- Email: [email protected]
Changes in lipids and biochemical properties of actomyosin from pre- and post-spawned hake (Merluccius hubbsi Marini)
Comp. Biochem. Physiol, Vol. lI2B, No. 4, pp. 743-748, 1995 Copyright © 1995 Elsevier Science Inc. Printed in Great Britain. All rights reserved 0305-0491/95 $9.50 + 0.00
Pergamon 0305-0491(95)00131-X
Changes in lipids and biochemical properties of actomyosin from pre- and post-spawned hake (Merluccius hubbsi Marini) Juan Pablo Busalmen, Sara In6s Roura, Hugo Roldan* and Marcos Crupkin The Centro de Investigaciones de Tecnologia Pesquera (CITEP)(INTI), Marcelo T. de Alvear 1168, 7600 Mar del Plata, Argentina The total lipid contents of muscle, the liver somatic index and the total lipid contents of actomyosin in hakes changed with the gonadal condition of the fish. Non-polar lipids in actomyosin from pre-spawned hake were 145% higher than in actomyosin from post-spawned hake; polar lipids were 30% higher. The relative percentage of phospholipids changed from 32% in pre-spawned hake to 48% in post-spawned hake. The Mg2+-ATPase activity in actomyosin increased from the pre-spawned to the post-spawned conditions, and this could be related to a higher phospholipids to neutral lipids ratio in post-spawned hake. Gradual decreases in both Ca2+-ATPase activity and myosin: actin ratio during the gonadal development of fish were found, suggesting a partial loss of myosin functionality of the actomyosin complex. Key words: Muscle lipids; Actomyosin complex; Merluccius hubbsi; Gonadal development; Actomyosin ATPases; Myosin: actin ratio; Myosin functionality. Comp. Biochem. Physiol. lI2B, 743-748, 1995.
Introduction
Changes in the actomyosin composition of mature Argentinian hake (Merluccius hubbsi Marini) are influenced by the metabolic state of the fish and are related to the reproductive cycle (Crupkin et al., 1988). Biochemical, physicochemical and functional properties of hake actomyosin reflect these changes in the actomyosin composition (Beas et al., 1988; Montecchia et al., 1990). The Mg2+-ATPase activity is related to the actin-myosin interaction and the Ca 2+ATPase activity is related to the myosin functionality in the actomyosin complex. Enzy__ Correspondence to: Marcos Crupkin, Centro de Investigaciones de Tecnologia Pesquera (CITEP)(INTI), Marcelo T. de Alvear 1168, 7600 Mar del Plata, Argentina. Tel. 023-80-2801; Fax 023-89-1324. * Professional of the comision de investigaciones cientificas de la PCIA. de Buenos Aires. Received 18 November 1994; revised 3 May 1995; accepted 12 May 1995. 743
matic activities as Mg2+-, Mg2+-(EGTA)- and Ca2+-ATPase in actomyosin of pre-spawned hake are three times lower than those in actomyosin of post-spawned hake (Roura et al., 1990), suggesting that the myosin functionality in the actomyosin complex could be modified. Actomyosin preparations usually contain lipids that bind to the complex and produce significant effects on the properties of actomyosin (Hamada et al., 1982). Lipids play a central role in preserving protein stability. T~aguchi and Ikeda (1968) and Taguchi (1970) reported that the addition of lecithin, the main lipid component usually found in myosin B preparation, produced an increase in the MgZ+-ATPase activity of actomyosin and in its substrate affinity. Although the changes in the biochemical, physicochemical and functional properties of hake actomyosin, related to the metabolic state and gonadal maturation of the fish, have
744
J. P. Busalmen et al.
been studied, information concerning the influence of lipids on the properties of the actomyosin complex is scarce. The purpose of the present work is to provide information on the lipids in actomyosin isolated from pre- and post-spawned hake and to investigate their influence on the protein composition and enzymatic activities of the actomyosin complex.
Material and Methods Fish source Hake (M. hubbsi Marini) were caught by commercial vessels in the Southeast Atlantic Ocean at latitudes from 36° to 53° South from October 1990 to May 1993. Fish were kept on ice until they reached the laboratory in an early post-rigor condition. Fish measuring from 35 to 45 cm from the snout to the tip of the mid-caudal fin were selected and stored in boxes with ice. Samples, consisting of six females, were removed for analysis at days 0, 3,7 and 10 of storage. The gonadal condition of the specimens was determined with the histological techniques described by Goldemberg et al. (1987). Liver-somatic index (LSI) determination The LSI for each fish was calculated as follows:
LSI:
ditions for each enzyme were 0.25 mg/ml of protein, 1 mM ATP and 10 mM CaCI2 for Ca2+-ATPase and 0.10 mg/ml of protein, 1 mM ATP and 1 mM MgC12 for Mg2+-ATPase. A final incubation volume of 2 ml was used. Incubation times were 4 min for Ca 2+-ATPase and 1 min for MgZ+-ATPase. Reactions were interrupted by addition of 0.75 ml of cold 40% trichloroacetic acid solution. Liberated phosphorus was determined to the method of Chen et al. (1956). SDS-Polyacrylamide (PAGE)
gel
electrophoresis
SDS-PAGE was carried out in 10% gels according to the procedure of Laemmli (1970) in a mini-slab Sigma vertical apparatus. The quantitative area of the bands was determined by scanning gels at 570 nm with a Shimadzu SC 91.0 apparatus. Determination of lipid contents of muscle and actomyosin Total lipids were extracted from muscle and from actomyosin with 20 ml of a mixture of chloroform and methanol (2: 1, v/v) and purified by the procedure of Folch et al. (1957). The solvent was removed by evaporation at 40-50°C under nitrogen, and the residual lip-
wet weight (liver minus gall bladder) × 100. wet weight (fish, including liver minus gonads)
Actomosin preparation Actomyosin was purified according to the method of Tsuchiya et al. (1975), with the modifications introduced by Crupkin et al. (1982). The myofibrillar protein extract in 0.6 M KCI-NaHCO3 0.003 M, pH 7.0, was centrifuged at 7500 g, and actomyosin was obtained by diluting the supernatant with cold water up to a final concentration of 0.2 M KCI. The precipitate was solubilized in 0.6 M KC1. The dilution-solubilization steps were repeated three times to obtain purified natural actomyosin (NAM).
ids were weighed. The results were related to the amount of protein in the sample and are expressed as mg lipid/100 mg protein. Lipid phosphorus was determined by the method of Chen et al. (1956), and the results were multiplied by 23.5 (Geigy, 1975) to express the results as phospholipids content (P6cora, 1991). The 23.5 factor comes from the ratio between the mean molecular weight of phospholipids and the amount of phosphorus of each phospholipid molecule (Geigy, 1975). The contents in non-polar lipid (NPL) were calculated as the difference between total lipids and polar lipids.
