- Email: [email protected]
Myofibrils of cooked meat are a continuum of gap filaments
,14eatSo¢nc¢ 7 (1982) 189-196
MYOFIBRILS OF COOKED MEAT ARE A C O N T I N U U M OF GAP FILAMENTS
R. H. LOCKER & D. J. C. WILD Meat Industry Research Institute o f New Zealand (Inc.). PO Box 617, Hamilton. New Zealand
(Received: 15 December, 1981)
SUMMARY
When cooked meat is subjected to high degrees of stretch it becomes apparent in high magnification electron micrographs that A-flaments hat'e ceased to exist. The Aband isflled with a coaguhtm of actomyosin. Fragmentation of this coagulum during stretch rereals an array of fine filaments (identified as gap flaments). This result is obtained irrespectire of rigor temperature, state of contraction or degree of cooking. if the meat isf r s t aged, the gap filaments surt'it'ing in the l-band are too weak to open up the A-band. The results show that myofibrils in cooked meat are entirely dependent on heat-stable gapflaments for structural continui O"and tensile strength. Theories of nwat temh'rness must be ret'ised accordhtgly.
INTRODUCTION
Recently we have paid much attention to the tensile and histological properties of muscle in both the raw and the cooked state, and to the way in which these properties may be modified by such factors as rigor temperature, state of contraction and ageing. While raw rigor muscle undergoes the marked and irreversible changes of'yielding" during extension (Locker & Wild, 1982a), cooked muscle appears superficially to behave in an elastic manner. It stretches more readily, to break cleanly at about 90 % extension, and then recovers almost to its original length (Locker et al., 1982). Both the heat-modified myofibrils and the collagen fibres, rendered elastic by denaturation, contribute substantially to this behaviour. Cooked muscle is, however, not truly elastic. There is a sudden irreversible change in extensibility near I kg/cm 2, attributable to the myofibrillar component (Locker et al., 1982), there is a degree of "creep' if loading is slow and, 189 Meat Science 0309-1740/82,000%0189; $02-75 ~, Applied Science Publishers Ltd, England, 1982 Printed in Great Britain
190
R. H. LOCKER, D. J. C. WILD
although most of the recovery occurs instantly at breakage, shortening takes some minutes for completion. Examination by light or electron microscopy of cooked muscle which has recovered after loading to breakage showed no obvious damage to the overall integrity of the myofibrils. However, close examination of high power micrographs of such broken strips, restretched before fixation, showed that the A-filaments had ceased to exist as such and were replaced by fine filaments embedded in fragments of coagulum. The effect was equally visible in cold shortened muscle. Brief reference has already been made to this result (Locker et al., 1982), which is here reported in detail.
MATERIALS AND METHODS
Ox sternomandibularis muscles were allowed to go into rigor mortis under various temperature regimes, when strips were cut and cooked under restraint as previously described (Locker et al., 1982). Some strips were stretched mechanically to i 5, 30, 50 and 75 % extension at 10 % a minute using our 'yieldmeter" (Locker & Wild, 1982b). Thin strips, l - 2 m m in diameter, were then cut from these and stretched carefully by hand back to the same degrees of extension, before taping to rods for fixation. Other strips were loaded by hand until they broke and were then allowed to recover at 2°C. Thin strips were again cut from these as above, and restretched by 50-70 % for fixation. Samples were fixed in 2.5 % glutaraldehyde in 0. I Mcacodylate buffer pH 7-1, then in 1% osmium tetroxide in the same buffer. They were then embedded in Spurr's resin. Sections were stained with uranyl acetate-lead aspartate.
