Electrophoresis isozymes

Figure 7-11. Normal and pathologic patterns of lactate dehydrogenase (LDH) isozymes in human serum. LDH isozymes of serum were separated by electrophoresis and visualized using the coupled reaction scheme shown on the left. (NBT, nitroblue tetrazolium PMS, phenazine methylsulfate). At right is shown the stained electropherogram. Pattern A is serum from a patient with a myocardial infarct B is normal serum and C is serum from a patient with liver disease. Arabic numerals denote specific LDH isozymes.

Isozymes are also a common presence in enzyme preparations and they can often be detected via polyacrylamide gel electrophoresis. The detected presence of isozymes may result in the need for further purification steps and the kinetic characterization of each isozyme. It may be necessary to use nondenaturing electrophoretic procedures to separate the different isozymes. See Isozymes Enzyme Concentration... 

Because of their structural differences, isozymes may often be distinguished by separation in an electric field (electrophoresis) or by reactivity with selective antibodies. 

One of the first enzymes found to have isozymes was lactate dehydrogenase (LDH) (p. 538), which, in vertebrate tissues, exists as at least five different isozymes separable by electrophoresis. All LDH isozymes contain four polypeptide chains (each of Mt 33,500), each type containing a different ratio of two lands of polypeptides. The M (for muscle) chain and the H (for heart) chain are encoded by two different genes. 

The biosynthesis in yeast of two enzymes that are D-mannoproteins has been studied. A membrane-associated isozyme of invertase (EC 3.2.1.26) has been shown to be a precursor of the external invertase.190 Its molecular weight, as determined by SDS-poly(acrylamide) gel electrophoresis, is 50,000, that is, smaller than that of the external invertase, and it correlates well with the presence of only the inner-core sugars of the final form. It is strictly bound to membranes, possibly those of the endoplasmic reticulum, and it can be completely split191 by endo-/3-N-acetylglucosaminidase H (EC 3.2.1.30). The addition of tunicamycin, which specifically inhibits formation of d-GIcNAc-PP-DoI, inhibits synthesis of external invertase, as well as further formation of the membrane-associated form, which completely disappears after addition of the antibiotic.190 In these aspects, the synthesis of this extracellular enzyme follows the pathway for secreted glycoproteins in animal systems. 

A comparison of spontaneously and experimentally released -lactamase preparations of B. licheniformis (40) indicates that the ends of the polypeptide chain can vary without affecting the properties of the enzyme. Although such preparations differ in the C- and N-terminus, they are serologically and catalytically indistinguishable (40,63). Similarly, B. licheniformis 749/C isozymes, separable by starch gel electrophoresis or DEAE-chromatography and believed to differ at the C- or N-terminus (40), have identical substrate specificities (Table V). 

Purified preparations of alkaline phosphatase from E. coli, judged homogeneous when examined in the analytical ultracentrifuge, contain several isozymes, because several bands which contain enzymic activity are obtained in starch-gel and disc-gel electrophoresis. Although most workers find three bands (38, 39, 41, 43, 69), four (44) and five (70) equally spaced bands have been found. 

Lazdunski and Lazdunski (43), separated three isozymes on DEAE-cellulose. They found that pure samples of either isozyme I (the isozyme with the least negative charge at pH 7.0 is referred to as isozyme I) or isozyme III, after dissociation and reassociation, gave only the original single band on disc-gel electrophoresis. Pure isozyme II, after dissociation and reassociation, gave three bands, corresponding to isozymes I, II, and III. It was later shown (44), that when monomers of isozymes I and III are mixed before reassociation, three bands are obtained. This... 

It appears reasonable that because several types of CYP are associated with the endoplasmic reticulum, various inducers may induce one or more of them. Because each of these types has a relatively broad substrate specificity, differences may be caused by variations in the extent of induction of different cytochromes. Now that methods are available for gel electrophoresis of microsomes and identification of specific isoforms by immunoblotting and isoforms-specific antibodies, the complex array of inductive phenomena is being more logically explained in terms of specific isozymes. 

Figure 2. Densitometer tracings illustrating the isozyme migration patterns of five EAP phenotypes during electrophoresis for 4 hr at ()°C. The s and s storage bands can also be seen.