Protein determination
Lipid distribution
Protein concentration was determined on aliquots of the extracts by the methods of Lowry et al. (1951), using bovine albumin as standard.
Total lipids were analyzed by thin-layer chromatography (TLC) on Silica gel G. TLC plates were developed using n-hexane/diethyl ether/formic acid (80:20:2 v/v) for NPL and chloroform/methanol/acetic acid/water (25: 15:4 : 2 v/v) for polar lipid as solvent systems. Spots were visualized and scanned according to the method of Kerenyi et al. (1980) in a Shimadzu SC 910 densitometer.
Actomyosin ATPase activities Enzymatic activities of actomyosin were measured at 37°C in a 30 mM Tris-maleate buffer (pH 6.8) with 50 mM KCI. Specific con-
745
Influence of lipid on hake actomyosin Table 1. Total lipid contents of muscle and NAM and LSI from pre- and post-spawned hake
Table 2. Total lipid (TL), NPL and polar lipid (PL) contents of pre- and post-spawned actomyosin
Pre-spawned
Muscle NAM LSI
Early (Nov)
Late (Dec/Jan)
Post-spawned (May)
8.6 9.2 5.3
2.6 4.0 3.4
5.3 2.1 5.4
Lipid contents was expressed as mg/100 mg of protein. Values are means of three samples.
Identification of the components was done by comparison of their relative mobility (Rf) with phospholipid standards (L-o~-phosphatidylcholine dipalmitoyl No. P5763 from egg yolk, L-a-phosphatidylethanolamine dipalmitoyl No. P0890 synthetic, L-a-phosphatidyl-Lserine No. P8518 from bovine brain, L-alysophosphatidylcholine No. L4129 from egg yolk, sphingomyelin No. $7004 from bovine brain -99%--contains primarily stearic and nervonic acids; all standards were from Sigma and standards of NPL (Mixture TLC-Icholesteryl oleate, methyloleate, oleic acid and triolein) were from Science Lab. Inc. Phospholipid and NPL standards were run on the same plate. Once the polar lipids were chromatographed and identified, their phosphorus content was determined by the method of Rouser et al. (1969).
Results and Discussion Lipids o f N A M f r o m hake muscle
Prcora (1991) reported seasonal variations in the lipid contents of hake muscle, with fat depletion during the summer. Roura et al. (1993) also reported differences in the lipid composition of hake muscle in pre- and postspawned conditions. The lipid contents in muscle and in actomyosin from hake in preand post-spawned conditions are shown in Table 1. The total lipid contents of muscle decreased from early pre-spawned hakes (November) to late pre-spawned hakes (December/January) and presented a partial recovery in post-spawned hakes (May). LSI values (Table 1) present a similar trend. These results are in agreement with those previously reported by Crupkin et al. (1988), who suggested that an energy demanding process like gonadal development (from September to March) involves the use of reserves in liver and muscles, with resulting decreases in the LSI. Actomyosin preparations from fish muscle contain lipids attached to the actomyosin complex (Ando et al., 1981; Taguchi and Ikeda, 1968). The total lipid contents of actomyosin
Pre-spawned actomyosin (Dec/Jan) Post-spawned actomyosin (May)
TL
NPL
PL
PL: NPL
4.0
2.7
1.3 (32)
0.48
2.1
1.1
1.0 (48)
0.91
Lipid contents was expressed as mg/100 mg of protein. Values are the mean value of three samples. Values in parenthesis represents the relative percentage of PL and TL.
changed with the gonadal condition of hake, reaching a minimum in the post-spawned condition (Table 1). This could indicate that to cover energy demands, hakes consume lipids not only from muscle reserves but also from those attached to the actomyosin complex. Although the lipid contents of muscles presented a partial recovery in the post-spawned condition, the lipid of the actomyosin complex did not. This would indicate that the recovery of fat reserves occurs first at tissue levels and later at the actomyosin level. The contents of total, polar and NPL in actomyosin from pre- and post-spawned hakes are shown in Table 2. It can be appreciated that the decrease in total lipids of actomyosin from the pre-spawned to the post-spawned conditions is mostly due to a decrease in NPL. The NPL in actomyosin from pre-spawned hake were 2.45 times those from postspawned hake, whereas the polar lipids in actomyosin from pre-spawned hake were only 1.3 times those from post-spawned hake. Beas et al. (1988) used differential scanning calorimetry to show the denaturation of intact muscle from pre-spawned hake. Shenouda and Piggot (1974) indicated that the denaturation of myosin in a myosin model system by different methods cause configurational changes that produce a noticeable increase in myosin-lipid interactions. On the other hand, Roura et al. (1992) reported that a loss in the filamentous structure of actomyosin from prespawned hake was related to a lesser affinity between myosin and actin. This decreased affinity was attributed to myosin denaturation. Hydrophobic interactions are altered during transition to the denatured state (Anglemier and Montgomery, 1976). Surface hydrophobicity of actomyosin is higher in pre-spawned than in post-spawned hake (Beas and Crupkin, 1990). Roura et al. (1992) postulated that the higher surface hydrophobicity would be evidence of a partially denatured or sensitized protein. The higher neutral lipid contents of actomyosin from pre-spawned hake (Table 2)
746
J. P. Busalmen et al.
7 0,25
0,20 0,15
2,00 PHE-SPAWNED
e~
1,75
o, Io
1,50
0,05
1,25
o,oo c*
0,75
&
rv
0,50[ 0,25 O,OOl
POST-SPAWNEI
NOV. DEC. JAN. FEB. IMAR. APR. MAY PRE-SPAWN'ED
PO~T-SPAWNED
Fig. 2. Mg2+-ATPase (Rq) and CaZ+-ATPase (+) activity during the reproductive cycle of fishes. Each point is the mean value of six fish. Bars indicate the standard deviations. I
i
EE
1
TG FFA
I
}
I
i
!
