Human Erythrocytic Acid Phosphatase

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. 

The enzyme solution was then treated with ammonium sulfate to 55% saturation. The precipitated enzyme was centrifuged, dissolved in a minimal volume of water (about one seventy-fifth that of the eluate) and dialyzed overnight against several hundred volumes of 0.01 M acetate, pH 5.0. Any precipitated protein was centrifuged off the color of the resulting solution was a dark brown red due chiefly to the presence of catalase and hemoglobin. The degree of purification was approximately 400-fold that of the crude hemolysate. 

The dialyzed enzyme solution was now subjected to a repetition of the preceding procedures admixture of suflBcient calcium phosphate gel to adsorb protein but leave the enzyme in solution centrifugation and addition of more gel to the supernatant to adsorb the enzyme elution of the enzyme from the gel with a mixture of 0.15 M acetate and 0.015 M citrate at pH 4.5 addition of solid ammonium sulfate to the eluate to 55% saturation and precipitation of the enzyme. At this stage, the purifications ranged from 650- to 1100-fold with a recovery of approximately 20-30% of the activity present in the crude red cell hemolysate. Solution of this precipitate, dialysis treatment with solid ammonium sulfate and collection of the precipitate appearing between 40 and 55% saturation yielded a preparation that represented a 1500-fold purification. The preparations were stable when left sedimented in the ammonium sulfate solution. 

A much purer preparation of acid phosphatase from horse erythrocytes was obtained by Ito et al. (12) by adding the DEAE-chromatography procedure to the method of Tsuboi and Hudson (T2). Since this procedure may be applicable to human erythrocytes, it will be mentioned briefly. One liter of horse erythrocytes was washed and lysed by the addition of 4 liters of distilled water. One liter of calcium phosphate gel suspension was added to the hemolysate to remove most of the nonenzymatic protein, and the mixture was centrifuged. Five liters of the gel suspension were added to the supernatant, resulting in the adsorption of the enzyme. The enzyme was eluted with citrate-acetate buffer, pH 4.5, and solid ammonium sulfate was added to the eluate up to 60% saturation. The precipitate was collected, dissolved in 40 ml of water, dialyzed against water at 5°C for 10 hours, and again subjected to calcium phosphate gel adsorption, elution, and precipitation with solid ammonium sulfate to 60% saturation. 

Georgatsos (Gl) failed to obtain any fractions upon applying whole hemolysates to Sephadex G-75 or G-100. However, when he precipitated the acid phosphatase with acetone, washed the precipitate twice in acetone, then extracted the resulting dry powder with 0.14 Jlf NaCl, he obtained an active preparation of acid phosphatase. Application of aliquots of this extract to Sephadex G-75 and elution with 0.14 M NaCl resulted in two peaks. The first peak had two pH optima, one at pH 5.0 and another at pH 6.0. It was activated by Mg optimally at a concentration of 6.6 mJlf. The second peak had a pH optimum at 5.0 and was not affected by Mg. Conversely, fluoride at a concentration of 10 mM inhibited the enzyme activity in the first peak to the extent of 47% but did not affect that in the second. As Georgatsos (Gl) has pointed out, the conflicting results obtained by different investigators may be due to the change in proportion of these two components as purification proceeds from the crude hemolysate. 

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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,... 

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). 

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). 

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. 

Another procedure to increase the specificity of acid phosphatase determinations for prostatic disease has involved the use of n- (-I-) -tartrate to distinguish between the enzyme from the prostate and other tissues. In a series of papers from 1947 to 1949, Abul-Fadl and King (Al, A2, A3, A4) studied the properties of various acid phosphatases and reported that 0.01 Af L- (4-) -tartrate inhibited the hydrolysis of phenyl phosphate by human prostatic acid phosphatase dissolved in normal saline or in plasma to the extent of 95%, but had no effect on the hydrolysis by acid phosphatase from erythrocytes. The inhibitions of acid phosphatases from other human tissues were as follows liver, 70% kidney, 80% spleen, 70%. 

Acid phosphatases are produced by erythrocytes, the liver, kidney, spleen, and prostate gland. The enzyme of the prostate gland is clinically important, because its increased activity in the blood can be an indication of prostate cancer. The phosphatase from the prostate gland is strongly inhibited by tartrate ion, but acid phosphatases from other tissues are not. How can this information be used to develop a specific procedure for measuring the activity of the acid phosphatase of the prostate gland in human blood serum ... 

