Erythrocytes isoenzymes

The clinical picture includes cramps and recurrent myoglobinuria following intense exercise. Aside from episodes of myoglobinuria, none of the patients was weak. Forearm ischemic exercise caused a 1.5-2.0-fold increase in venous lactate concentration, an abnormally low but not absent response. Muscle biopsy showed normal or only moderately increased glycogen concentration. Because other accessible tissues, such as erythrocytes, leukocytes and cultured fibroblasts, express a different isoenzyme, the diagnosis of PGM-M subunit deficiency must be established by biochemical studies of muscle. Four different... 

A different problem results from deficiency of enzymes of glycolysis such as phosphofructokinase (see Box 20-D), phosphoglycerate rnutase, and pyruvate kinase. Lack of one isoenzyme of phosphoglycerate rnutase in muscle leads to intolerance to strenuous exercise/ A deficiency in pyruvate kinase is one of the most common defects of glycolysis in erythrocytes and leads to a shortened erythrocyte lifetime and hereditary hemolytic anemia.s... 

Many isoenzymes have been identified from various human tissue sources however, our consideration will deal with six erythrocytic systems that have received routine crime laboratory status. These are phosphoglucomutase (PGM), adenylate kinase (AK), adenosine deaminase (ADA), glucose-6-phosphate dehydrogenase (G-6-PD), 6-phosphogluconate dehydrogenase (6-PGD) and erythrocytic acid phosphatase (EAP). 

Another isoenzyme with substantial interest is erythrocytic acid phosphatase (EAP) (8, 9, 10). This system has three autosomal allelic genes termed A, B and C. These can be homozygous or heterozygous giving rise to BA, CA and CB phenotypes. Each of these phenotypes is easily distinguished using starch gel electrophoresis with very useful population frequencies of approximately A - 13%, B - 35%, C - 0.2%, BA - 43%, CA - 3%,... 

Nixon PF, Kaczmarek MJ, Tate J, Kerr RA, and Price J (1984) An erythrocyte transketo-lase isoenzyme pattern associated with the Wernicke-Korsakoff syndrome. European Journal of Clinical Investigation 14, 278-81. 

The presence of acid phosphatase in the human erythrocyte was recognized in 1934 (D4) and properties of this enzyme were studied for almost thirty years (A4, K6, Tl, T2, T4, T5) before its role in human genetics was revealed (H13). This role will be described in detail later. The properties of crude preparations of erythrocytic acid phosphatase have been previously noted in this review. At this point, we shall describe methods of purification, and the nature of the isoenzymes, particularly as they are related to the phenomenon of polymorphism. 

A single polypeptide chain can in theory exist in an infinite number of different conformations. However, one specific conformation generally appears to be the most stable for any given sequence of amino acids, and this conformation is assumed by the chain as it is synthesized within the cell. Thus, the primary structure of the polypeptide chain also determines its three-dimensional secondary and tertiary structures. It is conceivable that in some cases there may be several alternative conformations ("conforraers ) of a single chain that are of nearly equal stabilities and therefore these alternative forms may coexist. This possibility was first suggested to account for the heterogeneity noted in preparations of the cytoplasmic and mitochondrial isoenzymes of malate dehydrogenase and has also been proposed as an explanation of the multiple electrophoretic zones of erythrocyte acid phosphatase. However, no multiple enzyme forms have been shown unequivocally to be due to conformational isomerism. 

LD activity is present in all cells of the body and is invariably found only in the cytoplasm of the cell. Enzyme concentrations in various tissues are about 500 times greater than those normally found in serum. Therefore leakage of the enzyme from even a small mass of damaged tissue increases the observed serum activity of LD to a significant extent. Different tissues show different isoenzyme composition. In cardiac muscle, kidneys, and erythrocytes, the... 

Hemolysis, if sufficiently severe, produces an LD isoenzyme pattern similar to that in myocardial infarction. Megaloblastic anemias, usually resulting from the deficiency of folate or vitamin cause the erythrocyte precursor cell to break down in the bone marrow (ineffective erythropoiesis), resulting in the release of large quantities of LD-1 and LD-2 isoenzymes. Marked elevations of the total LD activity in serum— up to 50 times the upper reference limit—have been observed in the megaloblastic anemias. These elevations rapidly return to normal after appropriate treatment. 

Serum is the preferred specimen for measuring LD activity. Plasma samples may be contaminated with platelets, which contain high concentrations of LD. Serum should be separated from the clot as soon as possible after the specimen has been obtained. Hemolyzed serum must not be used because erythrocytes contain 150 times more LD activity (particularly LD-1 and LD-2) than serum. The different isoenzymes vary in their sensitivity to cold, LD-4 and LD-5 being especially labile. Activity of LD-4 and LD-5 is lost if the samples are stored at -20 C. Thus serum specimens should be stored at room temperature, at which no loss of activity occurs for at least 3 days. 

Serum should be immediately separated from erythrocytes and stabilized by the addition of 50 p,L of acetic acid (5 moi/L) per milliliter of serum to lower the pH to 5.4, at which the enzyme is stable. Under these conditions, TR-ACP activity is maintained at room temperature for several hours, for up to a week if the serum is refrigerated, and for 4 months if stored at -20 °C. Hemolyzed serum specimens are contaminated with considerable amounts of the erythrocyte tartrate-resistant isoenzyme and should be rejected. 

Mukerji SK, Pimstone NR. Uroporphyrinogen decarboxylases from human erythrocytes purification, complete separation and partial characterization of the two isoenzymes. Int J Biochem 1992 24 105-19. 