RESULTS
Stretching muscle cooked in rigor at excised length Histological changes were studied in cooked muscle strips, stretched to various degrees. After going into rigor unrestrained at 15 °C, 2 °C, 37 °C and at 2 °C with the final stages at 37°C (2 ° + 37°C rigor, Locker et al., 1982), the strips were cooked under restraint for 40min at 80°C. Strips cut from muscle set in rigor at 15 °C were subjected after cooking to gradual mechanical extension to various degrees, and fixed at that degree of extension. The unstretched muscle (Fig. la) had the typical appearance of cooked muscle: an amorphous A-band with sparse filaments in a short I-band, somewhat obscured by coagulated material. On stretching by 15% or 30% (Figs 1 b and c), extension occurred almost entirely in the I-band, with the filaments and a double N-line becoming clearer. The A-band appeared unchanged. At 50% stretch distinct
Fig. I. Sections of ox sternomandibularis muscle, allowed to go into rigor unrestrained at 15 °C, cut into strips, cooked under restraint for 40 rain at 80°C (unless otherwise stated) and stretched to various degrees, etc. (all × 21,000). (a) Unstretched. (b) Stretched mechanically at I0~/~ a minute to 15"o extension. (c) As m (b), to 3 0 ~ extension. (d) As in (b), to 5 0 ~ extension. (e) Loaded by hand to breaking point and allowed to recover. (f) Aged I day at 15 ~C before cooking. Loaded to breaking point, recovered and re-stretched by 50 ~o- (g) As in (f) but aged 7 days at 2 °C. (h) Unaged rigor muscle cooked 3 h at 100°C. Loaded to breaking point, recovered and re-stretched by 5000 .
Fig. 2a. A 'relaxed" myofibril from muscle allowed to go into rigor unrestrained for 7 h at 37 °C, cooked for 40 rain at 80°C, loaded to breaking point, allowed to recover and re-stretched by 60 % ( × 62,000).
Fig. 2b. A myofibril showing'contraction bands" from muscle which was cold shortened by holding for 24 h at 2 °C, but in which rigor was completed in 3 h at 37 °C ('2 ° + 37 °C rigor', Locker et al., 1982). Then treated in the same way as in Fig. 2a ( x 61,000).
MYOFIBRILS OF COOKED MEAT ARE A CONTINUUM OF GAP FILAMENTS
193
changes were evident in the A-band, which had stretched markedly and assumed a speckled appearance (Fig. ld). On close inspection this appears to be due to fragments ofcoagulum superimposed on an underlying array of fine filaments, seen most clearly in the pseudo-H-zone. These changes in the A-band were more marked when strips were loaded by hand until they broke, allowed to recover, and smaller strips CUt from them were stretched back by 50-70 0,~ for fixation (Fig. 2a). The array of fine filaments through the broken coagulum was here quite distinct. The coagulum in the centre of the A-band did not fragment, although fine filaments were also discernible here. In places the coagulum appears to be arranged in illdefined rows with a period of just over 200 nm. When strips were loaded until they broke, they recovered to near the original dimensions and appearance (Fig. le) although the N-line was clearer. The changes in the A-band on stretching were seen in 'relaxed' sarcomeres whatever the rigor temperature. In muscle which had gone into rigor at 2 °, 2 ° + 37 ° or 37 °C many fibres showed "contraction bands', i.e. had sarcomeres of 1-5 lam or less. These responded to stretch in much the same way but, in the absence of an lband, the gap-filaments were continuous across the sarcomere, and again were embedded in fragmented coagulum (Fig. 2b). This effect was the same whether the contraction bands were induced by cold shortening at 2 °C or heat shortening at 37°C. Results were similar if the strip was subjected to severe cooking (3 h at 100°C). When loaded to breaking point and stretched back by 50'~.,, structure was surprisingly well preserved (Fig. lh). Such strips recovered in the same way (Fig. lc) as those cooked 40min at 80°C (Fig. le). Ageing of strips prior to normal cooking, followed by loading to breaking point and restretching by 50 ° o as above, produced different results. In strips aged I day at 15°C, response varied in individual fibres, some closely resembling unaged moderately stretched fibres as in Figs lb and lc, while others had clearly suffered profound change in those filaments of the l-band which had survived cooking (Fig. I f). These stretched while the A-band did not, but there was a high proportion of broken filaments. In places, short filaments, thicker than the intact ones, lie against the edge of the A-band (see arrow). These appear to be the result of springback after breakage. In a less aged sample (7 days at 2°C) the preservation of filaments was much better (Fig. lg). Here they are remarkably clean, and free of coagulated or N-line material, but notably sparser than normal I-filaments.