12C S~ 1~" PS+P1 PE
Fig. 1. Typical TLC profiles of non-polar and polar fractions from pre- and post-spawned actomyosin. EE, sterols esters; FFA, free fatty acid; TG, triglyceride; LPC, lysophosphatidylcholine; SPH, sphingomyelin; PC, phosphatidylcholine; PS, phosphatidylserine; PE, phosphatidylethanolamine.
is in correspondence with higher surface hydrophobicity resulting from conformational changes during the gonadal growth phase (Roura et al., 1992). The constituents of the non-polar fraction was investigated. Very hydrophobic molecules like triglycerides and sterol esters contributed over 90% of this fraction. Figure 1 represent a typical TLC profiles of non-polar and polar fractions from pre- and post-spawned actomyosin. The distribution of NPL in actomyosin did not change from the pre- to the post-spawned conditions (Fig. 1). The different decreases in the polar and non-polar fractions of actomyosin lipids from the pre- to the post-spawned conditions resulted in an increase in the relative percentage of phospholipids that were 32% in the prespawned stage and 48% in the post-spawned stage. As a result, the ratio of phospholipids to neutral lipids almost doubles after spawning (Table 2). The composition of the polar lipid fraction is shown in Fig. 1. Phosphatidylcholine and phosphatidylethanolamine are the main constituents; lysophosphatidylcholine, sphingomyelin, phosphatidylserine and phosphatidylinositol are present in lower quantities. The only difference found in the postspawned condition, with reference to the pre-spawned condition, was a 20% decrease
in the peak corresponding to the sum of phosphatidylserine and phosphatidylinositol. Lecithin and phosphatidylethanolamine in actomyosin constituted 90% of the polar lipids fraction in both gonadal stages of the fish (Fig. 1). Similar results were reported for phospholipids in hake myofibrils (Roura et al., 1993) and in hake muscle (P6cora, 1991). A TPase activities o f N A M
ATPases activities of actomyosin from preand post-spawned hake were measured in November, December, January, February and May. MgZ+-ATPase activity in post-spawned actomyosin was significantly higher (P < 0.01) than in pre-spawned actomyosin (Fig. 2). Similar results had been reported by Montecchia et al. (1990) and Roura et al. (1990). The Mg2+-ATPase activity and the affinity of fish actomyosin for ATP increase and the binding of myosin to actin becomes loose upon the addition of lecithin (Taguchi and Ikeda, 1968; Taguchi, 1970). Ando et al. (1981) obtained a more active precipitate rich in phospholipids from carp myosin B centrifuged at 25,000 g for 1 hr as compared with the original extract. As was mentioned above, the ratio of phospholipids to neutral lipids was higher in postspawned actomyosin (0.91) than in prespawned actomyosin (0.48) (Table 2). Because lecithin is the major constituent of polar lipids, the increased activity of MgZ+-ATPase in the post-spawned condition could be related to the change in the phospholipids : neutral lipids ratio. Roura et al. (1992) proposed two mecha-
Influence of lipid on hake actomyosin
747
~AWNED POST-SPA~D nisms for myosin-actin interactions in fish bekD fore and after spawning for in vitro experi19"1.0 ments. They also postulated that a decrease 117.0 91.8 in the affinity between myosin and actin could 72.7 be related to an in vivo increase in the proteo57.8 A lytic activity that would selectively degrade 40.8 the heavy myosin chain; however, the possiTm 34.1 bility that the differences in lipid distribution between pre- and post-spawned actomyosin MICe could be affected the macromolecular complex must not be rejected. The Ca2+-ATPase activity in the early preM/A 2.2 2.1 1.7 1.9 1.7 2.3 spawned condition was 0.23 mmol Pi/min/mg of protein, gradually decreased to a mini- Fig. 3. SDS-mini-slab-polyacrylamide gel (10%) electromum of 0.07 mmol Pi/min/mg of protein at phoretic patterns of actomyosin from pre- and postthe end of the gonadal maturation and then spawned period. M/A represents the myosin:actin ratios in actomyosin; MHC, myosin heavy chain; A, actin; Tr, increased to 0.21 mmol Pi/min/mg of protein troponin; Tm, tropomyosin; MLCs, myosin light chains; in the early post-spawned stage (Fig. 2). Re- ST, standards of molecular weight from Sigma kit No. sults reported by Roura et al. (1990) showed MW-SDS-BLUE; kD, molecular weights. Each electrothat the Ca2+-ATPase activity of actomyosin phoretical pattern corresponds to a different actomyosin sample. from post-spawned hake was about three times higher than that from pre-spawned hake. Present results would indicate a gradual Analysis of actomyosin patterns loss in the myosin functionality of the actomySDS-PAGE patterns of actomyosin from osin complex during gonadal development pre- and post-spawned hake are shown in Fig. with a gradual recovery after spawning. 3. The characteristic polypeptide bands of acThe actomyosin from pre-spawned hake has tomyosin (myosin heavy chain, actin, A, trosignificantly less heavy myosin and more actin pomyosin, Tm and myosin light chains) are than the actomyosin from the resting and post- present in both gonadal stages. The myosin: spawning periods (Crupkin et al., 1988). The actin ratio decreased during gonadal growth ultrastructure of hake actomyosin presents a from 2.2 in the early pre-spawned stage to 1.7 characteristic arrowhead filamentous struc- in the late pre-spawned stage. This ratio reture in post-spawned fish, whereas in pre- covered to 2.3 in the post-spawned stage. spawned hake it enfolds and aggregates into These results are in agreement with those reglobular masses. The loss of the filamentous ported by Crupkin et al. (1988). Changes in structure was attributed to lower affinity be- the actomyosin composition could lead to tween myosin and actin (Roura et al., 1992). modifications