrils with Triton X-100 treatment are studied. *Author to whom correspondence should be addressed.

The alterations found in these parameters would be directly related to the treatment of hake myofibrils with Triton X-100. MATERIALSAND METHODS Fish source Hake (Merluccius hubbsi) were caught by commercial vessels in the South-West Atlantic Ocean, from 36 to 53°S, from October through February. Fish were kept on ice until they reached the laboratory in an early post-rigor condition. Female specimensof 35-45 era length were selected, and the length from the snout to the tip of the mid-caudal ray was measured to the nearest millimeter. The gonadal condition of hakes was determined using histological techniques described by Goldemberg et al. (1987). Protein preparation Fish skeletal muscle actomyosin was prepared as previously described (Crupkin et al., 1982). Myofibrils were prepared as described by Yasui et al. (1975) according to the scheme reported in Fig. 1 (unless otherwise specified when ion calcium was added to the standard buffer). This solution was referred to as Ca-standard buffer. All steps were carried out at 4°C. Myosin B (actomyosin isolated from myofibrils) was prepared as described by Seki and Narita (1980). Protein determination Protein concentration was determinated on aliquots of the extracts by the Lowry methods (Lowry et al., 1951). Myofibrillar ATPase activity The Mg2+-ATPase activity of myofibrilswas measured in a solution containing 0.75 mM ATP, 30 mM Tris-maleate buffer (pH 7.0) (in some experimentsTris-maleate buffer pH 8.0 was used), 0.25 mg]ml-l of protein, and 2 mM MgClv For the inhibition of myofibrillar MgZ+-ATPase in the absence of Ca2+ ions, 0.5 mM EGTA was used. The mixture was incubated at 25°C for 3 rain. The reaction was stopped by addition of 0.5 ml 40% trichloroacetic acid, and the mixture was centrifuged. The inorganic phosphate liberated was determined in the supernatant fluid by the method of

923

924

SARAI. ROURAet al. Table 1. Mg2+-ATPase activity of myofibrils with and without Triton X-100 Mg 2+-ATPase #moles Pi/mg protein/min

FILLET ÷20raM TRIS -MALEATE STANDARD BUFFER, OAmM KCt pH 7.0 HOMOGENIZED t5 MIN AT 3000 x G

I SUPERNATART ~SCARDED

I PELLET r

I SUPERNATANT DISCARDED

RESU~IPENDEDIN STANDARD BUFFER AND CENTRIFUGED ISMIN AT 3000 x G

I

CONTROL

MYOFIBRILS REBUSPENOED IN STANDARD BUFFER WITH I%TRITON X - t 0 0 , CENTRIFUGED 45 MIN AT 3000 x G

I SUPERNATANT DISCARDED

pElLET TRITON X°'IOO WAS REMOVED WITH THREE WASHED WITH THE STANDARD BUFFER. CENTRIFUGED ~5 MIN AT 3000x

G

TRITON X-400 TREATED MYOFIBRILS

Fig. 1. Purification of hake myofibrils with and without Triton X-100. Chen et al. (1956). The specific activity was expressed as /~mol PJmg protein/min. For kinetic studies, the following substrate concentrations were used: 0.4, 0.5, 0.6, 0.75, 0.9 and 1.0 mM ATP. The value of the Hill coefficientnu was calculated as previously described (Farias et al., 1970). Reduced viscosity Myosin B viscosity was measured at 20 + 0.01°C using a Ubbelohde viscometer, as described previously (Crupkin et al., 1979). A TP response The adenosine triphosphate (ATP) response of the myosin B fraction was determined as described previously (Crupkin, 1982). Contraction response The contraction of myofibrils at room temperature was followed with a phase contrast microscope NU2 Carl Zeiss, Jena (magnification 500 x). The contraction buffer contained 50mM KC1, 5mM MgSO4, I mM ATP, 0.5mM DTT, 20 mM imidazole, and either 1 mM EGTA or 0.1 mM CaC12, (pH 7.0) (Wagner, 1986). The same area was photographed (Panatomic-SO 32 Kodak) before and 1-3 min after the addition of the contraction buffer. Statistical analysis The statistical significance of the differences between mean values was determined by an analysis of the variance, and using the Duncan's New Multiple Range Test (Steel and Torrie, 1960).

