Pressure-induced changes in the connectin/titin localization in the myofibrils revealed by immunoelectron microscopy
Introduction
The presence of connectin (also called titin) filaments within the sarcomere of vertebrate skeletal muscle is now well-established. Connectin, a giant structural protein that was first identified by Maruyama, Natori, and Nonomura (1976), is thought to be elastic in nature and could account for some of the elastic properties of skeletal muscle (Maruyama, 1986, Wang, 1985). The influence of connectin on meat tenderization during post-mortem conditioning has been investigated by many workers.
The gap filaments, which are considered to be composed of connectin, have been shown to be susceptible to protease by Locker and Leet (1976), and the disappearance of insoluble connectin from myofibrils during conditioning of meat has been reported by Takahashi and Saito (1979). Therefore, the degradation of connectin has been implicated as being responsible for the increasing meat tenderness that occurs during conditioning. On the other hand, King, Kurth, and Shorthose (1981) and King (1984) have denied any significance of the degradation of connectin in meat tenderness on the ground that the protein is destroyed by cooking, even though the degradation of connectin has been observed during conditioning of mutton. No disappearance of connectin from chicken muscle and no significant differences in the content and electrophoretic pattern of connectin isolated from chicken muscle during conditioning were reported by Locker (1984) and Suzuki, Sawaki, Hosaka, Ikarashi, and Nonami (1985), respectively. From these observations, connectin is unlikely to be responsible for the meat tenderization caused by conditioning. However, there are still some uncertainties about the fate and role of connectin during conditioning. As suggested by Suzuki et al. (1987), it is important to clarify qualitative change of connectin structure during post-mortem storage if we are to understand the role of connectin in meat tenderness.
In normal skeletal muscle, connectin exists as α-connectin (titin-1) together with a small amount of β-connectin (titin-2) (Maruyama et al., 1984, Wang et al., 1979). Alpha-connectin is easily degraded to β-connectin during post-mortem storage (Maruyama et al., 1977, Seki and Watanabe, 1984), and the extent of degradation is dependent upon time and temperature (Lusby, Ridpath, Parrish, & Robson, 1983). It has been suggested that the amount of titin is less in tender steaks and that the rate of titin degradation during post-mortem storage is accelerated in tender steaks compared to their less-tender counterparts (Paterson & Parrish, 1987). From these observations the amount of connectin and the conversion of α-connectin to β-connectin may be one of the causes of meat tenderization. Nevertheless Fritz, Mitchell, Marsh, and Greaser (1993) reported that titin content (sum of titins-1,-2 and-3), as determined by gel electrophoresis, did not distinguish ‘tough’ from ‘tender’ meat. The changes in the position of titin epitope in the sarcomere during posts mortem storage by using indirect immunofluorescent method have been reported by Ringkob, Marsh, and Greaser (1988) and Fritz and Greaser (1991). However, the changes in the titin epitope obtained by the phase microscopic observation were insufficient to clarify the relationship between the changes of connectinltitin filament and meat tenderization.
In our previous report, we revealed that a brief exposure of muscle to high hydrostatic pressure could induce the conversion of α-connectin to β-connectin, and that calpain was responsible for the pressure-induced conversion of α- to β-connectin (Kim et al., 1993, Kim et al., 1992). The increase in the amount of extractable connectin from the pressurized muscle, i.e. the increase of the conversion of α-connectin to β-connectin, is probably one of the causes of the pressure-induced tenderization of meat (Suzuki, Kim, Tanji, & Ikeuchi, 1998).
This paper describes high pressure effects on the connectin epitope in the sarcomere by using immunoelectron microscopy, in comparison with those naturally observed in the conditioned muscle.
Section snippets
Preparation of glycerinated muscle
The fiber bundles of chicken pectoralis profundus muscles prepared immediately after death and from the stored muscles were glycerinated in the usual manner for 10 weeks according to the procedure described by Kitazawa (1975).
