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Influence of Frozen Storage on Microsomal Phospholipase Activity in Myotomal Tissue of Atlantic Cod (Gadus morhua)
Con Inst. Food Sci. Technol. J. Vo!. 21, No. 4, pp. 399-402, 1988
RESEARCH
Influence of Frozen Storage on Microsomal Phospholipase Activity in Myotomal Tissue of Atlantic Cod (Gadus morhua) P. Chawla " B. MacKeigan, S.P. Gould and R.F. Ablete Department of Food Science and Technology Technical University of Nova Scotia P.O. Box 1000 Halifax, Nova Scotia B3J 2X4
ski et al., 1976). These changes are significant among commercially important species including Atlantic cod (Gadus morhua) for they determine both the storage life and overall acceptability of the frozen product. In part, textural changes associated with Gadoid species during frozen storage can be attributed to denaturation ofmyofibrillar proteins (Dyer, 1951). It is recognized that accumulation of free fatty acids (FFA) in frozen muscle tissue also contribute to textural changes by promotion of protein-lipid cross-linkages resulting in myofibriIlar denaturation (Sikorski et at., 1976). In Atlantic cod, the content of FFA in myotomal tissue is directly related to hydrolysis of the phospholipid fraction, which accounts for about 90 percent of the total lipid content (Tsukuda, 1976). Previous evidence indicates the phospholipid content of cod muscle declines rapidly under marginal frozen storage ( - 12 - - 20°C) and is accompanied by a commensurate elevation of FFA levels (Dyer and Dingle, 1961). The hydrolysis of phospholipids was shown to be specifically attributed to activity of endogenous phospholipase enzymes (Olley and Lovern, 1960; Olley et al., 1962). Close investigation has revealed in cod muscle stored at -12°C, the amount of FFA increase due to hydrolysis by endogenous phospholipase activity rises rapidly during the first 8 w of storage to about 200 mg per 100 g myotomal tissue but that the accumulation of FFA declines, thereafter (Anderson and Ravesi, 1970). To date, the impact of frozen storage on hydrolytic activity of phospholipase in gadoid species has been evaluated only from circumstantial evidence. This approach has precluded the possibility of determining if the observed trends of hydrolysis are influenced by either declining substrate availability or changes in the functional capacity of the phospholipase enzyme per se. Thus, there remains a need to directly evaluate frozen-state impacts on the hydrolytic capacity of the enzyme system. The present preliminary study was conducted to determine the influence of frozen storage
Abstract The impact of frozen storage on the detectable actIVity of endogenous phospholipase was evaluated in a microsomal fraction isolated from myotomal tissue of Atlantic cod (Gadus morhua) stored up to 12 weeks at - 30 o e. In comparison to initial levels, enhanced phospholipase activity was detected up to eight weeks of frozen storage at - 30 0 e but this was significantly reduced on further storage to 12 weeks. Under the same conditions and in comparison to 0 weeks, microsomal membrane-bound and cytosolic enzymes including 5' -nucleotidase, acid phosphatase and succinic dehydrogenase showed a linear decline of detectable activity over the same 12 week period. It is concluded that protein denaturative changes in the cellular matrix of myotomal tissue likely account for an overall decline of microsomal membrane-bound and cytosolic enzymes during frozen storage at - 30 o e, but that phospholipase showed a marked level of resilience to denaturation.
Resume L'impact de !'entreposage surgele sur I'activite decelable de la phospholipase endogene fut evalue dans une fraction microsomale isolee de tissu myotomal de la morue de I' Atlantique (Gadus morhua) entreposee jusqu'a 12 semaines a - 30°e. Par comparaison aux niveaux initiaux, l'activite accrue de la phospholipase fut decelee jusqu'a huit semaines d'entreposage surgele a - 30 0 e mais ceci a diminue significativement par la suite jusqu'a 12 semaines. Dans les memes conditions et par rapport a leur activite initiale, les enzymes cytosoliques et celles liees a la membrane microsomale dont la 5' - nucleotidase, la phosphatase acide et la dehydrogenase succinique montrerentune diminution lineaire d'activite decelable au cours de la meme periode de 12 semaines. 11 est conclu que les changements de denaturation proteique dans la matrice cellulaire du tissu myotomal sont probablemt;At responsables de la regression globale des enzymes cytosoliques et celles liees a la membrane microsomale au cours de l'entreposage surgele a - 30 o e, mais la phospholipase a montre un fort niveau de resistance a la denaturation.
