Human prostatic acid phosphatase has phosphotyrosyl protein phosphatase activity

The major secreted isoenzyme of human prostatic acid phosphatase (PAcP) (EC 3.1.3.2), which catalyses p-nitrophenyl phosphate (PNPP) hydrolysis at acid pH values, was found to have phosphotyrosyl protein phosphatase activity since it dephosphorylated three different phosphotyrosine-containing protein substrates. Several lines of evidence are presented to show that the phosphotyrosyl phosphatase and PAcP are the same enzyme. A highly purified PAcP enzyme preparation which contains a single N-terminal peptide sequence was used to test for the phosphotyrosyl phosphatase activity. Both activities comigrated during gel filtration by high performance liquid chromatography. Phosphotyrosyl phosphatase activity and PNPP acid phosphatase activity exhibited similar sensitivities to different effectors. Both phosphatase activities showed the same thermal stability. Specific anti-PAcP antibody reacted to the same extent with both phosphatase activities. PNPP acid phosphatase activity was competitively inhibited by the phosphotyrosyl phosphatase substrate. To characterize further the phosphotyrosyl phosphatase activity, the Km values using different phosphoprotein substrates were determined. The apparent Km values for phosphorylated angiotensin II, anti-pp60src immunoglobulin G and casein were in the nM range for phosphotyrosine residues, which was about 50-fold lower than the Km for phosphoserine residues in casein.

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An optimized continuous-monitoring procedure for semiautomated determination of serum acid phosphatase activity.

Clinical Chemistry ◽

10.1093/clinchem/22.12.2025 ◽

1976 ◽

Vol 22 (12) ◽

pp. 2025-2028 ◽

Cited By ~ 19

Author(s):

R Bais ◽

J B Edwards

Keyword(s):

Acid Phosphatase ◽

Phosphatase Activity ◽

Acid Phosphatase Activity ◽

Continuous Monitoring ◽

Prostatic Acid Phosphatase ◽

Monitoring Method ◽

Modified Method ◽

Nitrophenyl Phosphate ◽

Monitoring Procedure

Abstract A continuous-monitoring method for measuring acid phosphatase activity with alpha-naphthyl phosphate as the substrate was critically evaluated and modified. Using partially purified prostatic acid phosphatase, we show that certain conditions for the assay must be satisfied to ensure linearity. These conditions include maintaining the pH between 5.6 and 5.9 and the addition of detergent to sustain linearity. The results obtained with alpha-naphthyl phosphate have been compared with those obtained by using p-nitrophenyl phosphate as substrate. When used with an automatic rate analyzer, the modified method is as sensitive but more reproducible.

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The effect of antibody on human prostatic acid phosphatase activity—I: Temperature and pH stabilization of acid phosphatase enzyme activity by rabbit antibody to acid phosphatase

Molecular Immunology ◽

10.1016/0161-5890(75)90109-1 ◽

1975 ◽

Vol 12 (2) ◽

pp. 131-136

Author(s):

A Foti

Keyword(s):

Enzyme Activity ◽

Acid Phosphatase ◽

Phosphatase Activity ◽

Acid Phosphatase Activity ◽

Prostatic Acid Phosphatase ◽

Rabbit Antibody ◽

Phosphatase Enzyme

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Kinetic Method for Determining Acid Phosphatase Activity in Serum with Use of the "CentrifiChem"

Clinical Chemistry ◽

10.1093/clinchem/18.8.841 ◽

1972 ◽

Vol 18 (8) ◽

pp. 841-844 ◽

Cited By ~ 12

Author(s):

Diane L Fabiny-Byrd ◽

Gerhard Ertingshausen

Keyword(s):

Acid Phosphatase ◽

Phosphatase Activity ◽

Acid Phosphatase Activity ◽

Kinetic Method ◽

Reaction Rates ◽

Phosphatase Activities ◽

Fast Red ◽

Presence And Absence

Abstract Acid phosphatase activity is determined by splitting 1-naphthyl phosphate, concurrently diazotizing the released 1-naphthol with Fast Red TR, and measuring the resulting color. The test is performed in the presence and absence of tartrate. Reaction rates can be continuously monitored, and their difference is proportional to acid phosphatase activity that is inhibited by tartrate. Results for sera with normal and increased acid phosphatase activities are presented and three different methods for acid phosphatase are compared. The kinetic blank used in the reaction eliminates all nonenzymatic contributions to substrate splitting.

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Spectrophotometric and liquid-chromatographic studies of thymolphthalein monophosphate. Specifications for high-quality substrate for the measurement of prostatic acid phosphatase activity.

Clinical Chemistry ◽

10.1093/clinchem/27.8.1372 ◽

1981 ◽

Vol 27 (8) ◽

pp. 1372-1377 ◽

Cited By ~ 3

Author(s):

G N Bowers ◽

M Onoroski ◽

R S Schifreen ◽

L R Brown ◽

R E Klem ◽

...

