Acid phosphatase purification

V6. Vihko, P., Human prostatic acid phosphatases. Purification of a minor enzyme and comparisons of the enzymes. Invest. Urol. 16, 349-352 (1979). 

Pulse ultrasonic relaxation method, 32 18 Pump-and-probe techniques, 46 137 Purification, of actinide metals, see Actinide, metals, purification XjPj Purified protein, 36 94 Purple acid phosphatases, 40 371, 376, 43 362, 395-398, 44 243-245 biological function, 43 395 homology, 43 397... 

Milk acid phosphatase has been purified to homogeneity by various forms of chromaotgraphy, including affinity chromatography purification up to 40 000-fold has been claimed. The enzyme shows broad specificity on phosphate esters, including the phosphoseryl residues of casein. It has a molecular mass of about 42 kDa and an isoelectric point of 7.9. Many forms of inorganic phosphate are competitive inhibitors, while fluoride is a powerful non-competitive inhibitor. The enzyme is a glycoprotein and its amino acid composition is known. Milk acid phosphatase shows some similarity to the phosphoprotein phosphatase of spleen but differs from it in a number of characteristics. 

Lavallee and Rosenkrantz (54) studied the purification of dog prostatic acid phosphatase from prostatic secretion obtained from pilocarpine-stimulated dogs with cystopreputiostomy prostatic fistulas. A 45-... 

Fia. 14. Polyacrylamide gel electrophoresis at various purification steps. Gels 1 to 4 were stained for acid phosphatase activity gel 5 was stained for protein. A current of 4 mA/gel was applied to gels 1-3 for 2 hr and to gels 4 and 5 for 6 hr. Gel 1, homogenate 2, DEAE-cellulose peak II 3, DEAE-cellulose peak I 4 and 5, crystalline enzyme. From Igarashi and Hollander (4). 

Chersi et al. 103) have carried out extensive purification of spleen acid phosphatase. Spleen was fractionated to yield crude spleen nuclease II 104). This preparation was found to contain large quantities of non-... 

Seedlings are a rich source for nonspecific acid phosphatase. Newmark and Wenger (114) have reported on a 1000-fold purification from lupine seedlings. The purified enzyme hydrolyzes phosphate monoesters and pyrophosphate with p-nitrophenyl phosphate as substrate. The optimal activity was at pH 5.2-5.5, and Km was 3 X 10 4 M. Fluoride inhibition was noncompetitive. 

Shaw (115) reported a 300-fold purification of enzyme from tobacco leaves. Activity of the enzyme was optimal at pH 5.5-5.7, and divalent cations were not required for activity. The enzyme possessed high activity toward ribonucleoside 2 - and 5 -monophosphates and glucose 1-phosphate. There was no activity toward RNA or phosphodiesters. Fluoride acts as a noncompetitive inhibitor for this enzyme. This behavior of fluoride is in contrast to the behavior with prostatic acid phosphatase where the inhibition is strictly competitive. 

Acid phosphatase of S. aureus PS 55 is eluted from the cell surface by 1.0 M KC1 at pH 8.5. Gel filtration of this material gave a 44-fold purification. The protein seems homogeneous by gel filtration, starch block electrophoresis, and analytical ultracentrifugation with the weight of approximately 58,000 (12a). 

Paul, E.M. Williamson, V.M. (1987). Purification and properties of acid phosphatase-1 from a nematode-resistant tomato cultivar. Plant Physiology 84, 399-403. 

Hakalahti, L., and Vihko, P. (1989). Purification of monoclonal antibodies raised against prostate-specific acid phosphatase for use in vivo in radioimaging of prostatic cancer. ]. Immunol. Methods 117, 131-136. 

The dephosphorylation of 5-chloro and 5-bromo-D-xylulose-l-phosphate was carried out by the addition of acid phosphatase. After purification, 5-chloro-D-xylu-lose and 5-bromo-D-xylulose were recovered as pure compounds in 47 and 12% yields, respectively, from DHAP. In this study, we have shown that DHAP generated from glycidol 7 can be used in situ as a donor substrate of FruA in the presence of 2-halo-acetaldehydes 20 as acceptor substrates for the synthesis of 5-halo-D-xylulose 19. Given that DHAP aldolases display a broad specificity towards acceptor substrates, this strategy can be applied generally to the synthesis of various analogs of monosaccharides. 

Purification of Acid Phosphatase from Wheat Germ... 

The following solutions are required for purification of acid phosphatase. 

