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LDH isozyme profile of striated muscles and electrophoretically-typed single fibres from cane toad (Bufo marinus)

J.R. Babb, R. Blazev, J.G. Kemp and G.M.M. Stephenson, Muscle Cell Biochemistry Laboratory, School of Biomedical and Health Sciences, Victoria University, P.O. Box 14428, Melbourne, VIC 8001, Australia.

In mammalian tissues (other than gonads), lactate dehydrogenase (LDH) is a tetrameric enzyme (Tsuji et al., 1994) produced by five random combinations of two different subunits, A and B [LDH1 (B4)-lowest activity; LDH2 (A1B3); LDH3 (A2B2), LDH4 (A3B1); LDH5 (A4)-highest activity], which are encoded by different genes (LDH-A and LDH-B). The expression of these genes and therefore the distribution of LDH isozymes (LDHisoz) are tissue-specific. Using histochemically typed single fibre segments from rabbit skeletal muscles, Leberer & Pette (1984) found a predominance of B-type LDHisoz in type I fibres and of A-type LDHisoz in type II fibres, but found no difference in the LDHisoz profile between type IIA and IIB fibres. The authors concluded that the expression of the LDH subunits is not related to specific MHC isoforms (MHCi). Recently we reported that rectus abdominis muscle of the cane toad contains a wide range of MHC-based fibre types that include pure twitch, hybrid twitch (i.e., comprising one or several twitch MHCi, respectively) and hybrid tonic-twitch fibres (Nguyen & Stephenson, 2002) of sizes that allow both LDHisoz and MHCi analyses. Currently there is no information on the number of LDHisoz in cane toad (Bufo marinus) skeletal muscles or on their distribution among different muscles and fibre types. In this study we aimed to address this cognitive gap using whole muscle homogenates and single muscle fibres from adult cane toads.

Male and female toads (79-149g) were killed by double pithing in accordance with procedures approved by Victoria University. Crude extracts from several skeletal [rectus abdominis (n = 9), hyoglossus (n = 4), iliofibularis (n = 4), semimembranosus (n = 6), and sartorius (n = 5)] and cardiac (n = 7) muscles of the toad and from mouse soleus muscle (n = 4; used as a mammalian LDHisoz marker) were prepared in 0.9% NaCl and then further diluted in BPB-S solution [0.5 mg/ml bromphenol blue; 40% (w/v) sucrose; 0.1M Tris-HCl; pH 7.0] to a final concentration (w/v) of 0.45% (toad skeletal muscle), 1.2% (toad cardiac muscle), and 10.0% (mouse soleus muscle). Single fibre segments (1.8-85.3 nl), isolated from rectus abdominis muscles (n = 6), were placed in 10.5 μl BPB-S solution and frozen at -84oC for later analysis of LDHisoz and MHCi. LDHisoz (in tissue extracts and single fibres) and MHCi (in single fibres) were resolved by non-denaturing polyacrylamide gel electrophoresis (LDHisoz) and SDS-polyacrylamide gel electrophoresis (MHCi) using the protocol of Leberer & Pette (1984), with modifications, and the method described by O'Connell et al. (2006), respectively. LDHisoz were visualized by an activity-based gel staining procedure using a system of coupled reactions which link lactate oxidation to reduction of nitroblue tetrazolium to formazan. MHCi bands were visualized by silver staining and quantified densitometrically (Molecular Dynamics). Each fibre segment was examined first for LDHisoz and then for MHCi composition.

Five LDHisoz were detected in both toad cardiac muscle and mouse soleus muscle, but their electrophoretic mobilities were different. These isozymes are presumably formed by the tetrameric combination of species-specific A and B subunits. With the exception of the toad semimembranosus muscle samples, which displayed the toad equivalent of mammalian LDH4 and LDH5 (LDH5toad and LDH4toad) isozymes, all the toad skeletal muscle samples examined in this study displayed only one LDHisoz, viz. LDH5toad. The population of 100 single rectus abdominis fibres comprised 31 tonic-twitch hybrids, of which 14 fibres contained predominantly (> 80% MHCtotal) the tonic MHCi, and 69 pure and hybrid twitch fibres. Of the 14 predominantly tonic fibres, 13 fibres (93%) displayed only LDH5toad, regardless of the identity or the number of the twitch MHCi co-expressed with the tonic isoform, and one fibre contained both LDH5toad and LDH4toad. Similarly, 99% (68/69) of the pure and hybrid twitch fibres and 17 tonic-twitch hybrid fibres displayed only LDH5toad, with one hybrid twitch fibre containing both LDH5toad and LDH4toad. The LDHisoz composition of the two fibres containing both LDH5toad and LDH4toad was not unique to their MHC classification as other fibres of the same type showed only the LDH5toad isozyme. These results indicate that in cane toad skeletal muscles, like in rabbit skeletal muscles, there is no simple and tight relationship between the molecular forms of LDH (key enzyme in anaerobic metabolism) and those of MHC (molecular motor in muscle contraction).

Leberer E & Pette D. (1984) Histochemistry, 80: 295-8.

Nguyen LT & Stephenson GMM. (2002) Journal of Muscle Research and Cell Motility, 23: 147-156.

O'Connell B, Blazev R & Stephenson GMM. (2006) American Journal of Physiology, 290: C515-23.

Tsuji S, Qureshi MA, Hou EW, Fitch WM & Li SS. (1994) Proceedings of the National Academy of Sciences USA, 91: 9392-6.

This study was supported by the Australian Research Council.