DISCUSSION
There is already some evidence for disintegration of A-filaments on cooking. Schmitt & Parrish ( 1971) examined samples of beef muscle heated briefly to various internal temperatures. The A-filaments were quite intact after heating to 50°C,
194
R. H. LOCKER, D. J. C. WILD
while after reaching 60°C they remained visible in longitudinal, but not in transverse, section. They were no longer visible in muscle heated to 70 ° or 80 °C. In earlier work on ox sternomandibularis allowed to enter rigor at 15 °C and given a normal cook of 40 min at 80 °C, the A-band appeared featureless except for a somewhat speckled appearance (Locker et al., 1977). Stretching after cooking accentuated the speckling, with a suggestion of fine filamentous substructure (Locker et al., 1977; Figs 1,2). The micrographs presented here reveal more convincingly than previously the disappearance of A-filaments in favour of fine filaments. When muscle, cooked after rigor at 15 °C, is stretched by up to 30 ~ , extension of the sarcomere is confined almost entirely to the l-band, but by 500/o stretch the A-band has extended considerably, with a profound change in its fine structure. We interpret this change in the following way. The A-filaments depolymerise on heating along with the actin filaments, leading to the formation of a stoichiometric actomyosin gel, packed tightly around the surviving A-filament cores, which we have earlier claimed to be comprised of gap filaments (Locker & Leet, 1976). As stretch progresses the extending filaments are exposed as the gel begins to fragment. On release of tension, the elastic gap filaments close up the coagulum again, and the sarcomere reverts to its original appearance. The failure of the coagulum in the central region of the A-band to fragment is due to the overlap of the opposing actin filaments there, resulting in a denser gel. It has already been pointed out that the actin filaments in the l-band disintegrate early in cooking, leaving a sparser array of gap filaments (Locker et al., 1977). These are only half as numerous as the A-filaments (see the model of gap-filament connections in Locker &Leet, 1976). The I-band therefore is the more extensible, and the point of failure under tensile stress. Thus, within the sarcomere, structural continuity is entirely dependent on gap filaments although, strictly, they do not form a true continuum but terminate in the A-bands, when they rely on overlap to maintain continuity. The surrounding actomyosin gel and the apparent survival of the M-line suffice to prevent slippage of the filaments over each other. In contracted sarcomeres the weaker I-band link is missing and the structure amounts to a true continuum of gap filaments within the sarcomere. Previous attempts to explain the toughening induced by cold shortening have involved a continuum of thick filaments, fused end to end by cooking (Voyle, 1969: Marsh & Carse, 1974). This theory must now be restated more accurately in terms of a continuum of gap filaments. Since the gap filaments are twice as abundant as in the I-band, and are further reinforced by a tight actomyosin gel, a tensile strength of more than twice that of unshortened muscle may be expected, even allowing a little for lateral expansion of the lattice on contraction. The gap filaments themselves may well be thicker and stronger when cooked in a completely unstretched state, thus further enhancing toughness. On the basis of our model for gap filament connections half the gap filaments
MYOFIBRILS OF COOKED MEAT ARE A CONTINUUM OF GAP FILAMENTS
195
would be continuous through any Z-line to the Z-lines on either side. The other half would overlap by the same amount as the opposing A-filaments did after penetration of the Z-line. It should therefore be a stronger continuum than the notational A-filament continuum, based on a degree of overlap which may amount to little more than end-to-end fusion. It is surprising that the rigorous cooking at 100 °C has no effect on the results. The resolution of the micrographs equals those from standard cooking. Recovery is the same as before. This emphasises the great resistance of gap filaments to heat, already noted (Locker et al., 1977). The fact that shear force in the muscle maintains a steady state between 24 h and 100 h cooking at 80 °C (Davey et al., 1976) is an even more dramatic manifestation of this stability. The results of ageing reflect the vulnerability of gap filaments in the I-band to proteolytic attack (Davey & Graafhuis, 1976; Locker et al., 1977). The micrographs show a great variation in the capacity of