in the shape of the major actoMyosin exhibits hydrophobic surface prop- myosin complex (Roura et al., 1992). The erties and binds hydrophobic groups in a co- lower Ca 2+-ATPase activity in the actomyosin operative manner. Those hydrophobic bind- of pre-spawned hake may indicate that the ings make a substantial contribution to the myosin functionality decreases with the gostability of myosin filaments; therefore, the in- nadal development. The lower myosin content creasing tendency of myosin to aggregate par- in actomyosin from pre-spawned hake could allels the increase in the hydrophobic ligands be related to an in vivo increase of the proteo(Pinaev et al., 1982). The integrity of the thick lytic activity that selectively degrades heavy filament could then be affected by the lipid- myosin chain (Crupkin et al., 1988). actomyosin interactions a t the different gonadal stages. The high surface hydrophobicity (Beas and Crupkin, 1990), the loss in the natu- References ral structure of actomyosin and the biochemi- Ando S., Kozo T. and Koichi Z. (1981) Effect of gel fraccal properties in pre-spawned hake (Roura et tion on carp myosin B ATPase activity. Bull. Jap. Soc. Sci. Fish. 47, 1579-1583. al., 1992) are evidence of a partially denatured Anglemier A. F. and Montgomery M. W. (1976) Amino or sensitized protein. The greater amounts of acid peptides and proteins. In Principles of Food SciNPL found in actomyosin from pre-spawned ence, Part 1. Food Chemistry (Edited by Fennema hake could be the result of a denatured actoO. R.), p. 238. Marcel Dekker, New York. myosin complex. Natural actomyosin from Beas V. E. and Crupkin M. (1990) Protein-protein interactions in gels of pre- and post-spawning fish actomyosin. post-spawned hake presented better biochemIn Proceedings of the Freezing and Chilling of New ical, physicochemical and ultrastructural Fish Products, pp. 115-121. International Institute of properties; thus, full functionality of actomyoRefrigeration, Aberdeen, U.K. sin occurs with low lipid contents. Beas V. E., Crupkin M. and Trucco R. E. (1988) Gelling
748
J. P. Busalmen et al.
properties of actomyosin from pre- and post-spawning hake (Merluccius hubbsi). J. Food Sci. 53, 1322-1326. Chen P. S., Toribara T. and Wagner H. (1956) Microdeterminations of phosphorus. Analyt. Chem. 28, 17561758. Crupkin M., Barassi C. A., Arguello J. M. and Trucco R. E. (1982) Effect of post rigor fish storage on ice on physicochemical properties of actomyosin. J. Sci. Food Agric. 33, 1129-1134. Crupkin M., Montecchia C. L. and Trucco R. E. (1988) Seasonal variations in gonado-somatic index, liversomatic index and myosin/actin ratio in actomyosin of mature hake (Merluccius hubbsi). Comp. Biochem. Physiol. 89A, 7-10. Folch J., Lees M. and Sloane Stanley G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. biol. Chem. 226, 497. Geigy S. A. (1975) Scientific Tables, 6th edn. Ciba Geigy. Basilea Swiss. Goldemberg A. L., Par6n L. S. and Crupkin M. (1987) Acid phosphatase activity in pre- and post-spawning hake (Merluccius hubbsi). Comp. Biochem. Physiol. 87A, 845-849. Hamada I., Yabuno F., Furumatsu K. and Niwa E. (1982) The effect of lipid on the heat denaturation of actomyosin. Bull. Jap. Soc. Sci. Fish. 48, 189-193. Kerenyi L., Bartalos L. and Jobst K. (1980) lododensitometry: rapid quantitation of lipids mixtures after thin layer chromatography. Clin. Chem. Acta 100, 189-191. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, Lond. 227, 680. Lowry O. H., Rosenbrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Montecchia C. L., Crupkin M. and Trucco R. E. (1990) Seasonal variations in biochemical and physicochemical properties of actomyosin and energy content of the liver, gonads and muscle of mature Argentine hake, Merluccius hubbsi Marini. J. Fish Biol. 37, 837-843. Prcora R. P. (1991) Variaciones estacionales de los lipidos
del mlisculo de la merluza (Merluccius hubbsi) y su influencia en los cambios producidos durante la conservaci6n en hielo. Ph.D. thesis, U.N.M.dP, Argentina. Pinaev G., Tartakovsky A., Shanbhag V. P., Johansson G. and Backman L. (1982) Hydrophobic surface properties of myosin in solution as studied by partition in aqueous two-phase systems: effects of ionic strength, pH and temperature. Mol. cell. Biochem. 48, 65-69. Roura S. I., Montecchia C. L., Goldemberg A. L., Trucco R. E. and Crupkin M. (1990) Biochemical and physicochemical properties of actomyosin from pre- and postspawned hake (Merluccius hubbsi) stored on ice. J. Food Sci. 55, 688-692. Roura S. I., Pecci Saavedra J., Trucco R. E. and Crupkin M. (1992) Electron microscopical and biochemical studies of actomyosin from pre- and post-spawned hake. Comp. Biochem. Physiol. 101B, 361-365. Roura S. I., Rold~n H., Goldemberg A. L., Trucco R. E. and Crupkin M. (1993) Effect of Triton X-100 treatment on the lipid composition, biochemical and functional properties of hake (Merluccius hubbsi) myofibrils purified from pre- and post-spawned fish. Comp. Biochem. Physiol. 106B, 191-197. Rouser G., Siakotos A. N. and Fleischer S. (1969) Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots. Lipids 1, 85-86. Shenouda S. Y. K. and Pigott G. M. (1974) Lipid-protein interaction during aqueous extraction of fish protein: myosin lipid interaction. J. Food Sci. 39, 726-734. Taguchi T. (1970) Studies on the properties of fish actomyosin from fish muscle. V. Activation offish actomyosin ATPase by lecithin. Bull. Jap. Soc. Sci. Fish. 36, 612-616. Taguchi T. and Ikeda S. (1968) Studies on the properties of fish actomyosin. IV. Effect of lecithin on the ATPase activity of actomyosin from fish muscle. Bull. Jap. Soc. Sci. Fish. 34, 411-414. Tsuchiya T., Tsuchiya Y., Nonomura Y. and Matsumoto J. (1975) Prevention of freeze denaturation of carp actomyosin by sodium glutamate. J. Biochem. 77, 853-862.