Additions MgCI: (2mM) MgCI 2 (2 mM) + EGTA (0.5 mM)

Triton X-100 treated myofibrils

0.50_+ 0.15 a 0.23 + 0.06 b

0.90 + 0.30 c 0.30 + 0.06 d

Each value is the mean _+ SD of six determinations, a,b and c,d values are significantly different (P < 0.01) as calculated by an analysis of the variance and using the Duncan's New Multiple Range Test.

Mg2+-ATPase activity of both control and Triton X-100 treated myofibrils was reduced about 60% when the free Ca 2+ was reduced to less than 10 -8 M by the addition of EGTA (Table 1). The 60% inhibition of myofibrillar ATPase observed with EGTA is in agreement with results found by others in cardiac and skeletal myofibrillar ATPase (Solaro et al., 1971; Yasui et al., 1975; Seki and Narita, 1980; Fanburg et al., 1964). The activity of ATPase both in presence and absence of EGTA, was slightly higher in samples that had been treated with Triton X-100 than in control samples (Table 1). Similar results were reported in the Mg:+-ATPase activity of carp myosin B (Ando, 1983). Seki and Narita (1980), found low Ca 2÷ sensitivity and low intrinsic viscosity in myosin B from sardine myofibrils treated with Triton X-100. They attributed their results to changes in the protein during ice storage of fish (Seki and Narita, 1980). Hake actomyosin purified from muscle extracts showed the characteristic values of reduced viscosity and ATP response (Table 2). On the other hand myosin B obtained from control myofibrils and actomyosin isolated from hake muscle presented similar values of reduced viscosity and ATP response (Table 2). However, myosin B from myofibrils treated with Triton X-100 had lower reduced viscosity and lower ATP response than myosin B obtained from control myofibrils (Table 2). The decrease in these parameters would be the result of the action of Triton X-100 on the actomyosin complex in hake myofibrils. Considering that the MgZ+-ATPase activities are related to the actin/myosin interaction in the actomyosin complex, kinetic studies of the enzyme from myofibrils treated with Triton X-100 and from control myofibrils were performed. The experiments were Table 2. Reduced viscosity and ATP response of muscle actomyosin and of myosin B from myofibrils treated with Triton X-100 and control myofibrils Myosin B Biochemical properties Reduced viscosity

RESULTS AND DISCUSSION ATP response (%)

Kinetic study o f the myofibrillar Mg2 +-A TPase The activities of Mg2+-ATPase and Mg 2+(EGTA)-ATPase are related to the actin/myosin interaction in the actomyosin complex in presence and absence of endogenous calcium ions respectively.

Control myofibrils

Actomyosin muscle

Control myofibrils

Triton X-100 myofibrils

3.5 4.0 56.2 60.2

3.0 3.8 58.3 50.1

2.2 2.3 26.5 31.8

ATP response of actomyosin was determined measuring the reduced viscosity before and after addition of ATP and Mg 2+ up to I mM. Reduced viscosity and ATP response values of actomyosin purified from muscle extracts were previously reported (Crupkin, 1982).

Effect of Triton X-100 on hake myofibrils

925

Table3. Kineticconstantsat pH 7.0 and 8.0of the Mg2+-ATPasefromcontrolsand TritonX-100 treated myofibrils Mg2+-ATPase #moles Pi/mg protein/min pH 7.0 pH 8.0 Myofibrils Myofibrils Triton X-100 Triton X-100 Constants Control Treated Control Treated Km(mM) 0.46 4.26 1.55 12.00 Vm(#mol Pi/mgprotein/min) 0.06 0.56 0.44 2.80 n, values 1.00 1.50 n.d.* n.d. Standard assayconditionswereused.The concentrationof ATPw a s variedfrom0.0 to 1.0mM. Kmand Vmwerecalculatedfroma Lineweaver-Burkplot.Valuesgivenare meansof triplicate assays. *Not determined. done at pH 7.0 and 8.0, with increasing concentration of the substrate. Typical results are presented in Table 3. As can be seen, at both pH levels the values of Km and Vmincreased by a factor of nine, indicating that the formation and the breakdown of the enzyme-substrate complex were altered by the detergent. At pH 7.0 the Hill coefficient nH was 1.5 indicating a positive cooperative binding in the enzyme treated with Triton X-100, n n was only 1.0 in controls. These results support the idea that the number of ATP sites or their interaction is greater in enzymes from treated myofibrils than in the control ones. It is worth noting that structural changes on rabbit myosin due to Triton X-100 were also reported recently (Highsmith, 1990).