Place 1 qt methanol (tightly capped) in a - 20°C freezer overnight. 10-4. Place at least 2 liters glass-distilled water at 4°C overnight. Figure 10-17 is a schematic flow chart of the operations involved in partial purification of acid phosphatase. Supernatant solutions enclosed in rectangles on the flow chart are to be sampled for later assay of enzyme activity and protein concentration. 

Figure 10-17. Schematic flow chart describing the major steps in the partial purification of acid phosphatase from wheat germ.

Acid Phosphatases from Different Tissues Purification, Isoenzymes, and Properties... 

The purification of acid phosphatase from the human prostate was undertaken, and high degrees of purity were obtained, before any solid information was available concerning the intracellular distribution of this enzyme or its existence in multiple molecular forms or isoenzymes. Accordingly, in this review several methods of purification will be described first, and the other aspects will then be considered. 

This solution was then applied to a column (72 X 3.3 cm) of Dowex 50 X-2 which had been equilibrated with the citrate phosphate buffer (B23). Elution was carried out successively with a citrate phosphate buffer of pH 5.0 and 6.00. About 50 fractions with a volume of 5-7 ml were collected. Two sharp peaks of protein concentration were obtained at about tubes 5-15 and tubes 27-29. The acid phosphatase activity was localized only in the second peak and represented a 10-fold purification. 

The preceding description of the use of chromatographic methods in the purification of prostatic acid phosphatase (B24, 04) has already indicated that this enzyme exists in more than one molecular form, or isoenzyme. There is, in addition, immunological (S19) and starch gel electrophoretic evidence (L14, L15, S24, S31) of the existence of several forms. In order to ensure that no isoenzymes are lost during any purification, it is preferable to perform such studies on a homogenate of the whole tissue. It should be recognized that the isoenzymatic composition may not be characteristic of the prostatic cell per se, but may also represent components from blood cells, secretory ducts, connective tissue, and other sources. 

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. 

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. 

The purification of acid phosphatase from the human liver and the description of its properties do not appear to have been accomplished. Partly, this may be due to the inherent difiBculty of obtaining normal, fresh human material in amounts substantial enough for purification. However, because of the cellular and physiological importance of acid phosphatase, it is advisable to describe in the present section the purifications of the enzyme from rat and bovine liver. Moreover, since these purifications were accomplished with the awareness that acid phosphatase from this source might be present in multiple molecular forms, the descriptions will naturally involve a consideration of the isoenzymes and their properties. 

A 336-fold purification by column chromatography was achieved by Brightwell and Tappel (B32) from lysosomes obtained by differential and density sucrose gradients. The lysosomes were frozen and thawed several times, then centrifuged. The resulting soluble acid phosphatase fraction was dialyzed against suitable buffers and then applied to a DEAE-cellulose column or to a CM-cellulose column. Each column was eluted with a linear 0-1 M NaCl solution, the former at pH 7.2 and the latter at pH 5.6. Two peaks of acid phosphatase activity were ob-... 

The solution was divided into two 40-ml portions, and each portion was added to a column of Sephadex G-75 that had been equilibrated with 0.01 M sodium acetate, 1 mM EDTA, pH 4.8. Elution was continued in the same buffer. Gel filtration of a crude extract of bovine liver on Sephadex-75 had previously given two small peaks and a third large peak of acid phosphatase activity. Elution of the purified 35-50% ammonium sulfate fraction now gave a small peak of about 10% of the enzyme activity, no second peak, and a third peak that contained 90% of the enzyme. The third peak (acid phosphatase III) represented the low molecular weight component and constituted 30% of the total acid phosphatase present in the 15,000g supernatant starting material the degree of purification was 54-foId. 

This final acid phosphatase preparation had a specific activity of 468 and represented an approximately 1900-fold purification of the acid phosphatase in the starting crude spleen nuclease II. It contained no acid deoxyribonuclease, acid ribonuclease, exonuclease, and phosphodiesterase activities that could be detected in a 0.1-ml sample after 2 hours of incubation with the appropriate substrate. The relative rates of hydrolysis of various substrates were as follows p-nitrophenyl phosphate, 100 5 -AMP, 63 j8-glycerophosphate, 60 ATP, 0. With p-nitrophenyl phosphate as substrate, the pH optimum was broad and lay between pH 3.0 and pH 4.8. The Michaelis constant at 37°C was 7.25 X 10" mM. Phosphate and chloride ions acted as competitive inhibitors. 

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