[24] Phosphoprotein phosphatase

[24]

PHOSPHOPROTEIN PHOSPHATASE

211

[ 24] P h o s p h o p r o t e i n P h o s p h a t a s e Protein-P ~ H20 --~ Dephosphoprotein ~- P,

By

HELEN R. REVEL

A s s a y Method

Principle. The assay depends on measuring the formation of inorganic phosphate released from the phosphoprotein casein. Reagents 1 M sodium acetate buffer, pH 5.8. 0.1 M ascorbic acid, freshly prepared and neutralized to pH 5.8 with NaOH. Casein. Dissolve 10 g. of casein (Hammersten) in 84 ml. of water with the gradual addition of 16 ml. of 0.4 M NaOH to give a final pH of 7.0. Dialyze at 4 ° for about 20 hours against 20 vol. of 0.1 M sodium acetate buffer, pH 5.8. Assay the solution for organic phosphate and adjust to contain 20 micromoles of organic phosphate per milliliter. Enzyme. Dilute enzyme with buffer to obtain a solution containing 0.05 to 0.5 unit/ml. 20% trichloroacetic acid.

Procedure. The following solutions are placed into a 12-ml. centrifuge tube: 0.1 ml. of acetate buffer, 0.05 ml. of ascorbic acid, 0.4 ml. of dialyzed casein, 0.35 ml. of water, and finally 0.1 ml. of enzyme. The tubes are incubated at 37 ° for 15 or 30 minutes. The reaction is stopped by the addition of 1 ml. of ice-cold 20% trichloroacetic acid. The tubes are centrifuged to remove denatured protein, and the supernatant solution is analyzed for inorganic phosphate. 1 Definition of Unit and Specific Activity. The unit is defined as the amount of enzyme that catalyses the release of 1 micromole of inorganic phosphate from casein in 1 minute at 37 °. Specific activity is defined as units per milligram of protein as measured by the biuret reaction. ~ Application o/ the Assay Method to Crude Tissue Preparations. The presence of proteolytie enzymes, nonspecific phosphatases, and phosphorylated compounds other than casein in crude tissue preparations interferes with the assay for phosphoprotein phosphatase activity. With i K. Lohmann and L. Jendrassik, Biochem. Z. 178, 419 (1926). : H. W. Robinson and C. G. Hogden, J. Biol. Chem. 135, 727 (1940).

212

ENZYMES OF PHOSPHATE METABOLISM

[24]

appropriate controls, however, a rough estimate of the enzyme activity can be made. A control tube without casein indicates the amount of inorganic phosphate released from phosphorylated compounds present in the crude extract. A second control tube in which a-glycerol phosphate replaces casein as substrate permits an estimate of nonspecific phosphatase activity. Purification Procedure Beef spleen is the most abundant source known for the enzyme. 3 Phosphoprotein phosphatase activity was originally observed in frog's eggs4 and has been found in chick embryos, 5 mouse liver, 6 rat spleen, liver, and kidney, 7 and to a lesser extent in other tissues of the ratY The method described is the purification of phosphoprotein phosphatase from beef spleen by the author's modification s of the procedure of Sundararajan and Sarma a,s which results in a soluble stable enzyme preparation of high specific activity. A procedure for purification involving autolysis has been described by Singer and Fruton, TM but the preparation has somewhat different properties. All operations are carried out at 4 ° unless otherwise stated. Step 1. Preparation of Crude Extract. Six iced beef spleens are freed of capsular material and ground in a meat grinder. Six batches of 400 g. of minced tissue are homogenized with 1 1. of a 0.5 M sodium chloride-0.2 M acetate solution of pH 5.0 in a 2-1. Waring blendor. The homogenate is centrifuged immediately at 1000 X g for 15 minutes, and the supernatant solution is filtered through several layers of gauze (4600 ml.). Step 2. Ammonium Sulfate Fractionation. Solid ammonium sulfate (22.6 g. per 100 ml. of crude extract) is added, and after 30 minutes of stirring the mixture is centrifuged at 16,000 X g for 20 minutes. The precipitate is discarded, and 26 g. of solid ammonium sulfate per 100 ml. of supernatant solution are added. After 30 minutes the mixture is centrifuged as before, and the precipitate is taken up in a minimum volume of water (about 300 ml.). Dialysis with stirring against 8 1. of distilled water (five to six changes) is continued (about 48 hours) until * T. A. Sundararajan and P. S. Sarma, Biochem. J. 56, 125 (1954). D. L. Harris, Y. Biol. Chem. 165, 541 (1946). 5 M. Foote and C. A. Kind, Arch. Bioehem. Biophys. 46, 254 (1953). * K. Paigen, J. Biol. Chem. 233, 388 (1958). TB. Norberg, Aeta Chem. 8eand. 4, 1206 (1950). * H. R. Revel and E. Racker, Biochim. et Biophys. Acta 43, 465 (1960). ' T. A. Sundararajan and P. S. Sarma, Bioehem. J. 65, 261 (1957). 1oM. F. Singer and J. S. Fruton, J. Biol. Chem. 229, 111 (1957).