individual fibres to age. I n those that have aged moderately the filaments survive cooking and stretching but they break if ageing is more severe. In either case they have insufficient strength to extend the Aband, merely stretching or breaking themselves. This is in contrast to the raw state where they fracture cleanly after ageing (along with actin filaments) without extension (Locker & Wild, 1982a). The clean, well preserved filaments in the moderately aged fibre of Fig. ! g suggest from their sparseness that they must be gap filaments, as previously claimed (Locker et al., 1977). It is hard to imagine a minority of actin filaments surviving intact while the rest disappear without trace. Within the A-band the gap filaments are immune to catheptic attack because of their r61e as thick filament cores, where they are completely shielded by a sheath of close-packed and resistant myosin 'tails'. In sarcomeres shortened to 1.5 pm there is no I-band and protection of gap filaments is complete. This accounts for the observed failure of cold shortened meat to age (Davey et al., 1967). The break up of the actomyosin coagulum might offer some explanation for the sudden increases in extensibility as cooked muscle is extended (Locker et al., 1982). On stretching some muscles which have gone into rigor at 15 °C and been cooked under restraint there is a single change in extensibility at about 1.0 kg/cm 2 and 10 ~o extension, while, in other cases, two change points are discernible. While break up of the gel only becomes~clearly visible at about 50 ~o stretch, it is quite possible that the process begins much earlier, even at 1 0 ~ stretch. However, if this were the case, appreciable extension of the A-band might have been expected earlier than 30 ~o stretch of the strip. Since it does not seem possible to explain two changes in extensibility on this basis, the situation is probably more complex. In conclusion, this paper has shown that in the A-band, the survivors of cooking are not A-filaments but a set of much thinner filaments with a remarkable stability to heat. This situation applies whatever the rigor temperature or final state of contraction, although the filaments are clearly weakened by ageing. Evidence is accumulating that gap filaments are formed from the high molecular weight protein
196
R. H. LOCKER, D. J. C. WILD
called "connectin' b y M a r u y a m a et al. (1980) (see also L o c k e r & Daines, 1980; K i n g & K u r t h , 1980) a n d "titin' by W a n g et al. (1979). We have identified these filaments with the g a p filaments. Since there a p p e a r s to be no o t h e r c a n d i d a t e for m a i n t e n a n c e o f s t r u c t u r a l c o n t i n u i t y within the myofibril, it seems t h a t the m y o f i b r i l l a r c o n t r i b u t i o n to tenderness m u s t be expressed in terms o f g a p filaments alone. Such a claim has a l r e a d y been m a d e ( L o c k e r et al., i 977), a n d is s t r e n g t h e n e d by the present results. A l t h o u g h there has been no r e s p o n s e so far in the literature to this claim, it seems that a radical revision o f theories o f m e a t tenderness, as p r o p o s e d in the a b o v e discussion, is now inevitable.
REFERENCES DAVEY, C. L. & GRAAFHUIS, A. E. (1976). J. Sci. Fd Agric., 27, 301. DAVEY,C. L., KUTTEL,H. & GILBERT,K. V. (1967). J. Fd Technol., 2, 53. DAvEv, C. L., NtEDE~n, A, F. & GRAAF~UIS,A. E. (1976). J. Sci. Fd Agric., 27, 251. Kt~G, N. L. & KURTH, L. 0980). In Fibrous Proteins: Scientific, industrialand Medical Aspects, Vol. 2.
(Parry, D.A.D. & Creamer, L. K. (Eds)), Academic Press, London, p. 57. LOCKER,R. H. & DAINES,G..1. (1980). In Fibrous Proteins: Scientific, Industrial and Medical Aspects, Vol. 2. (Parry, D. A, D. & Creamer, L, K, (Eds)), Academic Press, London, p. 43. LOCKER,R. H., D^INES, G. J., CARSE,W. A. & LEET, N. G. 0977). Meat Sci., I, 87. LOCKER, R. H., DAINES,G..1. & WILD, D. J. C. (1982). Meat Sci., submitted for publication. LOCKER,R. H. & LEE'r, N. G. 0976). J. UItrastruct. Res., 55, 157. LOCKER, R. H. & WILD, D. J. C. (1982a). Meat Sci.. 7, 93-107. LOCKER,R. H. & WlLO, D, J. C. (1982b). J. Text. Studies. (In press.) MA~H, B. B. & CA~E, W. A. (1974). J. Fd Technol., 9, 129. MARUYAMA,K., KIMURA,S., TOYOTA,N. & OHASHI,K. (1980). In Fibrous Proteins: Scientific, Industrial and Medical Aspects, Vol. 2. (Parry, D. A. D. & Creamer, L. K. (Eds)), Academic Press, London, p. 33. SC~MtTT, J. G. & PARRISH,F. C. (1971). J. FdSci., 36, I10. VOVLE,C. A. (1969). J. Fd Technol., 4, 275. WANG, K., MCCLURE,J. & TU. A. (1979). Proc. Natl. Acad. Sci., USA, 76, 3698.
