Phosphatase-Human Prostatic Acid

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. 

The combined extracts, which represented a 22-fold purification from the prostate, were dialyzed for 24 hours against distilled water at room temperature. The material inside the cellophane bag separated into a precipitate, which accounted for 60-70% of the total protein, and a clear supernatant containing over 85% of the activity and representing a 41-fold purification. This supernatant was then mixed with calcium phosphate gel at pH 7.5 and filtered. The filter cake was eluted with 0.02 M sodium citrate at pH 7.0, and the cake was washed with distilled water. The combined eluate, which showed an 81-fold purification, was concentrated by lyophilization. The enzyme solution was 

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. 

A substantially different procedure for purification was employed by Ostrowski and his co-workers (03, 04). The frozen human prostate gland was sliced into sections and weighed, and 5 volumes of 0.01% Tween 80 solution in distilled water was added. The mixture was homogenized in a Waring Blendor for 30 seconds at 13,000 rpm, stored in the cold 

The enzyme solution, FI, was brought to pH 7.0 powdered ammonium sulfate was added up to 45% saturation, and the solution was adjusted with ammonia to pH 7.0. The solution was cooled in the refrigerator for 24 hours, then centrifuged for 20 minutes at 3000 rpm, and the precipitate was discarded. To the supernatant ammonium sulfate was added up to 65% concentration and the pH readjusted to 7.0. After refrigeration as before, the precipitate was collected by centrifugation at 7000 rpm for 20 minutes. 

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Lin M-F, Meng T-C, Rao PS, et al. Expression of human prostatic acid phosphatase correlates with androgen-stimulated cell proliferation in prostate cancer cell lines. J. Biol. Chem. 1998 273 5939-5947. 

Although detailed structural as well as mechanistic knowledge of an enzyme is desirable, it is by no means necessary in order to design a suicide substrate. This has been shown by Myers and Widlanski (1993) who have designed a simple inhibitor of human prostatic acid phosphatase (PAP), an enzyme that is believed to be involved in the regulation of androgen receptor activity in prostate cells. Since the enzyme shows a preference for hydrolysis of aryl phosphates, the 4-(fluoromethyl)-phenyl phosphate (FMPP) was prepared as a substrate that would, on hydrolysis by the... 

Table XI (73) shows the Stokes radii and frictional ratio obtained by the study of purified acid phosphatase. The preparations show molecular homogeneity during filtration on Sephadex G-100, in the analytical ultracentrifuge, and during immunolectrophoresis. These data obtained by chromatography on Sephadex G-200 indicate that human prostatic acid phosphatase has an effective Stokes radius of 47.1 A and a frictional ratio of 1.56, suggesting considerable molecular asymmetry.

Putrefactive hacteria, such as Clostridium welchii, which frequently invade human blood during the agonal period or immediately after death, produce the enzyme neuraminidase ( 9). Neuraminidase has been shown to effect the heterogeneity of electrophoretic banding patterns of the human prostate acid phosphatase (10). The effect of this enzyme on EAP is not known. 

Smith and Whitby (10) found that incubation of the human prostatic acid phosphatase in the presence of neuraminidase... 

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%. 

D3. Davidson, H. M., and Fishman, W. H., A simplified purification procedure for human prostatic acid phosphatase based on pH and ammonium sulfate fractionation. J. Biol. Chem. 234, 526-528 (1959). 

Vihko P (1978) Human prostatic acid phosphatase and its radioimmunoassay. Acta Universitatis Ouluensis, Series D Medica 33, Chnica Chemica 1 1-78... 

Luchter-Wasylewska E (2001) Cooperative kinetics of human prostatic acid phosphatase. Biochim Biophys Acta 1548 257-264... 

Peters C, Geier C, Pohlmarm R et al (1989) High degree of homology between primary structure of human lysosomal acid phosphatase and human prostatic acid phosphatase. Biol Chem Hoppe Seyler 370 177-181... 

Lin ME, DaVolio J, Garcia-Arenas R (1992) Expression of human prostatic acid phosphatase activity and the growth of prostate carcinoma cells. Cancer Res 52 4600-4607... 

Beers SA, Schwender CF, Loughney DA et al (1996) Phosphatase inhibitors-III. Benzylaminophosphonic acids as potent inhibitors of human prostatic acid phosphatase. Bioorg Med Chem 4 1693-1701... 

Ortlund E, LaCount MW, Lebioda L (2003) Crystal structures of human prostatic acid phosphatase in complex with a phosphate ion and alpha-benzylaminobenzylphosphonic acid update the mechanistic picture and offer new insights into inhibitor design. Biochemistry 42 383-389... 

Vihko P, Kontturi M, Korhonen LK (1978) Purification of human prostatic acid phosphatase by affinity chromatography and isoelectric focusing. Part I. Clin Chem 24 466-470... 

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

Prostatic acid phosphatase (PAP) PASE/4LJ 52-kD human prostatic acid phosphatase Purified prostatic acid phosphatase from human seminal plasma Ventana NA HIER... 

A minor isoenzyme of human prostatic acid phosphatase (pi 5.5) has been separated from the major isoenzyme (pi 4.9). Immunological similarities between the two enzymes were demonstrated. 

Tartaric acid (18) Affinity purification of human prostatic acid phosphatase 251... 

Phosphatase-Human Prostatic Add.—Incubation of human prostatic acid phosphatase with glutaraldehyde resulted in formation of soluble enzymatically active species of both higher and lower molecular weight than the native enzyme. The modified enzyme shows essentially unaltered affinity towards different substrates and inhibitors, but is more resistant toward heat de-naturation than the native enzyme. 

Van Etten, R.L., Davidson, R., Stevis, P.E., MacArthur, H., and Moore D.L., 1991, Covalent structure, disulfide bonding, and identification of reactive surface and active site residues of human prostatic acid phosphatase./. Biol. Chem. 266 2313-2319. 

Sharief, F.S., Lee, H., Leuderman, M.M., Lundwall, A., Deaven, L.L., Lee, C., and Li, S.S.L., 1989, Human prostatic acid phosphatase cDNA cloning, gene mapping and protein sequence homology with lysosomal acid phosphatase. Biochem. Biophys. Res. Commun. 160 79-86. 

Vihko, R, Virkkunen, R, Henttu, R, Roiko, K., Solin, T., and Huhtala, M.L., 1988, Molecular cloning and sequence analysis of cDNA encoding human prostatic acid phosphatase. FEBS Lett. 236 275-281. 

Tailor, P.G., Govindan, M.V, and Patel, P.C., 1990, Nucleotide sequence of human prostatic acid phosphatase determined from a full-length cDNA clone. Nucleic Acids Res. 18 4928. 

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