Introduction It is acknowledged that myotomal tissue of fish species, particularly those of the lean-fleshed Gadoid family, is prone to problems associated with textural toughening under conditions of frozen storage (SikorI
2
Department of Food Science, University of Guelph, Guelph, Ontario, NIG 2WI Author to whom correspondence should be sent Copyright
~
1988 Canadian Institute of Food Science and Technology
399
on directly determinable phospholipase activity in microsomes isolated from myotomal tissue of Atlantic cod. Our laboratory recently determined the microsomal fraction of myotomal tissue to yield directly measurable phospholipase activity and provide a suitable model to evaluate the influence of frozen-state storage on the hydrolytic capacity of the enzyme (Ablett et al., 1986; Chawla and Ablett, 1987). The objective of the present study was to investigate the influence of frozen storage on the determinable activity of phospholipase in the myotomal tissue stored for up to 12 w at - 30°C.
Materials and Methods
Animals Live Atlantic cod (Gadus morhua) weighing between 500-1500 g were obtained from the marine facilities of the Federal Department of Fisheries and Oceans, Scotia-Fundy Region Laboratory, Halifax. All fish were maintained in 2 m diameter circular tanks with constant flowing seawater (10 L/min) and fed ad libitum with a diet consisting of chopped herring. Immediately prior to experimentation, the fish were transported live to the Canadian Institute of Fisheries Technology, Halifax, in plastic tanks containing 10 L sea water.
Isolation of Microsomes All fish were killed by a cranial blow and immediately bled by severance of the caudal vein. Subsequently, caudal flank epaxial musculature was exposed and samples removed onto ice by excision with a surgical blade. Tissue for frozen storage studies was excised from an initial pooled sample of six fish, divided into 30 g sub-samples of intact muscle, randomized and wrapped in aluminum foil and held in a cold room at - 30°C with free circulating air for periods of 0,4,8, and 12 w. Pilot studies have determined all samples to be frozen under CIFT cold room conditions in a period not exceeding 4 h. Microsomes were prepared from myotomal tissue sub-samples allowed to thaw at 23°C as previously described (Ablett et al., 1986). Protein content of microsomes was determined according to the method of Lowry et al. (1951) and the suspended microsomal fraction, as a source of phospholipase, was further used to measure the activity of the enzyme system. Freshly prepared microsomes were retained at 4°C for a maximal period of 4 d prior to experimentation.
Determination of Phospholipase Activity Phospholipase activity was determined according to the radiolytic method of Neas and Hazel (1985) as modified and described for Atlantic cod by Chawla and Ablett (1987). In this technique, the recovery of intact radiolabelled phosphatidylcholine (PC) and free fatty acids (FFA) were determined following incubation of 1,2-dipalmitoyl- 9-1O-[3H]-phosphatidylcholine (New England Nuclear, Montreal, PQ) in the presence of the microsomal suspension. The assay medium was prepared by placing 0.1 microcurie of 1,2-dipalmitoyl400 / Chawla et af.
9-1O-[3H]-phosphatidylcholine in a test tube. Organic solvents were removed under nitrogen and following addition of 0.9 mL of assay medium (0.1 M Tris - acetate, 8 mM CaCI 2 , 0.1070 Triton X-lOO; pH 8.0 at 20°C), the mixture was sonicated for 2 min in a bath sonicator. All assays were initiated by the addition of an aliquot of microsomal protein (2.0 mg) in the appropriate resuspension buffer and incubated over a period of 24 h at 20°C. Assays were terminated by the addition of 2.5 mL methanol and 1.25 mL CHCl3 and samples were stored overnight at 4°C. Subsequently, 1.25 mL and 1.25 mL H 20 were added and the samples centrifuged for 30 min at 2,500 x g (Bligh and Dyer, 1959). The lipid layer was removed and dried under nitrogen, washed twice with methanol and twice with CHCL3 and dried under nitrogen. Lipid samples were dissolved in 100 j-tL CHCl 3 :methanol (2: lv/v) spotted on silica gel TLC plates along with standards of phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and oleic acid. Thin layer plates were developed in a solvent system of CHCI/ methanol/acetic acid/Hp (25:15:4:2 v/v) and lipid spots visualized under ultraviolet light following spraying with 2,7-dichlorofluorescein (0.2 percent by weight in 90 percent ethanol). Spots corresponding to PC, LPC and fatty acid were scraped directly into liquid scintillation vials and 0.5 mL Beckman Tissue Solubilizer was added. The mixture was incubated for 4 h at 4°C, then 5 mL non-aqueous scintillation cocktail containing acetic acid (7 mL/L) was added. Counts per minute (CPM) were measured on Beckman Model LS 1800 liquid scintillation counter and converted to disintegrations per minute (DPM) using an external standard and quench calibration curve.
Microsomal Integrity Studies The integrity of the microsomal fraction was monitored by conducting a range of marker enzyme assays on the same microsomes isolated from myotomal subsamples frozen stored at - 30°C for 0,4,8 and 12 w.
5' -Nucleotidase Activity 5' -nucleotidase, as a marker for cellular membranes, was measured in the microsomal fraction isolated according to the method of Michell and Hawthorne (1965). Inorganic phosphate was measured by the method of Fiske and Subbarow (1925). Following incubation at 23°C for a period of 20 min, samples were read against phosphate standards at 830 nm. Units of activity were defined as j-tmole of phosphate liberated/mg protein/h.