MYOFIBRILS OF COOKED MEAT ARE A C O N T I N U U M OF GAP FILAMENTS
R. H. LOCKER & D. J. C. WILD Meat Industry Research Institute o f New Zealand (Inc.). PO Box 617, Hamilton. New Zealand
(Received: 15 December, 1981)
SUMMARY
When cooked meat is subjected to high degrees of stretch it becomes apparent in high magnification electron micrographs that A-flaments hat'e ceased to exist. The Aband isflled with a coaguhtm of actomyosin. Fragmentation of this coagulum during stretch rereals an array of fine filaments (identified as gap flaments). This result is obtained irrespectire of rigor temperature, state of contraction or degree of cooking. if the meat isf r s t aged, the gap filaments surt'it'ing in the l-band are too weak to open up the A-band. The results show that myofibrils in cooked meat are entirely dependent on heat-stable gapflaments for structural continui O"and tensile strength. Theories of nwat temh'rness must be ret'ised accordhtgly.
INTRODUCTION
Recently we have paid much attention to the tensile and histological properties of muscle in both the raw and the cooked state, and to the way in which these properties may be modified by such factors as rigor temperature, state of contraction and ageing. While raw rigor muscle undergoes the marked and irreversible changes of'yielding" during extension (Locker & Wild, 1982a), cooked muscle appears superficially to behave in an elastic manner. It stretches more readily, to break cleanly at about 90 % extension, and then recovers almost to its original length (Locker et al., 1982). Both the heat-modified myofibrils and the collagen fibres, rendered elastic by denaturation, contribute substantially to this behaviour. Cooked muscle is, however, not truly elastic. There is a sudden irreversible change in extensibility near I kg/cm 2, attributable to the myofibrillar component (Locker et al., 1982), there is a degree of "creep' if loading is slow and, 189 Meat Science 0309-1740/82,000%0189; $02-75 ~, Applied Science Publishers Ltd, England, 1982 Printed in Great Britain
190
R. H. LOCKER, D. J. C. WILD
although most of the recovery occurs instantly at breakage, shortening takes some minutes for completion. Examination by light or electron microscopy of cooked muscle which has recovered after loading to breakage showed no obvious damage to the overall integrity of the myofibrils. However, close examination of high power micrographs of such broken strips, restretched before fixation, showed that the A-filaments had ceased to exist as such and were replaced by fine filaments embedded in fragments of coagulum. The effect was equally visible in cold shortened muscle. Brief reference has already been made to this result (Locker et al., 1982), which is here reported in detail.
MATERIALS AND METHODS
Ox sternomandibularis muscles were allowed to go into rigor mortis under various temperature regimes, when strips were cut and cooked under restraint as previously described (Locker et al., 1982). Some strips were stretched mechanically to i 5, 30, 50 and 75 % extension at 10 % a minute using our 'yieldmeter" (Locker & Wild, 1982b). Thin strips, l - 2 m m in diameter, were then cut from these and stretched carefully by hand back to the same degrees of extension, before taping to rods for fixation. Other strips were loaded by hand until they broke and were then allowed to recover at 2°C. Thin strips were again cut from these as above, and restretched by 50-70 % for fixation. Samples were fixed in 2.5 % glutaraldehyde in 0. I Mcacodylate buffer pH 7-1, then in 1% osmium tetroxide in the same buffer. They were then embedded in Spurr's resin. Sections were stained with uranyl acetate-lead aspartate.