Figure 3. Densitometer tracings and accompanying photographs of EAP phenotype B illustrating the loss of activity of the b isozyme and increase in activity of the c isozyme during storage at 25°C. Samples were taken from clotted blood. Electrophoresis was carried out for 15 hr at 5°C. The successive densitometer tracings shown were made from the EAP phenotype B pattern located on the left in the photograph.

Figure 6. The effect of neuraminidase on the EAP phenotype patterns obtained during electrophoresis for 4 hr at 0°C. Samples 1 4 contained no neuraminidase. Samples 5-9 contained 10 units of neuraminidase activity per ml. All samples were incubated at 25°C. The photograph taken at 114 hr was exposed for 6 min to enhance EAP activity observed in samples 1 4. A 2-min exposure was used for photographs taken at the other times. The only EAP activity observed after incubation with neuraminidase for 114 hr was the c isozyme band of sample 5 (EAP phenotype AC). The c isozyme is the most stable of the four EAP isozymes (5).

CB, AC, and C (the rare type C was not observed in this study). Electrophoresis for 4 hours at 0 C consistently gave sharper EAP banding patterns than electrophoresis for 15 hours at 5 C. The a isozyme of types AB and AC was well defined during 4 hour electrophoresis and made the typing of EAP phenotype AB reliable and simple. 

Fig. 6.4 Affinity electrophoresis of the recombinant phosphorylases from potato tuber.,3) A crude bacterial cell extract containing the type-L isozyme (lane 1), the chimeric enzyme (lane2), or the type-H isozyme (lane 3) was electrophoresed in 5% polyacrylamide gels supplemented with 0 (A), 50 (B), and 500 pg/ml (C) glycogen. After electrophoresis, the gels were stained for enzyme activity in KI-b solution. (Reproduced with permission from Goldsmith and Fletterick, Pure and Appl. Chern., 55(4), 583 (1983)).

A number of immunological techniques have been used in comparative studies.8,9 The most important of these is microcomplement fixation (MC F), a quantitative technique that has played a key role in many classic studies of molecular evolution and molecular systematics. By selecting proteins with different rates of evolution, a broad range of divergences can be examined. The cost of the technique is moderate, but biochemical expertise is required and the labor involved is substantial. Protein must be purified from some or all taxa for antibody production, and, for those taxa, a sizable tissue or serum sample is needed. Antibody production itself is usually done in rabbits, so an animal care facility must be available. Like isozyme electrophoresis, the large body of immunological distance data already available ensures the continued value of this technique for certain investigations. 

Item Isozyme electrophoresis MCF DNA-DNA hybridization Restriction analysis RAPD Sequencing PCR Cloning... 

The combination of horizontal slab-gel electrophoresis and in situ assay of enzyme activity is a versatile and powerful method for detecting protein variants (isozymes and allozymes) in plants. Such variants are potentially useful as genetic markers for mapping chromosomes and in studies of breeding systems,1 population structure,2,3 gene flow,4 polyploidy,5,6 and systematics.7-9... 

C. W. Morden, J. Doebley, and K. F. Schertz, A Manual of Techniques for Starch Gel Electrophoresis of Sorghum Isozymes. Texas Agricultural Experiment State Publication MP-1635, The Texas A M University System, College Station, Texas 77843, 1987. 

If a solution contains two isozymes of an enzyme in equal amounts (as determined by gel electrophoresis) and it is known that the fcca, of each is the same but that the Km for the substrate in one is 1 mmol L-1 and the other is 0.1 mmolL-1, comment on the appearance of the Lineweaver-Burk plot of initial velocity data obtained for nine substrate concentrations ranging from 0.02 to 5 mmol L-1. 

The enzyme can occur in a variety of multiple molecular forms, named isoenzymes or isozymes. Such isoenzymes have the same enzymatic activity but can be separated by electrophoresis. Nagle and Haard (1975) separated the isoperoxidases of bananas into six anionic and one cationic component by gel electrophoresis. By using other methods of separation, an even greater number of isoenzymes was demonstrated. 

Ettlinger et at (83) suggested that isozymes not stimulated by ascorbate also exist. Although the two forms separated by Tsuruo et at (81) were both stimulated by ascorbate, some of the 3-5 isozymes demonstrated by gel electrophoresis in extracts of Sinapis alba, Brassica napus,... 

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