Pergamon 0305-0491(95)00131-X
Changes in lipids and biochemical properties of actomyosin from pre- and post-spawned hake (Merluccius hubbsi Marini) Juan Pablo Busalmen, Sara In6s Roura, Hugo Roldan* and Marcos Crupkin The Centro de Investigaciones de Tecnologia Pesquera (CITEP)(INTI), Marcelo T. de Alvear 1168, 7600 Mar del Plata, Argentina The total lipid contents of muscle, the liver somatic index and the total lipid contents of actomyosin in hakes changed with the gonadal condition of the fish. Non-polar lipids in actomyosin from pre-spawned hake were 145% higher than in actomyosin from post-spawned hake; polar lipids were 30% higher. The relative percentage of phospholipids changed from 32% in pre-spawned hake to 48% in post-spawned hake. The Mg2+-ATPase activity in actomyosin increased from the pre-spawned to the post-spawned conditions, and this could be related to a higher phospholipids to neutral lipids ratio in post-spawned hake. Gradual decreases in both Ca2+-ATPase activity and myosin: actin ratio during the gonadal development of fish were found, suggesting a partial loss of myosin functionality of the actomyosin complex. Key words: Muscle lipids; Actomyosin complex; Merluccius hubbsi; Gonadal development; Actomyosin ATPases; Myosin: actin ratio; Myosin functionality. Comp. Biochem. Physiol. lI2B, 743-748, 1995.
Introduction
Changes in the actomyosin composition of mature Argentinian hake (Merluccius hubbsi Marini) are influenced by the metabolic state of the fish and are related to the reproductive cycle (Crupkin et al., 1988). Biochemical, physicochemical and functional properties of hake actomyosin reflect these changes in the actomyosin composition (Beas et al., 1988; Montecchia et al., 1990). The Mg2+-ATPase activity is related to the actin-myosin interaction and the Ca 2+ATPase activity is related to the myosin functionality in the actomyosin complex. Enzy__ Correspondence to: Marcos Crupkin, Centro de Investigaciones de Tecnologia Pesquera (CITEP)(INTI), Marcelo T. de Alvear 1168, 7600 Mar del Plata, Argentina. Tel. 023-80-2801; Fax 023-89-1324. * Professional of the comision de investigaciones cientificas de la PCIA. de Buenos Aires. Received 18 November 1994; revised 3 May 1995; accepted 12 May 1995. 743
matic activities as Mg2+-, Mg2+-(EGTA)- and Ca2+-ATPase in actomyosin of pre-spawned hake are three times lower than those in actomyosin of post-spawned hake (Roura et al., 1990), suggesting that the myosin functionality in the actomyosin complex could be modified. Actomyosin preparations usually contain lipids that bind to the complex and produce significant effects on the properties of actomyosin (Hamada et al., 1982). Lipids play a central role in preserving protein stability. T~aguchi and Ikeda (1968) and Taguchi (1970) reported that the addition of lecithin, the main lipid component usually found in myosin B preparation, produced an increase in the MgZ+-ATPase activity of actomyosin and in its substrate affinity. Although the changes in the biochemical, physicochemical and functional properties of hake actomyosin, related to the metabolic state and gonadal maturation of the fish, have
744
J. P. Busalmen et al.
been studied, information concerning the influence of lipids on the properties of the actomyosin complex is scarce. The purpose of the present work is to provide information on the lipids in actomyosin isolated from pre- and post-spawned hake and to investigate their influence on the protein composition and enzymatic activities of the actomyosin complex.
Material and Methods Fish source Hake (M. hubbsi Marini) were caught by commercial vessels in the Southeast Atlantic Ocean at latitudes from 36° to 53° South from October 1990 to May 1993. Fish were kept on ice until they reached the laboratory in an early post-rigor condition. Fish measuring from 35 to 45 cm from the snout to the tip of the mid-caudal fin were selected and stored in boxes with ice. Samples, consisting of six females, were removed for analysis at days 0, 3,7 and 10 of storage. The gonadal condition of the specimens was determined with the histological techniques described by Goldemberg et al. (1987). Liver-somatic index (LSI) determination The LSI for each fish was calculated as follows:
LSI:
ditions for each enzyme were 0.25 mg/ml of protein, 1 mM ATP and 10 mM CaCI2 for Ca2+-ATPase and 0.10 mg/ml of protein, 1 mM ATP and 1 mM MgC12 for Mg2+-ATPase. A final incubation volume of 2 ml was used. Incubation times were 4 min for Ca 2+-ATPase and 1 min for MgZ+-ATPase. Reactions were interrupted by addition of 0.75 ml of cold 40% trichloroacetic acid solution. Liberated phosphorus was determined to the method of Chen et al. (1956). SDS-Polyacrylamide (PAGE)
gel
electrophoresis
SDS-PAGE was carried out in 10% gels according to the procedure of Laemmli (1970) in a mini-slab Sigma vertical apparatus. The quantitative area of the bands was determined by scanning gels at 570 nm with a Shimadzu SC 91.0 apparatus. Determination of lipid contents of muscle and actomyosin Total lipids were extracted from muscle and from actomyosin with 20 ml of a mixture of chloroform and methanol (2: 1, v/v) and purified by the procedure of Folch et al. (1957). The solvent was removed by evaporation at 40-50°C under nitrogen, and the residual lip-
wet weight (liver minus gall bladder) × 100. wet weight (fish, including liver minus gonads)
Actomosin preparation Actomyosin was purified according to the method of Tsuchiya et al. (1975), with the modifications introduced by Crupkin et al. (1982). The myofibrillar protein extract in 0.6 M KCI-NaHCO3 0.003 M, pH 7.0, was centrifuged at 7500 g, and actomyosin was obtained by diluting the supernatant with cold water up to a final concentration of 0.2 M KCI. The precipitate was solubilized in 0.6 M KC1. The dilution-solubilization steps were repeated three times to obtain purified natural actomyosin (NAM).
ids were weighed. The results were related to the amount of protein in the sample and are expressed as mg lipid/100 mg protein. Lipid phosphorus was determined by the method of Chen et al. (1956), and the results were multiplied by 23.5 (Geigy, 1975) to express the results as phospholipids content (P6cora, 1991). The 23.5 factor comes from the ratio between the mean molecular weight of phospholipids and the amount of phosphorus of each phospholipid molecule (Geigy, 1975). The contents in non-polar lipid (NPL) were calculated as the difference between total lipids and polar lipids.