LDH (M.W. 134,000) oceurs as five tetrameric isoenzymes composed of two different types of subunits. Subunits M (for muscle) and H (for heart) are encoded by loci in chromosomes 11 and 12, respectively. Two subunits used in the formation of a tetramer yield five combinations H4(LDH-1), H3M(LDH-2), H2M2(LDH-3), HM3(LDH-4), and M4(LDH-5). The tissue distributiont of LDH isoenzymes is variable. For example, LDH-1 and LDH-2 are the principal isoenzymes in heart, kidney, brain, and erythrocytes LDH-3 and LDH-4 predominate in endocrine glands (e.g., thyroid, adrenal, pancreas), lymph nodes, thymus, spleen, leukocytes, platelets, and nongravid uterine muscle and LDH-4 and LDH-5 preponderate in liver and skeletal muscle. In tissue injury or insult, the appropriate tissue isoenzymes appear in plasma (Chapter 8) thus, determination of LDH isoenzyme composition has diagnostic value. 

This form may occur in anaerobic tissues, in which lactate is the end product of glycolysis. However, in vitro differences in kinetic properties of the isoenzyme may be inappropriate to explain actual physiological actions for several reasons differences in kinetic properties between LDH-1 and LDH-5 are less marked at 37°C (body temperature) than at 25°C, high intracellular concentrations of the enzyme are present, and differences in the actual ratio of ketopyruvate to enolpyruvate exist (the enol form may be the more potent inhibitor). Furthermore, the occurrence of similar isoenzyme patterns in widely different tissues with divergent metabolic goals (e.g., LDH-5 in liver and musele LDH-1 in heart and erythrocytes) points out our lack of understanding of their preeise role. 

Three genes coding for isoenzymes of carbonic anhydrase I, II, and III, all belonging to the same gene family, are clustered on chromosome 8q22. These are all zinc metalloenzymes, soluble, monomeric, and have molecular weights of 29,000. Isoenzyme I is found primarily in erythrocytes (Chapters 1 and 39) and III in skeletal muscle. There are yet other isoenzymes coded for by different genes. 

In humans, the pi-class isoenzyme is widely distributed and represents the most thoroughly characterized extrahepatic GST. Pi-class GST have been purified from a number of sources, including placenta (G13, K17, P2) and erythrocytes (M8). Although these enzymes are now commonly referred to as GST Ji, the forms isolated from placenta, lung, and erythrocytes were originally named GST jt, GST k, and GST p, respectively. 

Some controversy exists in the literature regarding the possible existence of more than one pi-class GST. The acidic isoenzymes isolated from erythrocytes, lung, and placenta have been shown to share immunological identity and have the same subunit M, value and isoelectric point (A19, H52, K16). However, using nondenaturing starch-gel electrophoresis, both Laisney et al. (L4) and Suzuki et al. (S49) have shown that an acidic isoenzyme present in erythrocytes has a mobility different from that of the GST 3 enzyme observed in other tissues. Other workers have described two pi-class isoenzymes present in skeletal muscle tissue that have minor differences in their isoelectric point (S25). 

At present the use of activity measurements to quantitate plasma, serum, or urinary levels of the GST are inadequate. With CDNB it is difficult to obtain sufficient sensitivity to allow the measurement of the levels in normal subjects (A4). In addition many drugs and endogenous substances may inhibit the activity to values that lie within the reference range. For example bile salts and bilirubin inhibit GST activity (H17) and since both of these nonsubstrate ligands are increased in liver disease their accumulation in plasma could theoretically suppress GST activity to within the reference range. An important problem with GST activity measurements concerns the ubiquitous nature of the GST since poor organ specificity will result unless specific isoenzymes are measured. For example, platelets, erythrocytes, and white cells contain high levels of the isoenzyme and these cells may release their GST into plasma prior to separation of the blood sample (G4, H52, L12, M8, Rll, S43). With the substrates that are available to date, the activity measurements are inadequate for clinical use. 

Zorzano A, Herrera E (1990) Differences in the kinetic properties and sensitivity to inhibitors of human placental, erythrocyte, and major hepatic aldehyde dehydrogenase isoenzymes. Biochem Pharmacol 39 873-878... 

Muscle is the only source of CK and by measuring the isoenzyme CK-MB one can determine whether or not cardiac muscle is involved. If the liver is involved then the serum yGT should be increased as this is one of the most sensitive indicators of liver disease. LDH isoenzyme analysis will help identify erythrocyte damage as a possible source for some of the LDH and AST activity. In haemolytic disorders, one would expect a reticulocytosis and intravascular haemolysis will lead to a low serum haptoglobin level. These investigations will help identify whether or not erythrocytes have contributed to the serum enzymes. 

PFK-1 exists as a group of tissue-specific isoenzymes whose regulatory features match the role of glycolysis in different tissues. Three different types of PFK-1 isoenzyme subunits exist M (muscle), L (liver), and C. The three subunits show variable expression in different tissues, with some tissues having more than one type. For example, mature human muscle expresses only the M subunit, the liver expresses principally the L subunit, and erythrocytes express both the M and the L subunits. The C subunit is present in highest levels in platelets, placenta, kidney, and fibroblasts but is relatively common to most tissues. Both the M and L subunits are sensitive to AMP and ATP regulation, but the C subunits are much less so. Active PFK-1 is a tetramer, composed of four subunits. Within muscle, the M4 form predominates but within tissues that express multiple isoenzymes of PFK-1 heterotetramers can form that have full activity. 

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