Acid Phosphatase Activity Acid phosphatase, as a marker for lysosomal contamination, was determined for the microsomal fraction according to the spectrophotometric method of Trouet (1974). Denatured protein was eliminated by filtration through Whatman #42 filter paper and the inorganic phosphate in the filtrate determined according to the method of Fiske and Subbarow (1925), as J. Inst. Can. Sci. Technol. Aliment. Vol. 21, No. 4, 1988
Table I. Change in activity of phospholipase on frozen storage. Frozen storage (weeks)
Pica moles FFA released/g protein/h 2
0.46 ± 0.19 1 0.75 2 ± 0.10 4 2 ± 0.11 0.91 8 0.38 ± 0.18 12 1 Values represent the mean ± s.d. of triplicate assays conducted on a pooled sample of six fish. 2 Values significantly different (p < 0.05) to 0 week samples.
o
described above. Units of activity were defined as jtmole of phosphate liberated/mg protein/h.
Succinic Dehydrogenase Activity Succinic dehydrogenase activity, as a marker for mitochondrial contamination, was determined in microsomal fractions according to the method of Ackrell et al. (1978). In accordance with this method the change in absorbance at 600 nm was monitored for samples containing 0.1 mL microsomal protein over a period of 5 min and the activity of succinate dehydrogenase was calculated using the millimolar extinction coefficient of 19.1 at 600 nm for DCIP. Units of activity were defined as jtmoles DCIP reduced/mg protein/h.
Statistics Differences between mean values of 0 time and frozen stored samples (4,8 and 12 w) were evaluated by students "t-test" (Steel and Torrie, 1980). Mean values were considered statistically significant with p < 0.05. Correlation coefficients were also used to compare the relationship between decline of marker enzymes through the duration of frozen storage (Lapin, 1980).
Results and Discussion In comparison to 0 weeks, determinable phospholipase activity of microsomes isolated from frozen stored myotomal tissue (- 30 o q, demonstrated an enhanced hydrolytic capacity (p < 0.05) following 4 and 8 w storage (Table 1). Following 12 w storage, phospholipase hydrolytic capacity had returned to the same level of activity (p >0.05) observed at 0 weeks (Table 1). These data indicate the capacity for phospholipid hydrolysis was somehow enhanced during an
initial period up to 8 wand then decreased. The observation is in agreement with the indirect evidence of previous authors, indicating that greatest phospholipase activity and hence FFA accumulation, is observed during the initial few weeks of frozen storage (Lovern and Olley, 1962; Anderson and Ravesi, 1970; Oshima et al., 1983). As shown in Table 2, in comparison to 0 weeks, the activity of 5 -nucleotidase, acid phosphatase and succinic dehydrogenase showed a significant reduction over the same frozen storage period of 12 wat - 30°C. In all instances, almost linear decreases in the activity of the marker enzymes were detected. Negative correlations existed between the duration of frozen storage and the activity of 5 I -nucleotidase ( - 0.99), acid phosphatase (-0.91) and succinic dehydrogenase (-0.95). The results of the present preliminary study strongly suggest that during the initial period of frozen storage, the microsomal phospholipase system of Atlantic cod demonstrates an enhanced hydrolytic capacity. In possible explanation of this observation, it was acknowledged in earlier studies that hydrolysis of phospholipids may actually be promoted in the frozen state and that disruptive changes in membrane permeability might provide favourable conditions for catalytic hydrolysis (Lovern and Olley, 1962). Moreover, previous evidence suggests ice-crystal damage could result in rupture of mitochondrial (Hunter et al., 1964) and microsomal (Tappel et al., 1963) membranes which contain phospholipase enzyme complexes. In turn, liberation of membrane-bound phospholipase, might favour enhanced phospholipid hydrolysis through promotion of enzyme:substrate contact. More recently, it was concluded that dehydration, as a consequence of ice formation due to freezing, also played an important role in the acceleration of PC decomposition in frozen carp muscle (Hanaoka and Toyomizu, 1979). In this present preliminary study the extent to which these physicochemical events may underlie the observed enhancement of phospholipase activity could not be determined. Nevertheless, the decline of marker enzyme activity in microsomes following frozen storage of myotomal tissue for 12 w, substantiates the likelihood that protein denaturative changes did indeed occur during frozen-state storage. Thus, it is likely the functional integrity of these membrane-bound and cytosolic enzymes was reduced during frozen storage by physical freeze-mediated changes in the subcelluI
Table 2. Change in activity of three marker enzymes (5' nucleotidase, acid phosphatase, and succinic dehydrogenase) on frozen storage. Marker Enzyme Activityl Frozen Storage (Weeks)
o 4 8 12
5' Nucleotidase (J.lmoles Pi/mg protein/h) 0.60 0.48 2 0.342 0.24 2
± 0.06 ± 0.01 ± 0.07 ± 0.01
Acid phosphatase (J.lmoles Pi/mg protein/h) 0.10 0.04 2 0.03 2 0.03 2
± 0.01 ± 0.01 ± 0.00 ± 0.01
Succinic dehydrogenase (J.lmoles DCIP reduced/mg/h) 294.69 285.18 276.15 2 268.202
± 3.01 ± 4.19 ± 3.88 ± 6.72
