Isoenzyme hybrids

The gene for myophosphoiylase has been assigned to chromosome 1 lql3. The enzyme is a dimer of two identical 97 IcDa subunits and is the sole isoform present in skeletal muscle. Heart and brain also contain this isoform in addition to a distinct brain isoenzyme and a hybrid muscle/brain isoform. Smooth muscle also contains a phosphorylase isoform distinct from the muscle isoenzyme. If regenerating muscle fibers are present they also contain phosphorylase activity due to the presence, in fetal and developing muscle, of an isoform said to be identical with brain phosphorylase. 

The extent of the dissociation in 8 M urea can, moreover, not be estimated from the electrophoretically observed hybrid formation between wheat germ (isoenzyme II) SOD and respectively yeast and bovine SOD. BESOD did namely not dissociate in 8 M urea at 25 °C for at least 72 h, as indicated by sedimentation equilibrium analysis (Af. 32,000). The reactivity of Cys-6 and of the histidine residues was not increased either in that medium... 

Another category of multiple molecular forms can arise when enzymes are ofigomeric and consist of molecules made up of subunits. The association of different types of subunits in various combinations gives rise to a range of active enzyme molecules. When the subunits are derived from different structural genes, either multiple loci or multiple alleles, the hybrid molecules so formed are called hybrid isoenzymes. The ability to form hybrid isoenzymes is evidence of considerable structural similarities between the different subunits. Hybrid isoenzymes can be formed in vitro, but they are also formed in vivo in cells in which the different types of constituent subunits are present in the same sub-ceUular compartment. 

The number of different hybrid isoenzymes that can be formed from two nonidentical protomers depends on the number of subunits in the complete enzyme molecule. For a dimeric enzyme, one mixed dimer (hybrid isoenzyme) can be formed. If the enzyme is a tetramer, three heteropoly-meric isoenzymes may be formed (Figure 8-1). Examples of... 

Figure 8-1 Diagram showing the origin of isoenzymes, assuming the existence of two distinct gene loci. When the active enzymes are poiymers containing more than one subunit, hybrid isoenzymes consisting of mixtures of different subunits may be formed. One such isoenzyme can be formed in the case of a dimeric enzyme, such as creatine kinase, and three if the enzyme is a tetramer (e.g., lactate dehydrogenase). In both cases, two homopoiymeric isoenzymes can also exist. (From Moss DW. Isoenzyme ona/ys/s. London The Chemical Society 1979.)...

The isoenzyme pattern of creatine kinase in normal and diseased muscle has been investigated by several workers. Two subunits are recognized, usually denoted M and B the enzyme is a dimer, hence three forms are possible MM, BB, and the hybrid MB. Most muscles contain largely MM with sometimes a proportion of BB (the latter is characteristic of brain). Normal fetal muscle, however, contains a preponderance of BB (Gil, S3). After denervation, there is a relative decrease in MM (S3). In muscle from patients with fascioscapulohumeral dystrophy and polymyositis there was an increase in MB relative to MM, but a normal ratio was found in Duchenne and limb-girdle dystrophies and in most cases of neurogenic atrophy BB occurred in some cases of myotonic dystrophy (Gll). 

Class I aldolases of mammals and other vertebrates can be subdivided into three distinct isoenzymes.143,331 Identification of the parental aldolases A, B, and C has been made from their substrate specificities (ratio of activity towards D-fructose 1,6-bisphosphate and towards D-fructose 1-phosphate), electrophoretic mobilities, tissue distribution, and specific immunological properties. Aldolase A is the major form, present in muscle aldolase B, the predominant form in liver and kidney and aldolase C, present in brain with aldolase A. In tissues where more than one aldolase isoenzyme occurs, a hybrid form is often observed.331... 

These combinatorics can also be applied to heterooligomers and yield similar equations. In the 1960s, hybridization experiments with enzymes were fashionable. You either produced a from a or you isolated an isoenzyme, whose monomers a differed from a in their charge and nevertheless hybridized with a into an oligomer. For the hybridization, the oligomers were mixed in a certain proportion, split into subunits, and afterward associated into oligomers again. With native gel electrophoresis or lEC, the experimenter determined the number of the hybrids and with it the number of subunits. 

Much of the recent progress in our knowledge of the genetics of the sphingolipidoses has come from cell hybridization experiments. Cultured skin fibroblasts have been used to study the isoenzyme abnormalities in Tay-Sachs disease and Sandhoff-Jatzkewitz disease. In the former, only hexosaminidase isoenzyme A is lacking (Okada and O Brien, 1969) while in the latter, both hexosaminidase A and hexosaminidase B are deficient (Sandhoff et al., 1968). The deficiency of hexosaminidase A in these two disorders is due to different mutations and activity can be restored by complementation techniques (e.g. Rattazzi et al, 1976). The studies have also provided support for the idea that hexosaminidase A is composed of two non-identical subunits aP)n and hexosaminidase B is ( )n) (cf. Brady, 1978). Hybridization studies have now identified... 

There seems to be simultaneous synthesis of embryonic and adult subunits in early stages of myotube differentiation. This is not unexpected since complete hybrid sets of both isoenzymes apparently formed by subunit association are found during normal in situ development (Eppen-berger et al., 1964 Lebherz, 1972a). Addition of actinomycin D at various intervals prior to fusion or shortly after fusion shows, on the one hand, that the required RNA is present in the cell at least 6 hours before fusion, and, on the other hand, that in fused cells the CPK increase proceeds after actinomycin D addition for about 12 more hours (Shainberg et al., 1971). This constitutes evidence that the cell is able to continue protein synthesis and thus indicates that stable mRNA must be present (Fig. 6). Transcription of at least part of the information required for subsequent differentiation must have already occurred when myoblasts entered the phase of fusion. 

Similar experiments for other polymeric isoenzymes have not yet been done, but the ease with which CPK hybrid dimers between different subunits can be formed in vitro (Dawson et al., 1967) suggests that subunit exchange among CPK isoenzymes might also occur in vivo. 

Fig. 22. Isoenzymes of the pH 7.5 esterase of maize. Separation by zone electrophoresis. The F/S enzyme is a hybrid enzyme. ...

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