Isozymes of LDH

Isoenzymes or isozymes are enzymes from a single species that have the same kind of enzymatic activity but differ in chemical structure. In addition, they may differ in quantitative characteristics such as possessing different Km s with the same substrate and may differ in response to temperature and effectors. Isozymes of more than 100 enzymes have been demonstrated in humans. The most important of these for diagnostic purposes are the isozymes of LDH, CK, alkaline phosphatase, leucine aminopeptidase, acid phosphatase, and aldolase. These have been exploited for differential organ diagnosis. 

D-7) Serum LDH (lactate dehydrogenase) eleva- tion. LDH is a widespread intracellular enzyme that acts at this step. Detection of high serum levels is rather nonspecific for localizing the site of damage, but its measurement is helpful in confirming myocardial infarction or injury to the liver, skeletal muscle, or certain other tissues. The presence of 5 different isozymes of LDH helps to further localize the injury, as they are fairly tissue specific. For instance, LDj and LD2 are elevated in myocardial infarction, LD2 and LD3 elevation occur in acute leukemia LDs elevation follows liver or skeletal muscle injury. 

There are five possible isozymes of LDH HHHH, HHHM, HHMM, HMMM, and MMMM. We could have calculated the number from Equation 7, which gives the total number of quantitatively different ways of arranging n objects in groups of r ... 

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.)... 

There would be 15 possible isozymes of LDH, combining three different subunits into combinations of four. Besides the five isozymes containing only M and H, there would also be C, CH, C H, C H, CH M, C HM, C M, CHM, C M, and CM. ... 

Figure 26 demonstrates the organospecific features of the distribution of the isozymes of LDH (Marker , 1964). These differences in distribution are also apparent at the cellular level. For instance, LDH-1 was identified in frog egg yolk plates and in the connective tissue of the oviduct. LDH-3 was found in the cytoplasm of these eggs, certain cells of the oviduct epithelium, and, also, in the oviduct secretory glands. LDH-2 was not found in the egg, but appeared in some of the epithelial cells of the oviduct (Nace et al., 1960 Nace, 1963). 

Neurons and glial cells in hippocamp and the medula of the rat differ in their LDH spectrum in the former fast isozymes are prevalent, and in the latter slow isozymes are the most common form (Alexidze and Chaglid, 1970 Korochkin, 1972 b Pevsner, 1972). The fast isozymes of LDH are in the majority in the ascending part of the loop of Henley in the kidney and in the distal convoluted tubules. Slow isozymes are prevalent in the collecting ducts and vasa recta (McMillan, 1967). 

Some authors (Wachsmuth et ah, 1969), using specific inhibitors for the A and B subunits of LDH, demonstrated histochemically that isozymes of LDH are distributed unevenly within liver lobes. In the middle portion of the lobe LDH-5 is prevalent, and on the periphery LDH-1, LDH-2, and LDH-3 also appeared in substantial concentrations. These data were confirmed in our laboratory with the im-munohistochemical method of Coons. Characteristically, various mouse strains demonstrated considerable variability in this parameter, especially in the activity of the LDH fast fractions (Leonova, 1974). 

Similar qualitative and, more often, quantitative features of the spatial distribution of isozymes in tissues and cells is peculiar not only to isozymes of LDH. Many, if not all, isozymes and enzymes, controlled by both allelic and nonallelic genes, display differences of this type. 

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.

Plasma hormones and enzymes of testicular origin were used as markers for evaluation of acute testicular toxicity in rats treated with 1,3-DNB. Lactate dehydrogenase isozyme C4 (LDH-C4) and ABP were both elevated after treatment with doses between 10 and 25 mg/kg of 1,3-DNB (Reader et al. 1991). Testosterone levels were reduced after treatment with 10 and 32 mg/kg of 1,3-DNB (Reader etal. 1991 Rehnberg et al. 1988). 

In skeletal muscle the predominant isozyme contains four M chains, and in heart the predominant isozyme contains four H chains. Other tissues have some combination of the five possible types of LDH isozymes ... 

Different stages of development in embryonic or fetal tissues and in adult tissues. For example, the fetal liver has a characteristic isozyme distribution of LDH, which changes as the organ develops into its adult form. Some enzymes of glucose catabolism in malignant (cancer) cells occur as their fetal, not adult, isozymes. 

Figure 5. Lactic acid dehydrogenase schematic. This schematic illustrates the value of LDH isozyme patterns for the identification of menstrual blood and seminal material.

For the diagnosis of myocardial infarction, it is not only the detection of CK 2-MB that is important but also the contribution of the CK 2 fraction to the total CK activity. Activities of CK 2 in excess of 5% of the total CK activity should be regarded with suspicion. CK 2 as well as total CK activity will increase within a few hours of the acute episode. Maximum activity of CK 2 may precede that of total CK and remain elevated until the third or fourth day. LDH 1 release occurs 1-2 days after the appearance of CK (Figure 5.16). LDH 2 activity is normally higher than that of LDH 1. However, in myocardial infarction, the activity of LDH 1 will exceed that of LDH 2 near the time the CK 2 returns to normal levels. Thus, the judicious choices of the proper enzymes and the appropriate time, coupled with isozyme determinations, can provide a considerable degree of diagnostic certainty. 

