Erythrocytes acid phosphatase

Chronic in vivo hemolysis produces serum lactic dehydrogenase elevations in patients with mitral or atrial valve cardiac prosthesis (J2). In a series of 11 such patients these increases ranged from 1.1 to 1.6 times the upper limit of normal (S29). Blood pH is altered in hemolyzcd specimens because carbonic anhydrase is liberated from the erythrocytes and presumably alters the distribution of H2CO3 and NaHCOs (B2). Hemolysis will effect acid phosphatase activity if the substrate is hydrolyzed by erythrocyte acid phosphatase. Thus, hemolysis would be of concern if phenyl phosphate was the substrate, but would have a negligible effect if )8-glycerophosphate, which is not hydrolyzed by red cell acid phosphatase, was used (Bl). 

The Relative Ease of Hydrolysis of Various Substrates by Prostatic and Erythrocytic Acid Phosphatases 1... 

Group Substrate Normal range (units/100 ml) Serum plus prostatic acid phosphatase Serum plus erythrocytic acid phosphatase Heated serum Relative specificity for prostatic acid phosphatase... 

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%,... 

CB - 6%. Erythrocytic acid phosphatase has been found to remain viable for many months after drying and successful typing can be performed on a minimum of several threads (9), (Figure 2). 

Figure 2. Erythrocytic acid phosphatase schematic. A schematic drawing illustrating typical results of an EAP determination in a 13%, 1mm starch gel prepared in 0.24M Nall TO, 0.15M trisodium citrate tannic acid buffer diluted 1 100. The electrophoresis is carried out for 41/2 hr at approximately 410 V. The gels are stained by the fluorescence produced after enzymatic hydrolysis by methylumbellifertjl phosphate at 37° C for 1V2 hr.

Effect of Environmental Factors on Starch Gel Electrophoretic Patterns of Human Erythrocyte Acid Phosphatase... 

Human erythrocyte acid phosphatase (EAP) polymorphism was first described by Hopkinson, Spencer and Harris (1). EAP can be classified by electrophoresis into six different phenotypes,... 

The erythrocytic acid phosphatase from man and several other species showed two pH optima, one at a range of pH 4.3-4.8 and the second at pH 5.0-5.7. A concentration of 0.01 M Mg inhibited these activities to the extent of about 30-50% at the lower pH levels and somewhat less so in the region of the higher pH optimum. Human prostatic acid phosphatase showed one clear pH optimum, at about 5.0-5.2, and the inhibition by 0.01 M Mg + was about 30% in this region. 

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. 

Substrate Relative activities as Erythrocytic acid phosphatase percent of maximum Prostatic acid phosphatase... 

We have already discussed the properties of human erythrocytic acid phosphatase (Section 3.3), and we pointed out that, like acid phosphate in other tissues, it may exist in several isoenzymatic forms. In 1963, Hopkinson et al. (H13) subjected hemolysates of human red cells from an English population to horizontal starch-gel electrophoresis for 17 hours at 5°C. The gels were then sliced horizontally, covered with 0.05 M phenolphthalein sodium diphosphate at pH 6.0, and allowed to incubate for 3 hours at 37°C. Five different electrophoretic patterns of acid phosphatase activity could be distinguished in different individuals. Shortly thereafter Lai and his associates (L2) confirmed these findings and discovered an additional sixth pattern which had been predicted by Hopkinson et al. (H13). The distribution of these patterns in various types of population was assiduously pursued within the next several years, and several new ones were discovered in Negro populations (G3, K2). 

The phenotypes of erythrocyte acid phosphatase not only exhibit differences in electrophoretic behavior, but also show variation in total acid phosphatase activity. Spencer et al. (S26) studied the distribution of red cell phosphatase activities in hemolysates from 275 individuals with various phenotypes. The assay was performed with 0.01 M disodium p-nitrophenyl phosphate as substrate at pH 6.0 in citrate buffer. The units of activity were expressed as /unoles of p-nitrophenol liberated in 0.5 hour at 37°C per gram of hemoglobin. These results are shown in Table 5. 

Differences in the electrophoretic patterns and activities between the phenotypes raised the possibility that they might also be characterized in a more specific and detailed biochemical manner. In 1949, Abul-Fadl and King (A4) observed that 0.5% formaldehyde inhibited erythrocyte acid phosphatase completely, whereas 0.01 Af l-( + )-tartrate had no effect. When 0.5% formaldehyde was added to the reaction mixture in gel electrophoresis (H13), it completely inhibited all variants of erythrocyte acid phosphatase, so that no zones of activity in any of the five types were visible after the 3-hour incubation. 

B27. Bottini, E., Lucarelli, P., Agostino, R., Palmarino, R., Businco, L., and Anto-gnoni, G., Favism Association with erythrocyte acid phosphatase phenotype. Science 171, 409-411 (1971). 

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. 

Samples for this assay may be cell supernatant from cell culture experiments, cell lysates, tissue lysates, or biological fluids such as serum or plasma. High degree of hemolysis in serum or blood in tissue samples interferes with the measurement due to the presence of erythrocyte acid phosphatase. 

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