Effect o f the Triton X-100 on the contractile myofibrillar response The control of the actin-myosin interaction in vertebrate striated muscles by Ca 2+ depends on the presence of troponin-tropomyosin (Tn-Tm) (Huxley, 1972). At levels of Ca 2+ low enough to cause relaxation, Tm, by binding to actin, may sterically block the actin-myosin interaction. With the Ca 2÷ activation, Tm moves out of the groove into a "nonblocking" configuration (Huxley, 1983). The contraction of control myofibrils and myofibrils treated with Triton X-100 in contraction buffers containing either 0.1 mM CaC12 or 1 mM EGTA was observed. Since the differences in contraction speed between control and treated myofibrils could not be measured, only the contraction response positive to the ATP addition was recorded. Control myofibrils contracted in the presence of 1 mM EGTA when ATP was added. The contraction was almost fully completed 1 min after adding ATP to the side of the cover slip (Table 4, Fig. 2, panels A and B). A similar effect in reconstituted ghost with skeletal muscle myosin was reported (Mitsuka et al., 1979; Wagner, 1986); this loss of Ca 2÷ sensitivity

would indicate that some of the T n - T m complex is extracted during the preparation of myofibrils. Myofibrils treated with Triton X-100 did not contract under the same conditions (Table 4, Fig. 3, panels A and B); this could be due to the action of Triton X-100 on the actin-myosin interaction sites which were not protected by a regulatory T n - T m complex. The contraction response in the contraction buffer containing 0.1 mM CaC12 was positive in myofibrils treated with Triton X-100 and in control myofibrils (Table 4). In this way, contraction in the presence of high levels of Ca 2+ could be explained by the existence of unaffected actin sites which were protected by the T n - T m complex during the treatment of the myofibrils with Triton X-100. Control and Triton X-100 treated myofibrils were purified with a Ca-standard buffer to expose all the actin-myosin interaction sites to the action of Triton X-100, and the contractile response was observed in the contraction buffer with CaC1v In this activated state contraction was observed in control myofibrils. On the other hand myofibrils treated with Triton X-100 did not contract (Table 4). It is possible that the great exposure of the actin-myosin interaction sites produced by the Ca-standard buffer, would clear the access of the detergent which could affect the properties of these sites. The results of this paper prove that Triton X-100 affects the functional properties of hake myofibrils and the kinetic properties of the myofibrillar Mg 2+ATPase by acting on some sites of the actin-myosin interaction in hake myofibrils. Also, the use of the Mg2÷-ATPase inhibition in the presence of EGTA to determine the condition of myofibrillar proteins that have been treated with Triton X-100 would be unsuitable. Moreover, it would be particularly questionable to use Triton X-100 to purify fish skeletal myofibrils. Recent data demonstrate that the mechanism by which T n - T m inhibits the reaction of cross-bridges between actin and myosin may involve not only a

Table 4. Contractileresponseof control and TritonX-100treated myofibrils Contractionresponse Buffer standard Ca-standard buffer Contraction Control Triton X-IO0 Control Triton X - I O 0 buffer myofibrils myofibrils myofibrils myofibrils 1mM EGTA + + 0.1 mM CaC12 + + + -

926

SARAI. ROURA et al.

Fig. 2. Contraction response of control myofibrils. The myofibrils were photographed before the addition of a contraction buffer with ATP, and the same area was rephotographed 1-3 min after the addition of ATP. Panel A, control myofibrils before the addition of ATP; panel B, after the addition of ATP.

Fig. 3. Contraction response of Triton X-100-treated myofibrils. Analytical procedures were the same as described in Fig. 2. Panel A, treated myofibrils before the addition of ATP; panel B, after the addition of ATP.