Figure 10.27. Isozymes of Lactate Dehydrogenase. (A) The rat heart LDH isozyme profile changes in the course of development. The H isozyme is represented by squares and the M isozyme by circles. The negative and positive numbers denote the days before and after birth, respectively. (B) LDH isozyme content varies by tissue. [(A) After W.-H. Li, Molecular Evolution (Sinauer, 1997), p. 283 (B) After K. Urich, Comparative Animal Biochemistry (S nngQrVQrlag, 1990), p. 542.]...

Enzymes, which are normally produced in cells, are released into the blood when cells are injured. For example, after a heart attack, there is an increase in blood levels of creatine phosphokinase (CPK) and other enzymes such as lactate dehydrogenase (LDH). The extent of damage and the rate of recovery can be estimated by periodically measuring the levels of these enzymes. Measurement of the MB isozyme of CPK is also used as an aid in diagnosis. 

Measurement of serum concentration of total CK and CK-2 (i.e., MB) have long been used to assess the extent of myocardial damage in suspected MI (Chapter 8). Measurement of LDH isozymes has been used similarly. In recent years, however, reliance on cardiac troponin assays has increased and use of CK and LDH assays will probably decrease. 

LDH is a tetrameric enzyme occurring in several isozymes. Knowledge of the total activity of LDH in serum is important for the differential diagnosis of heart and liver diseases and pernicious anemia. The normal levels are up to 240 U/l. 

These simple measurements of enzyme activity may not be completely specific. For example, damage to the liver or skeletal muscle will also raise LDH in serum. However, the LDH released from heart muscle is different from that released by the liver or skeletal muscle. The different enzymes are called isoenzymes, that is, they catalyze the same reaction. In the case of LDH, the isoenzymes (or isozymes) are regulated differently and match the metabolic requirements of the appropriate tissues. 

This isozyme is chemically and immunologically distinct from the more widely distributed forms of LDH (84,85) however, its subimits (designated C) can form enzymically active hybrids with H in vitro and with M in vivo and in vitro (88). It has been isolated from mouse testes in a crystalline form (85) that is suitable for X-ray diffraction studies. (87). 

Serum levels of lactate dehydrogenase (LDH) were formerly used to diagnose an acute Ml. LDH is present in cells as a tetramer of four identical, or nearly identical, subunits. Each subunit is a separate polypeptide chain with a molecular weight of 35 kD (approximately 35,000 g/mole). These subunits are present as two isoforms, the H isoform (for heart) and the M isoform (for skeletal muscle). Although the heart produces principally the H4 form (four H subunits combined into a tetramer) and skeletal muscles produce principally the M4 isoform, the heart, skeletal muscle, and other tissues produce several intermediate combinations (e.g., H3M, HjMj). These tetrameric isoforms all have similar activities, but the individual monomeric subunits are inactive. Measurements of LDH isozymes in the serum are no longer used for diagnosis of a recent Ml because the enzyme is large, released slowly, and the isozyme pattern is not as specific for the heart as is CK. 

Reflect and Apply Many species have a third type of LDH subunit that is found predominantly in the testes. If this subunit, called C, were expressed in other tissues and could combine with the M and H subunits, how many LDH isozymes would be possible What would their compositions be ... 

Goto A, Takeuchi S, Sugimura K, Maruo T (2002) Usefulness of Gd-DTPA contrast-enhanced dynamic MRI and serum determination of LDH and its isozymes in the differential diagnosis of leiomyosarcoma from degenerated leiomyoma of the uterus. Int J Gynecol Cancer 12 354-361... 

Initial data in the study of isozymes were received in the investigation of the isozymes of lactate dehydrogenase (LDH) whose genetic control had been sufficiently studied. This enzyme plays and important role in glycolysis, and catalyzes the reversible reaction. 

Fig. 26. Zymogram of LDH isozymes from tissues of the rhesus monkey. Note that each tissue contains distinctive proportions of the isozymes. All five are present in every tissue although quantities of LDH-5 in some of the tissues are too small to be evident on this photograph. (After Markert, 1964)...

In our laboratory a variation of microelectrophoretic technique was developed. This method, in contrast to other microelectrophoretic methods (Alexidze and Chaglid, 1970 Rosenberg, 1970), makes possible the determination of LDH isozymes in any size cell, not only in large cells but in small cells as well (Korochkin, 1972b,c Korochkin et al., 1972b). The principles of this technique are demon-... 

Specific peculiarities in the subcellular distribution of LDH isozymes were found. The fast fractions were obtained mainly in the mitochondria, however the... 

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