The results of that experiment allow one to synthesize a-D-ribose-l-[l80U]-phosphate which can be employed to determine the position of bond cleavage by other enzymes whose role is transfer of phosphate (Pj) to water or to another acceptor. We report results on a. the position of bond cleavage in R-l-P by PNP from human erythrocytes and E. coli as well as by alkaline phosphatase, acid phosphatase, formic acid andb. the position of bond making in ribose-5-phosphate by phosphoglucomutase. The earlier experiment from this laboratory employed the equilibration ... 

A more systematic study of the acid phosphatases of erythrocytes and of human prostate was undertaken in 1949 by Abul-Fadl and King (A4). The preparations were crude, the prostatic phosphatase being obtained by grinding human prostate with a 5-fold volume of 0.9% NaCl. The erythrocytic phosphatase consisted of centrifuged red cells, separated from white cells, washed twice with 0.9% NaCl and hemolyzed in 9 volumes of water. The buffer-substrate mixture consisted of equal volumes of acetate buffer (concentration not stated) and 0.02 M disodium phenyl phosphate. 

Comparative Actions op Highly Purified Preparations of Human Erythrocytic and Prostatic Acid Phosphatases on Various Substrates ... 

Polymorphism of Acid Phosphatase in Human Erythrocytes 5.1. Introduction... 

It is of interest that within several years after the observations of Hopkinson et al. (H13), other human erythrocytic enzymes such as phosphoglucomutase, glucose 6-phosphate dehydrogenase, phosphogluco-nate dehydrogenase, adenylate kinase, peptidase, and adenosine deaminase were explored intensively with respect to their polymorphism (H2, HU). However, we shall concern ourselves here only with acid phosphatase. 

In addition to the studies cited above, there are several others showing that phenyl phosphate is much more readily hydrolyzed than j3-glycerophosphate by acid phosphatase from human erythrocytes, whereas no such marked difference exists with respect to human prostatic phosphatase (B2, Tl, T3). Unfortunately, there do not appear to be any systematic investigations of the substrate-velocity relationship for the acid phosphatases of other human tissues. In general, the available data would indicate that /3-glycerophosphate is a more specific substrate than phenyl phosphate for the detection and assay of acid phosphatase coming from the prostate. 

Gl. Georgatsos, J. G., Acid phosphatases of human erythrocytes. Arch. Biochem. Biophys. no, 354-356 (1965). 

Scott, E. M., Kinetic comparisons of genetically different acid phosphatases of human erythrocytes. J. Biol. Chem. 241, 3049-3052 (1965). 

In humans, acid phosphatase from erythrocytes is polymorphic with several alleles expressing the enzyme. This particular... 

Abul-Fadl MA, King EJ (1949) Properties of the acid phosphatases of erythrocytes and of the human prostate gland. Biochem J 45 51-60... 

Abbreviations are LY, hen egg-white lysozyme CON A, demetallized concanavalin A TP, demetallized porcine trypsin tRNA, nonspecific yeast transfer ribonucleic acid CA, human erythrocyte carbonic anhydrase B Hb, human adult carbonmonoxyhemo-globin AP, E. coli alkaline phosphatase TF, demetallized human transferrin IG, human nonspecific -/-immunoglobulin AD, alcohol dehydrogenase from yeast CP, human ceruloplasmin HC1 / 20, l/IO(L), 1/10(0, 1/2, 1/1, various states of association of Helix pomatia hemocyanin. Dashed line m>calculated using Equation 3 with no adjustable parameteis, using the viscosity of pure water to compute v . The proteins were assumed spherical, and a 3J-A hydration layer was included in computing the hydrodynamic radii. After Ref. 7. 

Abul-Fadl, M. A. M., and E. J. King Properties of the acid phosphatase of erythrocytes and of human prostate gland. Biochem. J. 45, 51 (1949). 

A number of nonspecific phosphatases can act on NAN-9-PO4 to dephosphorylate it. However, it appears that in rat liver there is a specific sialic acid-9-phosphatase (Warren and Felsenfeld, 1962). A similar enzyme has been purified some 800-fold from human erythrocyte lysates (Jourdian et al., 1964) and has been shown to be highly specific for sialic acid 9-phosphate (both A/ -acetyl and A/ -glycolyl). This enzyme completes the synthesis of sialic acid in animal tissues. In bacteria, however, another pathway does exist and may be widespread. Extracts of Neisseria meningitidis synthesize sialic acid utilizing free N-acetyl-... 

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