Protein determination
Lipid distribution
Protein concentration was determined on aliquots of the extracts by the methods of Lowry et al. (1951), using bovine albumin as standard.
Total lipids were analyzed by thin-layer chromatography (TLC) on Silica gel G. TLC plates were developed using n-hexane/diethyl ether/formic acid (80:20:2 v/v) for NPL and chloroform/methanol/acetic acid/water (25: 15:4 : 2 v/v) for polar lipid as solvent systems. Spots were visualized and scanned according to the method of Kerenyi et al. (1980) in a Shimadzu SC 910 densitometer.
Actomyosin ATPase activities Enzymatic activities of actomyosin were measured at 37°C in a 30 mM Tris-maleate buffer (pH 6.8) with 50 mM KCI. Specific con-
745
Influence of lipid on hake actomyosin Table 1. Total lipid contents of muscle and NAM and LSI from pre- and post-spawned hake
Table 2. Total lipid (TL), NPL and polar lipid (PL) contents of pre- and post-spawned actomyosin
Pre-spawned
Muscle NAM LSI
Early (Nov)
Late (Dec/Jan)
Post-spawned (May)
8.6 9.2 5.3
2.6 4.0 3.4
5.3 2.1 5.4
Lipid contents was expressed as mg/100 mg of protein. Values are means of three samples.
Identification of the components was done by comparison of their relative mobility (Rf) with phospholipid standards (L-o~-phosphatidylcholine dipalmitoyl No. P5763 from egg yolk, L-a-phosphatidylethanolamine dipalmitoyl No. P0890 synthetic, L-a-phosphatidyl-Lserine No. P8518 from bovine brain, L-alysophosphatidylcholine No. L4129 from egg yolk, sphingomyelin No. $7004 from bovine brain -99%--contains primarily stearic and nervonic acids; all standards were from Sigma and standards of NPL (Mixture TLC-Icholesteryl oleate, methyloleate, oleic acid and triolein) were from Science Lab. Inc. Phospholipid and NPL standards were run on the same plate. Once the polar lipids were chromatographed and identified, their phosphorus content was determined by the method of Rouser et al. (1969).
Results and Discussion Lipids o f N A M f r o m hake muscle
Prcora (1991) reported seasonal variations in the lipid contents of hake muscle, with fat depletion during the summer. Roura et al. (1993) also reported differences in the lipid composition of hake muscle in pre- and postspawned conditions. The lipid contents in muscle and in actomyosin from hake in preand post-spawned conditions are shown in Table 1. The total lipid contents of muscle decreased from early pre-spawned hakes (November) to late pre-spawned hakes (December/January) and presented a partial recovery in post-spawned hakes (May). LSI values (Table 1) present a similar trend. These results are in agreement with those previously reported by Crupkin et al. (1988), who suggested that an energy demanding process like gonadal development (from September to March) involves the use of reserves in liver and muscles, with resulting decreases in the LSI. Actomyosin preparations from fish muscle contain lipids attached to the actomyosin complex (Ando et al., 1981; Taguchi and Ikeda, 1968). The total lipid contents of actomyosin
Pre-spawned actomyosin (Dec/Jan) Post-spawned actomyosin (May)
TL
NPL
PL
PL: NPL
4.0
2.7
1.3 (32)
0.48
2.1
1.1
1.0 (48)
0.91
Lipid contents was expressed as mg/100 mg of protein. Values are the mean value of three samples. Values in parenthesis represents the relative percentage of PL and TL.
changed with the gonadal condition of hake, reaching a minimum in the post-spawned condition (Table 1). This could indicate that to cover energy demands, hakes consume lipids not only from muscle reserves but also from those attached to the actomyosin complex. Although the lipid contents of muscles presented a partial recovery in the post-spawned condition, the lipid of the actomyosin complex did not. This would indicate that the recovery of fat reserves occurs first at tissue levels and later at the actomyosin level. The contents of total, polar and NPL in actomyosin from pre- and post-spawned hakes are shown in Table 2. It can be appreciated that the decrease in total lipids of actomyosin from the pre-spawned to the post-spawned conditions is mostly due to a decrease in NPL. The NPL in actomyosin from pre-spawned hake were 2.45 times those from postspawned hake, whereas the polar lipids in actomyosin from pre-spawned hake were only 1.3 times those from post-spawned hake. Beas et al. (1988) used differential scanning calorimetry to show the denaturation of intact muscle from pre-spawned hake. Shenouda and Piggot (1974) indicated that the denaturation of myosin in a myosin model system by different methods cause configurational changes that produce a noticeable increase in myosin-lipid interactions. On the other hand, Roura et al. (1992) reported that a loss in the filamentous structure of actomyosin from prespawned hake was related to a lesser affinity between myosin and actin. This decreased affinity was attributed to myosin denaturation. Hydrophobic interactions are altered during transition to the denatured state (Anglemier and Montgomery, 1976). Surface hydrophobicity of actomyosin is higher in pre-spawned than in post-spawned hake (Beas and Crupkin, 1990). Roura et al. (1992) postulated that the higher surface hydrophobicity would be evidence of a partially denatured or sensitized protein. The higher neutral lipid contents of actomyosin from pre-spawned hake (Table 2)
746
J. P. Busalmen et al.
7 0,25
0,20 0,15
2,00 PHE-SPAWNED
e~
1,75
o, Io
1,50
0,05
1,25
o,oo c*
0,75
&
rv
0,50[ 0,25 O,OOl
POST-SPAWNEI
NOV. DEC. JAN. FEB. IMAR. APR. MAY PRE-SPAWN'ED
PO~T-SPAWNED
Fig. 2. Mg2+-ATPase (Rq) and CaZ+-ATPase (+) activity during the reproductive cycle of fishes. Each point is the mean value of six fish. Bars indicate the standard deviations. I
i
EE
1
TG FFA
I
}
I
i
!
