Electrophoresis lactate dehydrogenase

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.

Rosalki, S. B. Standardization of isoenzyme assays with special reference to lactate dehydrogenase isoenzyme electrophoresis. Clin. Biochem. (1974), 2> 29-40. 

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. 

Lactate dehydrogenase exists in the cytoplasm of humans and most animals as five forms which are easily separable by electrophoresis and are evenly spaced on electropherograms.8 This enzyme is a tetra-mer made of two kinds of subunits. Isoenzyme 1, which has the highest electrophoretic mobility, consists of four identical type B subunits. The slowest moving tetramer (isoenzyme 5) consists of four type A subunits, while the other three forms, AB3, A2B2, and A3B, contain... 

Lactate dehydrogenase occurs as a tetramer with two kinds of subunits designated H for heart and M for muscle. Five different LDHs are separable and identifiable by electrophoresis. The composition of these and their major tissue locations are as follows ... 

Agarose gel electrophoresis (typically 0.7-1 % agarose w/v) is increasingly used in clinical laboratories in place of cellulose acetate, for example in zone electrophoresis of serum proteins, isoenzyme analysis of lactate dehydrogenase and creatine kinase, and immunoelectrophoresis. 

Enzyme activity can be assayed using the same detection methods discussed above to determine metabolite concentrations. Spectroscopic methods are very important since they allow the progress of reaction to be monitored directly, which is much more convenient than sampling methods which may be based on chromatography, electrophoresis or the use of radioactive isotopes. As an example, the activity of lactate dehydrogenase can be assayed by following the reduction of NAD+ to NADH from the absorbance increase at 340 nm... 

Disc Gel Electrophoresis of Lactate Dehydrogenase Using Nitroblue Tetrazolium for Enzyme Visualization... 

Enzyme catalyzed reactions have also been studied at the single molecule level. Earlier work involved measurement of beta-galactosidase activity in droplets after a 10-15 h incubation. In a recent study, detection of fluorescent product generated by individual molecules of lactate dehydrogenase after a 1 hr incubation has been achieved using capillary electrophoresis . The activity of individual molecules were reproducible but activity of different molecules showed a 5-fold range. The differences in activity were suggested to reflect differences in conformation. 

Enzymes which exist in multiple forms within a single species of organism or even in a single cell are called isoenzymes or isozymes. Such multiple forms can be detected and separated by gel electrophoresis of cell extracts. Since they are coded by different genes, they differ in amino acid composition and thus in their isoelectric pH values. Lactate dehydrogenase is an example for the isoenzymes which occur as five different forms in the tissues of the human and other vertebrates. All the five isozymes catalyze the same reaction. 

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). 

Reflect and Apply The M and H subunits of lactate dehydrogenase have very similar sizes and shapes but differ in amino acid composition. If the only difference between the two were that the H subunit had a glutamic acid in a position where the M subunit had a serine, how would the five isozymes of LDH separate on electrophoresis using a gel at pH 8.6 (See Chapter 5 for details on electrophoresis.)... 

Wieland Theodor bora in 1913 in Munich, he studied chemistry in Freiburg Brsg. and Munich where he received his Dr. phil. in 1937. From 1937 until 1946 he was assistant, (Lecturer 1941), with Richard Kuhn at the Kaiser-Wilhelm/Max-Planck-Institute in Heidelberg. In 1946 he became Prof, at the Univ. of Mainz. Between 1951 and 1968 he was Prof, and Director of the Institute of Organic Chemistry at the Univ. of Frankfurt. After R. Kuhn s death in 1967, he was appointed Director of the Department of Chemistry, later Natural Products, of the Heidelberg Institute until 1981. He is hon. prof, of the Universities of Frankfurt and Heidelberg. His contributions include electrophoresis on paper of amino acids, peptides and proteins, isoenzymes of lactate dehydrogenase (with G. Pfleiderer), mixed anhydride method of peptide synthesis (p. 79) peptides of Amanita mushroonis (p. 211). 

Probably the most fully studied group of isoenzymes is that of lactate dehydrogenase (LDH). The quaternary structure of the enzyme consists of four subunits that are of two different types. One is labeled H because it is the predominant subunit present in the LDH enzyme found in heart muscle cells. The other, labeled M, predominates in other muscle cells. There are five possible ways to combine these four subunits to form the enzyme (see Figure 20.14). Each combination has slightly different properties, which allows them to be separated and identified by electrophoresis. 

Fig. 81. Iliotograph of starch gel after electrophoresis of crystalline preparations of lactate dehydrogenase from bovine muscles (special reacticn for enzyme LDH-1, LDH-2, etc., isozyme fractions) (Markert and Appella, 1963).

A typical spectrum of individual lactate dehydrogenases (LDH) of muscles after electrophoresis on starch gel is shown in Fig. 81 (Markert and Appella, 1963). These five fractions, as mentioned previously, are determined by (he existence of two genetic loci for synthesis of two types of protein subunits, specific as regards amino acid composition and antigenicity, designated A and B, and by the presence of a tetramer structure of the principal protein (AAAA-LDH-5 AAAB-LDH-4 AABB-LDH-3 ABBB-LDH-2 BBBB-LDH-1). 

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