Keyword(s):

Acid Phosphatase ◽

Phosphatase Activity ◽

Acid Phosphatase Activity ◽

Prostatic Acid Phosphatase ◽

Enzymic Activity ◽

Disodium Salt ◽

Critical Importance ◽

High Quality ◽

Activity Measurements ◽

Liquid Chromatographic

Abstract Fourteen lots of thymolphthalein monophosphate (TMP), disodium salt, obtained from 10 commercial suppliers were compared spectrophotometrically at 445 and 595 nm, liquid-chromatographically with monitoring at 254 nm, and enzymically by measurements of activity of prostatic acid phosphatase in human serum. Eight lots were classified as "unacceptable," six as "acceptable." Spectrophotometric testing revealed four lots with excessive thymolphthalein and three lots with grossly deficient amounts of TMP. In general, the chromatographic results paralleled those obtained by spectrophotometry, and both results correlated well with enzymic activity. Changing water content in this hygroscopic salt was a major problem, which resulted in great uncertainty as to the formula weight and therefore as to the moles of TMP actually taken. From these studies, specifications for high-quality TMP were determined. The critical importance of simultaneous enzymic activity measurements in comparisons with other "acceptable" lots in defining an adequate TMP substrate is stressed. Use of these specifications for selecting TMP for acid phosphatase activity measurements should improve intra- and inter-laboratory analytical performance.

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Comparison of the substrate specificities of protein phosphatases involved in the regulation of glycogen metabolism in rabbit skeletal muscle

Biochemical Journal ◽

10.1042/bj1620423 ◽

1977 ◽

Vol 162 (2) ◽

pp. 423-433 ◽

Cited By ~ 155

Author(s):

J F Antoniw ◽

H G Nimmo ◽

S J Yeaman ◽

P Cowen

Keyword(s):

Phosphatase Activity ◽

Protein Phosphatase ◽

Glycogen Synthase ◽

Protein Phosphatases ◽

Gel Filtration ◽

Beta Subunit ◽

Alpha Subunit ◽

Phosphorylase Kinase ◽

Substrate Specificities ◽

Phosphatase Activities

Muscle extracts were subjected to fractionation with ethanol, chromatography on DEAE-cellulose, precipitation with (NH4)2SO4 and gel filtration on Sephadex G-200. These fractions were assayed for protein phosphatase activities by using the following seven phosphoprotein substrates: phosphorylase a, glycogen synthase b1, glycogen synthase b2, phosphorylase kinase (phosphorylated in either the alpha-subunit or the beta-subunit), histone H1 and histone H2B. Three protein phosphatases with distinctive specificities were resolved by the final gel-filtration step and were termed I, II and III. Protein phosphatase-I, apparent mol.wt. 300000, was an active histone phosphatase, but it accounted for only 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities and 2-3% of the phosphorylase kinase phosphatase and phosphorylase phosphatase activity recovered from the Sephadex G-200 column. Protein phosphatase-II, apparent mol.wt. 170000, possessed histone phosphatase activity similar to that of protein phosphatase-I. It possessed more than 95% of the activity towards the alpha-subunit of phosphorylase kinase that was recovered from Sephadex G-200. It accounted for 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activity, but less than 5% of the activity against the beta-subunit of phosphorylase kinase and 1-2% of the phosphorylase phosphatase activity recovered from Sephadex G-200. Protein phosphatase-III was the most active histone phosphatase. It possessed 95% of the phosphorylase phosphatase and beta-phosphorylase kinase phosphatase activities, and 75% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities recovered from Sephadex G-200. It accounted for less than 5% of the alpha-phosphorylase kinase phosphatase activity. Protein phosphatase-III was sometimes eluted from Sephadex-G-200 as a species of apparent mol.wt. 75000(termed IIIA), sometimes as a species of mol.wt. 46000(termed IIIB) and sometimes as a mixture of both components. The substrate specificities of protein phosphatases-IIA and -IIB were identical. These findings, taken with the observation that phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities co-purified up to the Sephadex G-200 step, suggest that a single protein phosphatase (protein phosphatase-III) catalyses each of the dephosphorylation reactions that inhibit glycogenolysis or stimulate glycogen synthesis. This contention is further supported by results presented in the following paper [Cohen, P., Nimmo, G.A. & Antoniw, J.F. (1977) Biochem. J. 1628 435-444] which describes a heat-stable protein that is a specific inhibitor of protein phosphatase-III.

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Polyphosphate body and acid phosphatase localization in Nostoc sp.

Canadian Journal of Microbiology ◽

10.1139/m84-002 ◽

1984 ◽

Vol 30 (1) ◽

pp. 8-15 ◽

Cited By ~ 3

Author(s):

John D. DuBois ◽

Keith R. Roberts ◽

Lawrence A. Kapustka

Keyword(s):

Enzyme Activity ◽

Acid Phosphatase ◽

Phosphatase Activity ◽

Acid Phosphatase Activity ◽

Energy Stress ◽

Nitrophenyl Phosphate ◽

Carbon Compounds ◽

Electron And Light Microscopy ◽

Polyphosphate Bodies ◽

Hydrolysis Of

Polyphosphate bodies and acid phosphatase activity were characterized in Nostoc sp. to determine if the hydrolysis of polyphosphate bodies occurs during dark (energy stress) periods. Electron and light microscopy were used to locate polyphosphate bodies. Acid phosphatase activity was measured using p-nitrophenyl phosphate as the substrate to determine net changes in the level of the enzyme activity. To induce energy stress, Nostoc sp. cells were kept in the dark for 72 h to deplete stored carbon compounds. Cells incubated in the light for 72 h (controls) showed acid phosphatase activity localized around the perimeter of polyphosphate bodies. When cells were incubated in the dark, acid phosphatase activity occurred throughout the polyphosphate body matrix. However, complete hydrolysis of the polyphosphate body did not occur and the rate of acid phosphatase activity was not affected.

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