IYalues represent the mean ± s.d. of triplicate assays conducted on a pooled sample of six fish. 2Yalues in columns were significantly different (p < 0.05) to 0 week samples. Can. Inst. Food Sci. Technol. J. Vo!. 21, No. 4, 1988
Chawla et al. / 401
lar matrix of myotomal tissue. Moreover, the observations recorded for 5' - nucleotidase, a specific membrane marker, likely reflects the microenvironment of the phospholipase enzyme. How far these observed changes are reflective of endogenous phospholipase activity is not known, but they might account for a decline in the capacity of the enzyme to hydrolyse substrate after 8 w of frozen storage at - 30°C. The paradoxical observation of enhanced phospholipase activity up to 8 w storage in comparison to initial activity values may eventually prove of significance to the problem of frozen storage induced myotomal toughening, in that the results may indicate a resilience of this enzyme to denaturation under frozen-state conditions. Previous studies have determined the presence of up to 7 disulphide linkages in mammalian phospholipase and these bonds likely serve an important role in maintaining the physical integrity of the enzyme (Dennis, 1973). To date, no studies have evaluated the physical structure of phospholipase in Gadoids but it may be speculated that the enzyme demonstrates similar disulphide bonding characteristics. In summary, the results of the present study provide the first direct evaluation of frozen storage impact on determinable phospholipase activity in Atlantic cod myotomal tissue. The observation of enhanced phospholipase capacity during the first 8 w of frozen storage, strongly indicates the physico-chemical environment of frozen-state conditions somehow facilitates the catalytic capacity of the enzyme. Subsequently, and in parallel with other membraneintegral and cytosolic enzyme systems, the hydrolytic capacity apparently declined. Protein denaturation and hence a decreased overall functional integrity of the enzyme systems themselves might underlie this observation. Future studies will closely examine the response of the phospholipase enzyme system under different frozen storage regimes. Particular attention will be focused on structural membrane integrity changes occurring in the microsomal model under defined frozen-state conditions and the localized accumulation of FFA in the associated microenvironment.
References
Anderson, M.L. and Ravesi, E.M. 1970. On the nature of the association of protein in frozen-stored cod muscle. J. Food Sci. 35:551. Bligh, E.G. and Dyer, W.J. 1959. A rapid method of total lipid extraction and purification. Can. 1. Biochem. Physiol. 37:911. Chawla, P. and Ablett, R.F. 1987. Detection of microsomal phospholipase activity in myotomal tissue of Atlantic cod (Gadus morhua). J. Food Sci. 52:1194. Dennis, E.A. 1973. Kinetic dependence of phospholipase A2 activity on the detergent Triton X-lOO. J.Lipid Res. 14: 152. Dyer, W.1. 1951. Protein denaturation in frozen and stored fish. Food Res. 16:522. Dyer, W.J. and Dingle, J.R. 1961. Fish proteins with special reference to freezing. In: Fish as Food. G. Borgstrom (Ed.). Academic Press, New York, NY. Fiske, C.H. and Subbarow, Y. 1925. The colorimetric determination of phosphorus. 1. BioI. Chem. 66:375. Hanaoka, K. and Toyomizu, M. 1979. Acceleration of phospholipid decomposition in fish muscle by freezing. Bull. Jap. Soc. Sci. Fish. 45:465. Hunter, E.P., Scott, A., Hoffsten, P.E., Gebicki, J.M., Weinstein, J., and Schneider, A. 1964. Studies on the mechanism of swelling, lysis and disintegration of isolated liver mitochondria exposed to mixture of oxidized and reduced glutathione. J. BioI. Chem. 239:614. Lapin, L.L. 1980. Statistics - Meaning and method. 2nd ed. Harcourt Brace Jovanovich Inc., New York, NY. Lovern, J .A. and alley, J. 1962. Inhibition and promotion of postmortem lipid hydrolysis in the flesh of fish. J. Food Sci. 27:551. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. 1951. Protein measurement with Folin-phenol reagent. J. BioI. Chem. 193:265. Michell, R.H. and Hawthorne, J.N. 1965. The site of diphosphoinositide synthesis in rat liver. Biochem. Biophy. Res. Comm.21:333. Neas, N.P. and Hazel, 1.R. 1985. Phospholipase A2 from liver microsomal membrane of thermally acclimated rainbow trout. 1. Exp. Zool. 233:51. Oshima, T., Wada, S. and Koizumi, C. 1983. Enzymatic hydrolysis of phospholipids in cod flesh during cold storage. Bull. Jap. Soc. Sci. Fish. 49:1397. alley, 1. and Lovern, J .A. 1960. Phospholipid hydrolysis in cod flesh stored at various temperatures. J. Sci. Food Agric. 11:644. alley, J., Pirie, R. and Watson, H. 1962. Lipase and phospholipase activity in fish skeletal muscle and its relationship to protein denaturation. 1. Sci. Food Agric. 13:501. Steel, R.G.D. and Torrie, J .H. (1980). Principles and Procedures of Statistics. McGraw-Hill Book Co. Inc., New York, NY. Sikorski, Z., alley, J., Kostuch, S. 1976. Protein changes in frozen fish. Crit. Rev. Food Sci. and Nutr. 8:97. Tappel, A.L., Sawant, P.L. and Shibko, S. 1963. In: The Lysosomes. A.V.S. De Reuck and M.P. Cameron (Eds). J.A. Churchill Ltd., London. Trouet, A. 1974. Isolation of modified liver lysosomes. In: Methods in Enzymology. Vol. 31. S. F1eisher and L. Packer (Eds.). Academic Press, New York, NY. Tsukuda, N. 1976). Changes in the lipids of frozen fish. 11. Changes in the lipids of chum salmon and cod muscle during frozen storage. Bull Tokai. Reg. Fish. Res. Lab. 87: I.