Pressurization of the muscle
Pressurization of fiber bundles was performed as described previously (Kim et al., 1992). Briefly, high hydrostatic pressures of 100–300 MPa were applied to fiber bundles prepared immediately after death and fixed to glass rod in 50% glycerol containing 5 mM potassium
Experiment 1: changes in the connectin epitope during post-mortem storage
Immunoelectron micrographs showing the localization of connectin epitope in sarcomere during post-mortem storage of chicken pectoralis profundus muscles are shown in Fig. 1. The anti-connectin monoclonal antibody, 1D11, strongly labeled both sides of thick filaments near the H-zone and weakly labeled both sides of Z-line in the sarcomere of fiber bundles prepared immediately after death (Fig. 1b) as compared with that of the untreated fiber (Fig. 1a). In this paper, we are going to discuss the
References (29)
- et al.
Titin content of beef in relation to tenderness
Meat Science
(1993) Viscoelasticity and function of connectin/titin filaments in skinned muscle fibers
Advances in Biophysics
(1996)- et al.
Pressure-induced conversion of α-connectin to β-connectin
Meat Science
(1992) Breakdown of connectin during cooking of meat
Meat Science
(1984)- et al.
Proteolytic degradation of connectin, a high molecular weight myofibrillar protein, during heating of meat
Meat Science
(1981) - et al.
Histology of highly stretched beef muscle. II Further evidence on the location and nature of gap filaments
Journal of Ultrastructure Research
(1976) Connectin, an elastic filamentous protein of striated muscle
International Review of Cytology
(1986)- et al.
Chicken leg muscle α-connectin as stained by a monoclonal antibody to the 1200 kDa fragment
Comparative Biochemistry and Physiology
(1992) - et al.
Post-mortem changes of connectin in chicken skeletal muscle
Meat Science
(1985) - Fritz, J. D. & Greaser, M. L. (1991). Changes in titin and nebulin in postmortem bovine muscle revealed by gel...
The organization of titin filaments in the half-sarcomere revealed by monoclonal antibodies in immunoelectron microscopy: a map of ten repetitive epitopes starting at the Z line extends close to the M line
Journal of Cell Biology
Elastic behavior of connectin filaments during thick filament movement in activated skeletal muscle
Journal of Cell Biology
The difference between connectin and titin revealed by monoclonal antibody
Bulletin of Faculty of Agriculture, Niigata University
Effect of high hydrostatic pressure on the conversion of α-connectin to β-connectin
Journal of Biochemistry
Cited by (8)
High pressure processing of fresh meat - Is it worth it?
2013, Meat ScienceCitation Excerpt :Thus high pressure treatment of post-rigor meat at ambient temperatures, though of academic interest, does not seem to have any commercial value, other than that of extending shelf life. However if combined with elevated temperatures it may have merit as it was shown many years ago that moderate pressures (< 200 MPa) combined with elevated temperatures (> 40 °C) led to marked tenderisation in mutton, beef (Ma & Ledward, 2004; Macfarlane, 1985) and poultry (Suzuki et al., 2001; Zamri, Ledward, & Frazier, 2006). Bouton et al. (1977) found that pressure treatment of about 100 MPa for 2.5 min at 40 to 60 °C of post-rigor muscle caused significant increases in tenderness and Beilken, Macfarlane, and Jones (1990) showed that treatment at 150 MPa and 40 to 80 °C for up to 4 h prevents the development of myofibrillar toughness but has no effect on the connective component of toughness.
Protein interactions in muscle foods
2016, Ingredient Interactions: Effects on Food Quality, Second EditionApplication of time-intensity method to assess the sensory properties of Iberian dry-cured ham: Effect of fat content and high-pressure treatment
2014, European Food Research and TechnologyEffect of High Pressure on Physicochemical Properties of Meat
2013, Critical Reviews in Food Science and NutritionEffects of thermal processing combined with high pressure on the characteristics of cooked pork
2008, Korean Journal for Food Science of Animal ResourcesEffects of pressurization on structure, water distribution, and sensory attributes of cured ham: Can pressurization reduce the crucial sodium content?
2006, Journal of Agricultural and Food Chemistry