RESULTS
Stretching muscle cooked in rigor at excised length Histological changes were studied in cooked muscle strips, stretched to various degrees. After going into rigor unrestrained at 15 °C, 2 °C, 37 °C and at 2 °C with the final stages at 37°C (2 ° + 37°C rigor, Locker et al., 1982), the strips were cooked under restraint for 40min at 80°C. Strips cut from muscle set in rigor at 15 °C were subjected after cooking to gradual mechanical extension to various degrees, and fixed at that degree of extension. The unstretched muscle (Fig. la) had the typical appearance of cooked muscle: an amorphous A-band with sparse filaments in a short I-band, somewhat obscured by coagulated material. On stretching by 15% or 30% (Figs 1 b and c), extension occurred almost entirely in the I-band, with the filaments and a double N-line becoming clearer. The A-band appeared unchanged. At 50% stretch distinct
Fig. I. Sections of ox sternomandibularis muscle, allowed to go into rigor unrestrained at 15 °C, cut into strips, cooked under restraint for 40 rain at 80°C (unless otherwise stated) and stretched to various degrees, etc. (all × 21,000). (a) Unstretched. (b) Stretched mechanically at I0~/~ a minute to 15"o extension. (c) As m (b), to 3 0 ~ extension. (d) As in (b), to 5 0 ~ extension. (e) Loaded by hand to breaking point and allowed to recover. (f) Aged I day at 15 ~C before cooking. Loaded to breaking point, recovered and re-stretched by 50 ~o- (g) As in (f) but aged 7 days at 2 °C. (h) Unaged rigor muscle cooked 3 h at 100°C. Loaded to breaking point, recovered and re-stretched by 5000 .
Fig. 2a. A 'relaxed" myofibril from muscle allowed to go into rigor unrestrained for 7 h at 37 °C, cooked for 40 rain at 80°C, loaded to breaking point, allowed to recover and re-stretched by 60 % ( × 62,000).
Fig. 2b. A myofibril showing'contraction bands" from muscle which was cold shortened by holding for 24 h at 2 °C, but in which rigor was completed in 3 h at 37 °C ('2 ° + 37 °C rigor', Locker et al., 1982). Then treated in the same way as in Fig. 2a ( x 61,000).
MYOFIBRILS OF COOKED MEAT ARE A CONTINUUM OF GAP FILAMENTS
193
changes were evident in the A-band, which had stretched markedly and assumed a speckled appearance (Fig. ld). On close inspection this appears to be due to fragments ofcoagulum superimposed on an underlying array of fine filaments, seen most clearly in the pseudo-H-zone. These changes in the A-band were more marked when strips were loaded by hand until they broke, allowed to recover, and smaller strips CUt from them were stretched back by 50-70 0,~ for fixation (Fig. 2a). The array of fine filaments through the broken coagulum was here quite distinct. The coagulum in the centre of the A-band did not fragment, although fine filaments were also discernible here. In places the coagulum appears to be arranged in illdefined rows with a period of just over 200 nm. When strips were loaded until they broke, they recovered to near the original dimensions and appearance (Fig. le) although the N-line was clearer. The changes in the A-band on stretching were seen in 'relaxed' sarcomeres whatever the rigor temperature. In muscle which had gone into rigor at 2 °, 2 ° + 37 ° or 37 °C many fibres showed "contraction bands', i.e. had sarcomeres of 1-5 lam or less. These responded to stretch in much the same way but, in the absence of an lband, the gap-filaments were continuous across the sarcomere, and again were embedded in fragmented coagulum (Fig. 2b). This effect was the same whether the contraction bands were induced by cold shortening at 2 °C or heat shortening at 37°C. Results were similar if the strip was subjected to severe cooking (3 h at 100°C). When loaded to breaking point and stretched back by 50'~.,, structure was surprisingly well preserved (Fig. lh). Such strips recovered in the same way (Fig. lc) as those cooked 40min at 80°C (Fig. le). Ageing of strips prior to normal cooking, followed by loading to breaking point and restretching by 50 ° o as above, produced different results. In strips aged I day at 15°C, response varied in individual fibres, some closely resembling unaged moderately stretched fibres as in Figs lb and lc, while others had clearly suffered profound change in those filaments of the l-band which had survived cooking (Fig. I f). These stretched while the A-band did not, but there was a high proportion of broken filaments. In places, short filaments, thicker than the intact ones, lie against the edge of the A-band (see arrow). These appear to be the result of springback after breakage. In a less aged sample (7 days at 2°C) the preservation of filaments was much better (Fig. lg). Here they are remarkably clean, and free of coagulated or N-line material, but notably sparser than normal I-filaments.