Ablett, R.F., Chawla, P., and Gould, S.P. 1986. Evaluation of microsomes isolated from fresh and frozen myotomal tissue of Atantic cod (Gadus morhua). Comp. Biochem, Physiol. 84B:457. Ackrell, B.A.C., Kearney, F.B., and Singer, T.P. 1978. Mammalian succinate dehydrogenase. In: Methods in Enzymology. Vol. 53. S. F1eisher and L. Packer (Eds.). Academic Press, New York, NY.
Submitted October 5, 1987 Revised February 29, 1988 Accepted March 31, 1988
Acknowledgements This work was supported by an Operating grant (R.F.A.) made available from Natural Sciences and Engineering Research Council, Canada. The authors wish to thank Ms. Denise Berry for her assistance in preparation of this manuscript.
402 / Chawla et al.
J. Inst. Can. Sci. Technol. Aliment. Vol. 21, No. 4, 1988
RESEARCH
Influence of Frozen Storage on Microsomal Phospholipase Activity in Myotomal Tissue of Atlantic Cod (Gadus morhua) P. Chawla " B. MacKeigan, S.P. Gould and R.F. Ablete Department of Food Science and Technology Technical University of Nova Scotia P.O. Box 1000 Halifax, Nova Scotia B3J 2X4
ski et al., 1976). These changes are significant among commercially important species including Atlantic cod (Gadus morhua) for they determine both the storage life and overall acceptability of the frozen product. In part, textural changes associated with Gadoid species during frozen storage can be attributed to denaturation ofmyofibrillar proteins (Dyer, 1951). It is recognized that accumulation of free fatty acids (FFA) in frozen muscle tissue also contribute to textural changes by promotion of protein-lipid cross-linkages resulting in myofibriIlar denaturation (Sikorski et at., 1976). In Atlantic cod, the content of FFA in myotomal tissue is directly related to hydrolysis of the phospholipid fraction, which accounts for about 90 percent of the total lipid content (Tsukuda, 1976). Previous evidence indicates the phospholipid content of cod muscle declines rapidly under marginal frozen storage ( - 12 - - 20°C) and is accompanied by a commensurate elevation of FFA levels (Dyer and Dingle, 1961). The hydrolysis of phospholipids was shown to be specifically attributed to activity of endogenous phospholipase enzymes (Olley and Lovern, 1960; Olley et al., 1962). Close investigation has revealed in cod muscle stored at -12°C, the amount of FFA increase due to hydrolysis by endogenous phospholipase activity rises rapidly during the first 8 w of storage to about 200 mg per 100 g myotomal tissue but that the accumulation of FFA declines, thereafter (Anderson and Ravesi, 1970). To date, the impact of frozen storage on hydrolytic activity of phospholipase in gadoid species has been evaluated only from circumstantial evidence. This approach has precluded the possibility of determining if the observed trends of hydrolysis are influenced by either declining substrate availability or changes in the functional capacity of the phospholipase enzyme per se. Thus, there remains a need to directly evaluate frozen-state impacts on the hydrolytic capacity of the enzyme system. The present preliminary study was conducted to determine the influence of frozen storage
Abstract The impact of frozen storage on the detectable actIVity of endogenous phospholipase was evaluated in a microsomal fraction isolated from myotomal tissue of Atlantic cod (Gadus morhua) stored up to 12 weeks at - 30 o e. In comparison to initial levels, enhanced phospholipase activity was detected up to eight weeks of frozen storage at - 30 0 e but this was significantly reduced on further storage to 12 weeks. Under the same conditions and in comparison to 0 weeks, microsomal membrane-bound and cytosolic enzymes including 5' -nucleotidase, acid phosphatase and succinic dehydrogenase showed a linear decline of detectable activity over the same 12 week period. It is concluded that protein denaturative changes in the cellular matrix of myotomal tissue likely account for an overall decline of microsomal membrane-bound and cytosolic enzymes during frozen storage at - 30 o e, but that phospholipase showed a marked level of resilience to denaturation.
