Muscle electrophoretic pattern

The SDS-gel electrophoretic pattern of tropomyosin showed two bands, designated a and /8, around 35,000 Da (Cummins and Perry, 1973). The ratio of the two subunits varied from tissue to tissue mostly a subunit in fast skeletal muscle, equimolar a and )8 subunits in slow skeletal muscle, and exclusively a subunit in cardiac muscle (Bronson and Schachat, 1982). The tropomyosin molecule consists of aa or ajS dimers. A )8/8 molecule produced in vitro showed the same Ca -sensitizing action as that of aj8- or aa-tropomyosin (Cummins and Perry, 1973). Hence physiologically both a and /3 subunits are identical. 

Fig. 4. Ascending electrophoretic patterns of extracts of low ionic strength of carp (left) and codling (right) muscles. Upper part whole extracts. Lower part albumin fraction (after hamoir, 1955 and connell, 1953a).

Fig. 6. Ascending electrophoretic patterns of two 1-hour extracts at /i 0.15 and pH 7 of carp red muscles. Upper pari whole extracts. Lower part corresponding albumin fractions (after hamoib, 1955).

Fig. 8. Electrophoretic pattern of a 10-minute extract of carp muscles at n 0.5 and pH 6.9 (after hamoir, 1955).

Beckman et al. (28) have studied the electrophoretic separation of the acid phosphatase activity in tissue extracts on starch gel at pH 8. They described four electrophoretic bands A, B, C, and D. Table IV (28) shows the distribution of activity in different organ extracts. The ABD pattern predominated in kidney BD in liver, intestine, heart, and skeletal muscle B in skin and D in pancreas. The C component was present in a large number of placentae but not in other adult organs. All four electrophoretic components were inhibited by d-(- -)-tartrate A contained sialic acid, D had a lower pH optimum and was more heat resistant than A, B, and C. Components C and D showed parallel electrophoretic behavior. In human skin fibroblasts grown in tissue culture, the acid phosphatase was generally high and the most common pattern was BD. Almost every culture showed some activity. The BD... 

An example of an enzyme which has different isoenzyme forms is lactate dehydrogenase (LDH) which catalyzes the reversible conversion of pyruvate into lactate in the presence of the coenzyme NADH (see above). LDH is a tetramer of two different types of subunits, called H and M, which have small differences in amino acid sequence. The two subunits can combine randomly with each other, forming five isoenzymes that have the compositions H4, H3M, H2M2, HM3 and M4. The five isoenzymes can be resolved electrophoretically (see Topic B8). M subunits predominate in skeletal muscle and liver, whereas H subunits predominate in the heart. H4 and H3M isoenzymes are found predominantly in the heart and red blood cells H2M2 is found predominantly in the brain and kidney while HM3 and M4 are found predominantly in the liver and skeletal muscle. Thus, the isoenzyme pattern is characteristic of a particular tissue, a factor which is of immense diagnostic importance in medicine. Myocardial infarction, infectious hepatitis and muscle diseases involve cell death of the affected tissue, with release of the cell contents into the blood. As LDH is a soluble, cytosolic protein it is readily released in these conditions. Under normal circumstances there is little LDH in the blood. Therefore the pattern of LDH isoenzymes in the blood is indicative of the tissue that released the isoenzymes and so can be used to diagnose a condition, such as a myocardial infarction, and to monitor the progress of treatment. 

The protein components of a membrane can be readily visualized by SDS-polyacrylamide gel electrophoresis. As discussed earlier (Section 4.1.4). the electrophoretic mobility of many proteins in SDS-containing gels depends on the mass rather than on the net charge of the protein. The gel-electrophoresis patterns of three membranes—the plasma membrane of erythrocytes, the photoreceptor membrane of retinal rod cells, and the sarcoplasmic reticulum membrane of muscle—are shown in Figure 12.16. It is evident that each of these three membranes contains many proteins but has a distinct protein composition. In general, membranes performing different functions contain different repertoires of proteins. 

The patterns of several sets of isoenzymes change during normal development in tissues from many species. For example, changes in the relative proportions of several isoenzymes are noted during the embryonic development of skeletal muscle. The proportions of the electrophoretically more cathodal isoenzymes, of both LD and CK, progressively increase in this tissue, until approximately the sixth month of intrauterine life, when the pattern resembles that of differentiated muscle. Smaller quantitative changes in isoenzyme distribution may continue to birth and into early postnatal life. 

Considerable interest was aroused by the finding of Wieme and Lauryssens (W16) in 1962 that there is a change in the electrophoretic isoenzyme pattern of lactate dehydrogenase in diseased human muscle. The major isoenzyme of lactate dehydrogenase in most normal muscles moves slowest on electrophoresis (LDH 5), but in myopathic muscle the proportion of LDH 5 may be considerably reduced. This finding has been confirmed and extended by numerous workers, utilizing various techniques for isoenzyme differentiation (e.g., BIO, E5). The abnormal pattern is seen in most, but not all, cases of Duchenne dystrophy and in a variety of other muscular disorders. It may be evident in the very early stages of Duchenne dystrophy (P2) and is seen even in some female carriers of the disease (E3). 

The isoform diversity in striated muscles and their electrophoretic gel patterns are further complicated by partial phosphorylation of serine 283, the penultimate COOH-terminal residue (Ribolow and Barany, 1977 Mak etal., 1978 Montarras cfal., 1981,1982 Heeley cf al., 1982,1985). The extent of phosphorylation is highest for the a isoform in embryonic and neonatal tissue, dropping to much lower levels in adult muscle. Heeley et al. (1982,1989) and Heeley (1994) have compared the phosphorylated and nonphosphorylated forms of rabbit skeletal aa TM and observed significant differences... 

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