DISCUSSION
There is already some evidence for disintegration of A-filaments on cooking. Schmitt & Parrish ( 1971) examined samples of beef muscle heated briefly to various internal temperatures. The A-filaments were quite intact after heating to 50°C,
194
R. H. LOCKER, D. J. C. WILD
while after reaching 60°C they remained visible in longitudinal, but not in transverse, section. They were no longer visible in muscle heated to 70 ° or 80 °C. In earlier work on ox sternomandibularis allowed to enter rigor at 15 °C and given a normal cook of 40 min at 80 °C, the A-band appeared featureless except for a somewhat speckled appearance (Locker et al., 1977). Stretching after cooking accentuated the speckling, with a suggestion of fine filamentous substructure (Locker et al., 1977; Figs 1,2). The micrographs presented here reveal more convincingly than previously the disappearance of A-filaments in favour of fine filaments. When muscle, cooked after rigor at 15 °C, is stretched by up to 30 ~ , extension of the sarcomere is confined almost entirely to the l-band, but by 500/o stretch the A-band has extended considerably, with a profound change in its fine structure. We interpret this change in the following way. The A-filaments depolymerise on heating along with the actin filaments, leading to the formation of a stoichiometric actomyosin gel, packed tightly around the surviving A-filament cores, which we have earlier claimed to be comprised of gap filaments (Locker & Leet, 1976). As stretch progresses the extending filaments are exposed as the gel begins to fragment. On release of tension, the elastic gap filaments close up the coagulum again, and the sarcomere reverts to its original appearance. The failure of the coagulum in the central region of the A-band to fragment is due to the overlap of the opposing actin filaments there, resulting in a denser gel. It has already been pointed out that the actin filaments in the l-band disintegrate early in cooking, leaving a sparser array of gap filaments (Locker et al., 1977). These are only half as numerous as the A-filaments (see the model of gap-filament connections in Locker &Leet, 1976). The I-band therefore is the more extensible, and the point of failure under tensile stress. Thus, within the sarcomere, structural continuity is entirely dependent on gap filaments although, strictly, they do not form a true continuum but terminate in the A-bands, when they rely on overlap to maintain continuity. The surrounding actomyosin gel and the apparent survival of the M-line suffice to prevent slippage of the filaments over each other. In contracted sarcomeres the weaker I-band link is missing and the structure amounts to a true continuum of gap filaments within the sarcomere. Previous attempts to explain the toughening induced by cold shortening have involved a continuum of thick filaments, fused end to end by cooking (Voyle, 1969: Marsh & Carse, 1974). This theory must now be restated more accurately in terms of a continuum of gap filaments. Since the gap filaments are twice as abundant as in the I-band, and are further reinforced by a tight actomyosin gel, a tensile strength of more than twice that of unshortened muscle may be expected, even allowing a little for lateral expansion of the lattice on contraction. The gap filaments themselves may well be thicker and stronger when cooked in a completely unstretched state, thus further enhancing toughness. On the basis of our model for gap filament connections half the gap filaments
MYOFIBRILS OF COOKED MEAT ARE A CONTINUUM OF GAP FILAMENTS
195
would be continuous through any Z-line to the Z-lines on either side. The other half would overlap by the same amount as the opposing A-filaments did after penetration of the Z-line. It should therefore be a stronger continuum than the notational A-filament continuum, based on a degree of overlap which may amount to little more than end-to-end fusion. It is surprising that the rigorous cooking at 100 °C has no effect on the results. The resolution of the micrographs equals those from standard cooking. Recovery is the same as before. This emphasises the great resistance of gap filaments to heat, already noted (Locker et al., 1977). The fact that shear force in the muscle maintains a steady state between 24 h and 100 h cooking at 80 °C (Davey et al., 1976) is an even more dramatic manifestation of this stability. The results of ageing reflect the vulnerability of gap filaments in the I-band to proteolytic attack (Davey & Graafhuis, 1976; Locker et al., 1977). The micrographs show a great variation in the capacity of