Resume L'impact de !'entreposage surgele sur I'activite decelable de la phospholipase endogene fut evalue dans une fraction microsomale isolee de tissu myotomal de la morue de I' Atlantique (Gadus morhua) entreposee jusqu'a 12 semaines a - 30°e. Par comparaison aux niveaux initiaux, l'activite accrue de la phospholipase fut decelee jusqu'a huit semaines d'entreposage surgele a - 30 0 e mais ceci a diminue significativement par la suite jusqu'a 12 semaines. Dans les memes conditions et par rapport a leur activite initiale, les enzymes cytosoliques et celles liees a la membrane microsomale dont la 5' - nucleotidase, la phosphatase acide et la dehydrogenase succinique montrerentune diminution lineaire d'activite decelable au cours de la meme periode de 12 semaines. 11 est conclu que les changements de denaturation proteique dans la matrice cellulaire du tissu myotomal sont probablemt;At responsables de la regression globale des enzymes cytosoliques et celles liees a la membrane microsomale au cours de l'entreposage surgele a - 30 o e, mais la phospholipase a montre un fort niveau de resistance a la denaturation.
Introduction It is acknowledged that myotomal tissue of fish species, particularly those of the lean-fleshed Gadoid family, is prone to problems associated with textural toughening under conditions of frozen storage (SikorI
2
Department of Food Science, University of Guelph, Guelph, Ontario, NIG 2WI Author to whom correspondence should be sent Copyright
~
1988 Canadian Institute of Food Science and Technology
399
on directly determinable phospholipase activity in microsomes isolated from myotomal tissue of Atlantic cod. Our laboratory recently determined the microsomal fraction of myotomal tissue to yield directly measurable phospholipase activity and provide a suitable model to evaluate the influence of frozen-state storage on the hydrolytic capacity of the enzyme (Ablett et al., 1986; Chawla and Ablett, 1987). The objective of the present study was to investigate the influence of frozen storage on the determinable activity of phospholipase in the myotomal tissue stored for up to 12 w at - 30°C.
Materials and Methods
Animals Live Atlantic cod (Gadus morhua) weighing between 500-1500 g were obtained from the marine facilities of the Federal Department of Fisheries and Oceans, Scotia-Fundy Region Laboratory, Halifax. All fish were maintained in 2 m diameter circular tanks with constant flowing seawater (10 L/min) and fed ad libitum with a diet consisting of chopped herring. Immediately prior to experimentation, the fish were transported live to the Canadian Institute of Fisheries Technology, Halifax, in plastic tanks containing 10 L sea water.
Isolation of Microsomes All fish were killed by a cranial blow and immediately bled by severance of the caudal vein. Subsequently, caudal flank epaxial musculature was exposed and samples removed onto ice by excision with a surgical blade. Tissue for frozen storage studies was excised from an initial pooled sample of six fish, divided into 30 g sub-samples of intact muscle, randomized and wrapped in aluminum foil and held in a cold room at - 30°C with free circulating air for periods of 0,4,8, and 12 w. Pilot studies have determined all samples to be frozen under CIFT cold room conditions in a period not exceeding 4 h. Microsomes were prepared from myotomal tissue sub-samples allowed to thaw at 23°C as previously described (Ablett et al., 1986). Protein content of microsomes was determined according to the method of Lowry et al. (1951) and the suspended microsomal fraction, as a source of phospholipase, was further used to measure the activity of the enzyme system. Freshly prepared microsomes were retained at 4°C for a maximal period of 4 d prior to experimentation.
Determination of Phospholipase Activity Phospholipase activity was determined according to the radiolytic method of Neas and Hazel (1985) as modified and described for Atlantic cod by Chawla and Ablett (1987). In this technique, the recovery of intact radiolabelled phosphatidylcholine (PC) and free fatty acids (FFA) were determined following incubation of 1,2-dipalmitoyl- 9-1O-[3H]-phosphatidylcholine (New England Nuclear, Montreal, PQ) in the presence of the microsomal suspension. The assay medium was prepared by placing 0.1 microcurie of 1,2-dipalmitoyl400 / Chawla et af.