12C S~ 1~" PS+P1 PE
Fig. 1. Typical TLC profiles of non-polar and polar fractions from pre- and post-spawned actomyosin. EE, sterols esters; FFA, free fatty acid; TG, triglyceride; LPC, lysophosphatidylcholine; SPH, sphingomyelin; PC, phosphatidylcholine; PS, phosphatidylserine; PE, phosphatidylethanolamine.
is in correspondence with higher surface hydrophobicity resulting from conformational changes during the gonadal growth phase (Roura et al., 1992). The constituents of the non-polar fraction was investigated. Very hydrophobic molecules like triglycerides and sterol esters contributed over 90% of this fraction. Figure 1 represent a typical TLC profiles of non-polar and polar fractions from pre- and post-spawned actomyosin. The distribution of NPL in actomyosin did not change from the pre- to the post-spawned conditions (Fig. 1). The different decreases in the polar and non-polar fractions of actomyosin lipids from the pre- to the post-spawned conditions resulted in an increase in the relative percentage of phospholipids that were 32% in the prespawned stage and 48% in the post-spawned stage. As a result, the ratio of phospholipids to neutral lipids almost doubles after spawning (Table 2). The composition of the polar lipid fraction is shown in Fig. 1. Phosphatidylcholine and phosphatidylethanolamine are the main constituents; lysophosphatidylcholine, sphingomyelin, phosphatidylserine and phosphatidylinositol are present in lower quantities. The only difference found in the postspawned condition, with reference to the pre-spawned condition, was a 20% decrease
in the peak corresponding to the sum of phosphatidylserine and phosphatidylinositol. Lecithin and phosphatidylethanolamine in actomyosin constituted 90% of the polar lipids fraction in both gonadal stages of the fish (Fig. 1). Similar results were reported for phospholipids in hake myofibrils (Roura et al., 1993) and in hake muscle (P6cora, 1991). A TPase activities o f N A M
ATPases activities of actomyosin from preand post-spawned hake were measured in November, December, January, February and May. MgZ+-ATPase activity in post-spawned actomyosin was significantly higher (P < 0.01) than in pre-spawned actomyosin (Fig. 2). Similar results had been reported by Montecchia et al. (1990) and Roura et al. (1990). The Mg2+-ATPase activity and the affinity of fish actomyosin for ATP increase and the binding of myosin to actin becomes loose upon the addition of lecithin (Taguchi and Ikeda, 1968; Taguchi, 1970). Ando et al. (1981) obtained a more active precipitate rich in phospholipids from carp myosin B centrifuged at 25,000 g for 1 hr as compared with the original extract. As was mentioned above, the ratio of phospholipids to neutral lipids was higher in postspawned actomyosin (0.91) than in prespawned actomyosin (0.48) (Table 2). Because lecithin is the major constituent of polar lipids, the increased activity of MgZ+-ATPase in the post-spawned condition could be related to the change in the phospholipids : neutral lipids ratio. Roura et al. (1992) proposed two mecha-
Influence of lipid on hake actomyosin
747
~AWNED POST-SPA~D nisms for myosin-actin interactions in fish bekD fore and after spawning for in vitro experi19"1.0 ments. They also postulated that a decrease 117.0 91.8 in the affinity between myosin and actin could 72.7 be related to an in vivo increase in the proteo57.8 A lytic activity that would selectively degrade 40.8 the heavy myosin chain; however, the possiTm 34.1 bility that the differences in lipid distribution between pre- and post-spawned actomyosin MICe could be affected the macromolecular complex must not be rejected. The Ca2+-ATPase activity in the early preM/A 2.2 2.1 1.7 1.9 1.7 2.3 spawned condition was 0.23 mmol Pi/min/mg of protein, gradually decreased to a mini- Fig. 3. SDS-mini-slab-polyacrylamide gel (10%) electromum of 0.07 mmol Pi/min/mg of protein at phoretic patterns of actomyosin from pre- and postthe end of the gonadal maturation and then spawned period. M/A represents the myosin:actin ratios in actomyosin; MHC, myosin heavy chain; A, actin; Tr, increased to 0.21 mmol Pi/min/mg of protein troponin; Tm, tropomyosin; MLCs, myosin light chains; in the early post-spawned stage (Fig. 2). Re- ST, standards of molecular weight from Sigma kit No. sults reported by Roura et al. (1990) showed MW-SDS-BLUE; kD, molecular weights. Each electrothat the Ca2+-ATPase activity of actomyosin phoretical pattern corresponds to a different actomyosin sample. from post-spawned hake was about three times higher than that from pre-spawned hake. Present results would indicate a gradual Analysis of actomyosin patterns loss in the myosin functionality of the actomySDS-PAGE patterns of actomyosin from osin complex during gonadal development pre- and post-spawned hake are shown in Fig. with a gradual recovery after spawning. 3. The characteristic polypeptide bands of acThe actomyosin from pre-spawned hake has tomyosin (myosin heavy chain, actin, A, trosignificantly less heavy myosin and more actin pomyosin, Tm and myosin light chains) are than the actomyosin from the resting and post- present in both gonadal stages. The myosin: spawning periods (Crupkin et al., 1988). The actin ratio decreased during gonadal growth ultrastructure of hake actomyosin presents a from 2.2 in the early pre-spawned stage to 1.7 characteristic arrowhead filamentous struc- in the late pre-spawned stage. This ratio reture in post-spawned fish, whereas in pre- covered to 2.3 in the post-spawned stage. spawned hake it enfolds and aggregates into These results are in agreement with those reglobular masses. The loss of the filamentous ported by Crupkin et al. (1988). Changes in structure was attributed to lower affinity be- the actomyosin composition could lead to tween myosin and actin (Roura et al., 1992). modifications