individual fibres to age. I n those that have aged moderately the filaments survive cooking and stretching but they break if ageing is more severe. In either case they have insufficient strength to extend the Aband, merely stretching or breaking themselves. This is in contrast to the raw state where they fracture cleanly after ageing (along with actin filaments) without extension (Locker & Wild, 1982a). The clean, well preserved filaments in the moderately aged fibre of Fig. ! g suggest from their sparseness that they must be gap filaments, as previously claimed (Locker et al., 1977). It is hard to imagine a minority of actin filaments surviving intact while the rest disappear without trace. Within the A-band the gap filaments are immune to catheptic attack because of their r61e as thick filament cores, where they are completely shielded by a sheath of close-packed and resistant myosin 'tails'. In sarcomeres shortened to 1.5 pm there is no I-band and protection of gap filaments is complete. This accounts for the observed failure of cold shortened meat to age (Davey et al., 1967). The break up of the actomyosin coagulum might offer some explanation for the sudden increases in extensibility as cooked muscle is extended (Locker et al., 1982). On stretching some muscles which have gone into rigor at 15 °C and been cooked under restraint there is a single change in extensibility at about 1.0 kg/cm 2 and 10 ~o extension, while, in other cases, two change points are discernible. While break up of the gel only becomes~clearly visible at about 50 ~o stretch, it is quite possible that the process begins much earlier, even at 1 0 ~ stretch. However, if this were the case, appreciable extension of the A-band might have been expected earlier than 30 ~o stretch of the strip. Since it does not seem possible to explain two changes in extensibility on this basis, the situation is probably more complex. In conclusion, this paper has shown that in the A-band, the survivors of cooking are not A-filaments but a set of much thinner filaments with a remarkable stability to heat. This situation applies whatever the rigor temperature or final state of contraction, although the filaments are clearly weakened by ageing. Evidence is accumulating that gap filaments are formed from the high molecular weight protein
196
R. H. LOCKER, D. J. C. WILD
called "connectin' b y M a r u y a m a et al. (1980) (see also L o c k e r & Daines, 1980; K i n g & K u r t h , 1980) a n d "titin' by W a n g et al. (1979). We have identified these filaments with the g a p filaments. Since there a p p e a r s to be no o t h e r c a n d i d a t e for m a i n t e n a n c e o f s t r u c t u r a l c o n t i n u i t y within the myofibril, it seems t h a t the m y o f i b r i l l a r c o n t r i b u t i o n to tenderness m u s t be expressed in terms o f g a p filaments alone. Such a claim has a l r e a d y been m a d e ( L o c k e r et al., i 977), a n d is s t r e n g t h e n e d by the present results. A l t h o u g h there has been no r e s p o n s e so far in the literature to this claim, it seems that a radical revision o f theories o f m e a t tenderness, as p r o p o s e d in the a b o v e discussion, is now inevitable.
REFERENCES DAVEY, C. L. & GRAAFHUIS, A. E. (1976). J. Sci. Fd Agric., 27, 301. DAVEY,C. L., KUTTEL,H. & GILBERT,K. V. (1967). J. Fd Technol., 2, 53. DAvEv, C. L., NtEDE~n, A, F. & GRAAF~UIS,A. E. (1976). J. Sci. Fd Agric., 27, 251. Kt~G, N. L. & KURTH, L. 0980). In Fibrous Proteins: Scientific, industrialand Medical Aspects, Vol. 2.
(Parry, D.A.D. & Creamer, L. K. (Eds)), Academic Press, London, p. 57. LOCKER,R. H. & DAINES,G..1. (1980). In Fibrous Proteins: Scientific, Industrial and Medical Aspects, Vol. 2. (Parry, D. A, D. & Creamer, L, K, (Eds)), Academic Press, London, p. 43. LOCKER,R. H., D^INES, G. J., CARSE,W. A. & LEET, N. G. 0977). Meat Sci., I, 87. LOCKER, R. H., DAINES,G..1. & WILD, D. J. C. (1982). Meat Sci., submitted for publication. LOCKER,R. H. & LEE'r, N. G. 0976). J. UItrastruct. Res., 55, 157. LOCKER, R. H. & WILD, D. J. C. (1982a). Meat Sci.. 7, 93-107. LOCKER,R. H. & WlLO, D, J. C. (1982b). J. Text. Studies. (In press.) MA~H, B. B. & CA~E, W. A. (1974). J. Fd Technol., 9, 129. MARUYAMA,K., KIMURA,S., TOYOTA,N. & OHASHI,K. (1980). In Fibrous Proteins: Scientific, Industrial and Medical Aspects, Vol. 2. (Parry, D. A. D. & Creamer, L. K. (Eds)), Academic Press, London, p. 33. SC~MtTT, J. G. & PARRISH,F. C. (1971). J. FdSci., 36, I10. VOVLE,C. A. (1969). J. Fd Technol., 4, 275. WANG, K., MCCLURE,J. & TU. A. (1979). Proc. Natl. Acad. Sci., USA, 76, 3698.