9-1O-[3H]-phosphatidylcholine in a test tube. Organic solvents were removed under nitrogen and following addition of 0.9 mL of assay medium (0.1 M Tris - acetate, 8 mM CaCI 2 , 0.1070 Triton X-lOO; pH 8.0 at 20°C), the mixture was sonicated for 2 min in a bath sonicator. All assays were initiated by the addition of an aliquot of microsomal protein (2.0 mg) in the appropriate resuspension buffer and incubated over a period of 24 h at 20°C. Assays were terminated by the addition of 2.5 mL methanol and 1.25 mL CHCl3 and samples were stored overnight at 4°C. Subsequently, 1.25 mL and 1.25 mL H 20 were added and the samples centrifuged for 30 min at 2,500 x g (Bligh and Dyer, 1959). The lipid layer was removed and dried under nitrogen, washed twice with methanol and twice with CHCL3 and dried under nitrogen. Lipid samples were dissolved in 100 j-tL CHCl 3 :methanol (2: lv/v) spotted on silica gel TLC plates along with standards of phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and oleic acid. Thin layer plates were developed in a solvent system of CHCI/ methanol/acetic acid/Hp (25:15:4:2 v/v) and lipid spots visualized under ultraviolet light following spraying with 2,7-dichlorofluorescein (0.2 percent by weight in 90 percent ethanol). Spots corresponding to PC, LPC and fatty acid were scraped directly into liquid scintillation vials and 0.5 mL Beckman Tissue Solubilizer was added. The mixture was incubated for 4 h at 4°C, then 5 mL non-aqueous scintillation cocktail containing acetic acid (7 mL/L) was added. Counts per minute (CPM) were measured on Beckman Model LS 1800 liquid scintillation counter and converted to disintegrations per minute (DPM) using an external standard and quench calibration curve.
Microsomal Integrity Studies The integrity of the microsomal fraction was monitored by conducting a range of marker enzyme assays on the same microsomes isolated from myotomal subsamples frozen stored at - 30°C for 0,4,8 and 12 w.
5' -Nucleotidase Activity 5' -nucleotidase, as a marker for cellular membranes, was measured in the microsomal fraction isolated according to the method of Michell and Hawthorne (1965). Inorganic phosphate was measured by the method of Fiske and Subbarow (1925). Following incubation at 23°C for a period of 20 min, samples were read against phosphate standards at 830 nm. Units of activity were defined as j-tmole of phosphate liberated/mg protein/h.
Acid Phosphatase Activity Acid phosphatase, as a marker for lysosomal contamination, was determined for the microsomal fraction according to the spectrophotometric method of Trouet (1974). Denatured protein was eliminated by filtration through Whatman #42 filter paper and the inorganic phosphate in the filtrate determined according to the method of Fiske and Subbarow (1925), as J. Inst. Can. Sci. Technol. Aliment. Vol. 21, No. 4, 1988
Table I. Change in activity of phospholipase on frozen storage. Frozen storage (weeks)
Pica moles FFA released/g protein/h 2
0.46 ± 0.19 1 0.75 2 ± 0.10 4 2 ± 0.11 0.91 8 0.38 ± 0.18 12 1 Values represent the mean ± s.d. of triplicate assays conducted on a pooled sample of six fish. 2 Values significantly different (p < 0.05) to 0 week samples.
o
described above. Units of activity were defined as jtmole of phosphate liberated/mg protein/h.
Succinic Dehydrogenase Activity Succinic dehydrogenase activity, as a marker for mitochondrial contamination, was determined in microsomal fractions according to the method of Ackrell et al. (1978). In accordance with this method the change in absorbance at 600 nm was monitored for samples containing 0.1 mL microsomal protein over a period of 5 min and the activity of succinate dehydrogenase was calculated using the millimolar extinction coefficient of 19.1 at 600 nm for DCIP. Units of activity were defined as jtmoles DCIP reduced/mg protein/h.
Statistics Differences between mean values of 0 time and frozen stored samples (4,8 and 12 w) were evaluated by students "t-test" (Steel and Torrie, 1980). Mean values were considered statistically significant with p < 0.05. Correlation coefficients were also used to compare the relationship between decline of marker enzymes through the duration of frozen storage (Lapin, 1980).