Effect of Triton X-100 on hake myofibrils steric hindrance, but also the inhibition of an elementary step in the hydrolysis of A T P by actin and myosin (E1-Saleh and Potter, 1985). It may be possible that Triton X-100 alters some step in the abovementioned mechanisms. REFERENCES

Ando S. (1983) Removal of lipid from myosin B preparation by Triton X-100 treatment. Bull. Jap. Soc. Sci. Fish. 49, 927-932. Chen P. S., Toribara T. Y. and Warner H. (1956) Microdeterminations of phosphorus. Analyt. Chem. 28, 1756-1758. Crupkin M. (1982) Cambios en las proteinas miofibrilares de merluza (Merluccius hubbsi) durante el almacenamiento en frio. Doctoral Thesis, U.N.T., Argentina. Crupkin M., Barassi C. A., Martone C. B. and Trucco R. E. (1979) Effect of storing hake (Merluccius hubbsi) on ice on the viscosity of the extract of soluble muscle proteins. J. Sci. Food Agrie. 30, 911-913. Crupkin M., Barassi C. A., Arguello J. M. and Trueeo R. E. (1982) Effect of post-rigor fish storage on ice on physicochemical properties of actomyosin. J. Sci. Food Agric. 33, 1129-1134.

DuUey J. R. and Grieve P. A. (1975) A simple technique for eliminating interference by detergents in the Lowry method of protein determination. Analyt. Biochem. 64, 136-141. EI-Saleh S. C. and Potter J. D. (1985) Calcium-insensitive binding of heavy meromyosin to regulated actin at physiological ionic strength. J. biol. Chem. 260, 14775-14779. Fanburg B., Finkel R. M. and Martonosi A. (1964) The role of calcium in the mechanism of relaxation of cardiac muscle. J. biol. Chem. 239, 2298-2300. Farias R. N., Goldemberg A. L. and Trucco R. E. (1970) The effect of fat deprivation on the allosteric inhibition by fluoride on the (Mg2+)-ATPase and (Na + + K+)-ATPase from rat erythrecytes. Archs Biochem. Biophys. 139, 38-44.

CBPB 102/4--5

927

Highsmith S. (1989) Detergent modification of myosin function and structure in solution. Biochemistry 28, 6745-6750. Highsmith S. (1990) On the mechanism of detergent modification of myosin structure and function. J. Bioehem. 107, 554-558. Goldemberg L. A., Paron L. and Crupkin M. (1987)Acid phosphatase activity in pre- and post-spawning hake (Merluccius hubbsi). Comp. Biochem. Physiol. 89A, 845 -849. Goll D. E., Young R. B. and Stromer M. H. (1975) Separation of subcellular organeles by differential and density--gradient centrifugation. Proc. 27th Annual Reciprocal Meats Conf., National Livestock and Meat Board, Chicago, III. Huxley H. E. (1983) In Muscle and Nonmuscle Motility (Edited by Stracher A.), Vol. 1, pp. 1-104. Academic Press, New York. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Mitsuka M., Yamamda T. and Shimizu H. (1979) On the contraction of myosin-extracted skinned single fibers with active myosin fragments. J. Biochem. (Tokyo) 85, 559-565. Seki N. and Narita N. (1980) Changes in ATPase activities and other properties of carp myofibrillar proteins during ice-storage. Bull. dap. Soc. Sci. Fish. 46, 207-213. Solaro R. J., Pang D. C. and Briggs F. N. (1971) The purification of cardiac myofibrils with Triton X-100. Biochim. biophys. Aeta 245, 259-262. Steel R. D. G. and Torrie J. J. (1960) Principles and Procedure of Statistics. MacGraw-Hill, New York. Wang C. and Smith R. L. (1974) Lowry determination of protein in the presence of Triton X-100. Analyt, Biochem. 63, 414-417. Wagner P. D. (1986) Unphosphorylated calf thymus and aorta myosins contract ghost myofibrils. J. biol. Chem. 32, 14863-14866. Yasui T., Sumita T. and Tsunogae S. (1975) Stability of myofibrillar EDTA-ATPase in rabbit psoas fiber bundles. J. Agric. Food Chem. 6, 1163-1168.