in the shape of the major actoMyosin exhibits hydrophobic surface prop- myosin complex (Roura et al., 1992). The erties and binds hydrophobic groups in a co- lower Ca 2+-ATPase activity in the actomyosin operative manner. Those hydrophobic bind- of pre-spawned hake may indicate that the ings make a substantial contribution to the myosin functionality decreases with the gostability of myosin filaments; therefore, the in- nadal development. The lower myosin content creasing tendency of myosin to aggregate par- in actomyosin from pre-spawned hake could allels the increase in the hydrophobic ligands be related to an in vivo increase of the proteo(Pinaev et al., 1982). The integrity of the thick lytic activity that selectively degrades heavy filament could then be affected by the lipid- myosin chain (Crupkin et al., 1988). actomyosin interactions a t the different gonadal stages. The high surface hydrophobicity (Beas and Crupkin, 1990), the loss in the natu- References ral structure of actomyosin and the biochemi- Ando S., Kozo T. and Koichi Z. (1981) Effect of gel fraccal properties in pre-spawned hake (Roura et tion on carp myosin B ATPase activity. Bull. Jap. Soc. Sci. Fish. 47, 1579-1583. al., 1992) are evidence of a partially denatured Anglemier A. F. and Montgomery M. W. (1976) Amino or sensitized protein. The greater amounts of acid peptides and proteins. In Principles of Food SciNPL found in actomyosin from pre-spawned ence, Part 1. Food Chemistry (Edited by Fennema hake could be the result of a denatured actoO. R.), p. 238. Marcel Dekker, New York. myosin complex. Natural actomyosin from Beas V. E. and Crupkin M. (1990) Protein-protein interactions in gels of pre- and post-spawning fish actomyosin. post-spawned hake presented better biochemIn Proceedings of the Freezing and Chilling of New ical, physicochemical and ultrastructural Fish Products, pp. 115-121. International Institute of properties; thus, full functionality of actomyoRefrigeration, Aberdeen, U.K. sin occurs with low lipid contents. Beas V. E., Crupkin M. and Trucco R. E. (1988) Gelling
748
J. P. Busalmen et al.
properties of actomyosin from pre- and post-spawning hake (Merluccius hubbsi). J. Food Sci. 53, 1322-1326. Chen P. S., Toribara T. and Wagner H. (1956) Microdeterminations of phosphorus. Analyt. Chem. 28, 17561758. Crupkin M., Barassi C. A., Arguello J. M. and Trucco R. E. (1982) Effect of post rigor fish storage on ice on physicochemical properties of actomyosin. J. Sci. Food Agric. 33, 1129-1134. Crupkin M., Montecchia C. L. and Trucco R. E. (1988) Seasonal variations in gonado-somatic index, liversomatic index and myosin/actin ratio in actomyosin of mature hake (Merluccius hubbsi). Comp. Biochem. Physiol. 89A, 7-10. Folch J., Lees M. and Sloane Stanley G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. biol. Chem. 226, 497. Geigy S. A. (1975) Scientific Tables, 6th edn. Ciba Geigy. Basilea Swiss. Goldemberg A. L., Par6n L. S. and Crupkin M. (1987) Acid phosphatase activity in pre- and post-spawning hake (Merluccius hubbsi). Comp. Biochem. Physiol. 87A, 845-849. Hamada I., Yabuno F., Furumatsu K. and Niwa E. (1982) The effect of lipid on the heat denaturation of actomyosin. Bull. Jap. Soc. Sci. Fish. 48, 189-193. Kerenyi L., Bartalos L. and Jobst K. (1980) lododensitometry: rapid quantitation of lipids mixtures after thin layer chromatography. Clin. Chem. Acta 100, 189-191. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, Lond. 227, 680. Lowry O. H., Rosenbrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Montecchia C. L., Crupkin M. and Trucco R. E. (1990) Seasonal variations in biochemical and physicochemical properties of actomyosin and energy content of the liver, gonads and muscle of mature Argentine hake, Merluccius hubbsi Marini. J. Fish Biol. 37, 837-843. Prcora R. P. (1991) Variaciones estacionales de los lipidos
del mlisculo de la merluza (Merluccius hubbsi) y su influencia en los cambios producidos durante la conservaci6n en hielo. Ph.D. thesis, U.N.M.dP, Argentina. Pinaev G., Tartakovsky A., Shanbhag V. P., Johansson G. and Backman L. (1982) Hydrophobic surface properties of myosin in solution as studied by partition in aqueous two-phase systems: effects of ionic strength, pH and temperature. Mol. cell. Biochem. 48, 65-69. Roura S. I., Montecchia C. L., Goldemberg A. L., Trucco R. E. and Crupkin M. (1990) Biochemical and physicochemical properties of actomyosin from pre- and postspawned hake (Merluccius hubbsi) stored on ice. J. Food Sci. 55, 688-692. Roura S. I., Pecci Saavedra J., Trucco R. E. and Crupkin M. (1992) Electron microscopical and biochemical studies of actomyosin from pre- and post-spawned hake. Comp. Biochem. Physiol. 101B, 361-365. Roura S. I., Rold~n H., Goldemberg A. L., Trucco R. E. and Crupkin M. (1993) Effect of Triton X-100 treatment on the lipid composition, biochemical and functional properties of hake (Merluccius hubbsi) myofibrils purified from pre- and post-spawned fish. Comp. Biochem. Physiol. 106B, 191-197. Rouser G., Siakotos A. N. and Fleischer S. (1969) Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots. Lipids 1, 85-86. Shenouda S. Y. K. and Pigott G. M. (1974) Lipid-protein interaction during aqueous extraction of fish protein: myosin lipid interaction. J. Food Sci. 39, 726-734. Taguchi T. (1970) Studies on the properties of fish actomyosin from fish muscle. V. Activation offish actomyosin ATPase by lecithin. Bull. Jap. Soc. Sci. Fish. 36, 612-616. Taguchi T. and Ikeda S. (1968) Studies on the properties of fish actomyosin. IV. Effect of lecithin on the ATPase activity of actomyosin from fish muscle. Bull. Jap. Soc. Sci. Fish. 34, 411-414. Tsuchiya T., Tsuchiya Y., Nonomura Y. and Matsumoto J. (1975) Prevention of freeze denaturation of carp actomyosin by sodium glutamate. J. Biochem. 77, 853-862.