Results and Discussion In comparison to 0 weeks, determinable phospholipase activity of microsomes isolated from frozen stored myotomal tissue (- 30 o q, demonstrated an enhanced hydrolytic capacity (p < 0.05) following 4 and 8 w storage (Table 1). Following 12 w storage, phospholipase hydrolytic capacity had returned to the same level of activity (p >0.05) observed at 0 weeks (Table 1). These data indicate the capacity for phospholipid hydrolysis was somehow enhanced during an
initial period up to 8 wand then decreased. The observation is in agreement with the indirect evidence of previous authors, indicating that greatest phospholipase activity and hence FFA accumulation, is observed during the initial few weeks of frozen storage (Lovern and Olley, 1962; Anderson and Ravesi, 1970; Oshima et al., 1983). As shown in Table 2, in comparison to 0 weeks, the activity of 5 -nucleotidase, acid phosphatase and succinic dehydrogenase showed a significant reduction over the same frozen storage period of 12 wat - 30°C. In all instances, almost linear decreases in the activity of the marker enzymes were detected. Negative correlations existed between the duration of frozen storage and the activity of 5 I -nucleotidase ( - 0.99), acid phosphatase (-0.91) and succinic dehydrogenase (-0.95). The results of the present preliminary study strongly suggest that during the initial period of frozen storage, the microsomal phospholipase system of Atlantic cod demonstrates an enhanced hydrolytic capacity. In possible explanation of this observation, it was acknowledged in earlier studies that hydrolysis of phospholipids may actually be promoted in the frozen state and that disruptive changes in membrane permeability might provide favourable conditions for catalytic hydrolysis (Lovern and Olley, 1962). Moreover, previous evidence suggests ice-crystal damage could result in rupture of mitochondrial (Hunter et al., 1964) and microsomal (Tappel et al., 1963) membranes which contain phospholipase enzyme complexes. In turn, liberation of membrane-bound phospholipase, might favour enhanced phospholipid hydrolysis through promotion of enzyme:substrate contact. More recently, it was concluded that dehydration, as a consequence of ice formation due to freezing, also played an important role in the acceleration of PC decomposition in frozen carp muscle (Hanaoka and Toyomizu, 1979). In this present preliminary study the extent to which these physicochemical events may underlie the observed enhancement of phospholipase activity could not be determined. Nevertheless, the decline of marker enzyme activity in microsomes following frozen storage of myotomal tissue for 12 w, substantiates the likelihood that protein denaturative changes did indeed occur during frozen-state storage. Thus, it is likely the functional integrity of these membrane-bound and cytosolic enzymes was reduced during frozen storage by physical freeze-mediated changes in the subcelluI
Table 2. Change in activity of three marker enzymes (5' nucleotidase, acid phosphatase, and succinic dehydrogenase) on frozen storage. Marker Enzyme Activityl Frozen Storage (Weeks)
o 4 8 12
5' Nucleotidase (J.lmoles Pi/mg protein/h) 0.60 0.48 2 0.342 0.24 2
± 0.06 ± 0.01 ± 0.07 ± 0.01
Acid phosphatase (J.lmoles Pi/mg protein/h) 0.10 0.04 2 0.03 2 0.03 2
± 0.01 ± 0.01 ± 0.00 ± 0.01
Succinic dehydrogenase (J.lmoles DCIP reduced/mg/h) 294.69 285.18 276.15 2 268.202
± 3.01 ± 4.19 ± 3.88 ± 6.72
IYalues represent the mean ± s.d. of triplicate assays conducted on a pooled sample of six fish. 2Yalues in columns were significantly different (p < 0.05) to 0 week samples. Can. Inst. Food Sci. Technol. J. Vo!. 21, No. 4, 1988
Chawla et al. / 401
lar matrix of myotomal tissue. Moreover, the observations recorded for 5' - nucleotidase, a specific membrane marker, likely reflects the microenvironment of the phospholipase enzyme. How far these observed changes are reflective of endogenous phospholipase activity is not known, but they might account for a decline in the capacity of the enzyme to hydrolyse substrate after 8 w of frozen storage at - 30°C. The paradoxical observation of enhanced phospholipase activity up to 8 w storage in comparison to initial activity values may eventually prove of significance to the problem of frozen storage induced myotomal toughening, in that the results may indicate a resilience of this enzyme to denaturation under frozen-state conditions. Previous studies have determined the presence of up to 7 disulphide linkages in mammalian phospholipase and these bonds likely serve an important role in maintaining the physical integrity of the enzyme (Dennis, 1973). To date, no studies have evaluated the physical structure of phospholipase in Gadoids but it may be speculated that the enzyme demonstrates similar disulphide bonding characteristics. In summary, the results of the present study provide the first direct evaluation of frozen storage impact on determinable phospholipase activity in Atlantic cod myotomal tissue. The observation of enhanced phospholipase capacity during the first 8 w of frozen storage, strongly indicates the physico-chemical environment of frozen-state conditions somehow facilitates the catalytic capacity of the enzyme. Subsequently, and in parallel with other membraneintegral and cytosolic enzyme systems, the hydrolytic capacity apparently declined. Protein denaturation and hence a decreased overall functional integrity of the enzyme systems themselves might underlie this observation. Future studies will closely examine the response of the phospholipase enzyme system under different frozen storage regimes. Particular attention will be focused on structural membrane integrity changes occurring in the microsomal model under defined frozen-state conditions and the localized accumulation of FFA in the associated microenvironment.
References
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Submitted October 5, 1987 Revised February 29, 1988 Accepted March 31, 1988
Acknowledgements This work was supported by an Operating grant (R.F.A.) made available from Natural Sciences and Engineering Research Council, Canada. The authors wish to thank Ms. Denise Berry for her assistance in preparation of this manuscript.
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