[24]

PHOSPHOPROTEIN PHOSPHATASE

213

the dialyzate is free of sulfate ion. The precipitate formed during dialysis contains the major portion of the enzyme and is separated from the supernatant solution by centrifugation. The precipitate is extracted twice with 0.5 M sodium chloride-0.2 M acetate solution of pH 5.0, spinning after each extraction at 100,000 X g for 45 minutes in a Spinco centrifuge. The ratio of the ultraviolet extinction of the combined extracts (178 ml.) at 280 m~ to that at 260 m~ is 0.65 to 0.72, indicating the presence of some 11 to 14% nucleic acid. 11 Step 3. Heat and Protamine Sul]ate Treatment. The reddish solution is divided into 15-ml. aliquots and heated for 5 minutes in test tubes in a water bath maintained at 70 °. The solutions are then quickly chilled in an ice bath and centrifuged to remove denatured protein. The clear amber supernatant solution contains over 90% of the total activity of the unheated enzyme and about 25% of the protein. To 164 ml. of the heated enzyme 15 ml. of a 2% protamine sulfate solution are added at room temperature, and after 10 minutes the mixture is centrifuged at 8000 X g for 15 minutes. From the supernatant solution, which retains all the activity, the enzyme is precipitated by the addition of 56 g. of solid ammonium sulfate per 100 ml. The precipitate is taken up in about 8 ml. of water and is dialyzed for about 20 hours against 3 1. of 0.005 M sodium chloride-0.002 M acetate solution of pH 6.0. A precipitate which forms contains no activity and is discarded. The clear yellow-brown supernatant solution (17.8 ml.) has a ratio of ultraviolet extinction at 280 m~ to that at 260 m~ of 1.35. Step 4. Fractionation on a DEAE-CeUulose Column. The dialyzed enzyme solution is applied to DEAE-cellulose anion exchanger (5 X 3.5 SUMMARY OF PURIFICATIONPROCEDUREa

Step

Volume, ml.

Protein, mg.

Activity, units

Specific activity, units/mg. protein

1 2 3 4

4600 178 17.8 33

165,000 2,300 178 96

3550 1000 600 600

0.022 0.44 3.4 6.3

°It. R. Revel and E. Racker, Biochim. et Biophys. Acta 43, 465 (1960).

cm.) which has been equilibrated with 0.005 M sodium chloride-0.002 M acetate solution of pH 6.0. Fractions of 10 ml. are collected. The enzyme does not adhere to the cellulose and is eluted in tubes 4, 5 ,and 6 on zl O. Warburg and W. Christian, Biochem. Z. 310, 384 (1941).

214

ENZYMES OF PHOSPHATE METABOLISM

[24]

washing with the equilibrating buffer solution. The ratio of ultraviolet extinction at 280 m~ to that at 260 m~ of the purified enzyme at this stage is 1.45. A summary of the purification proccdure is given in the table. Properties

Specificity. In addition to its action on the phosphoproteins such as casein and phosvitin, the purified beef spleen enzyme catalyzes the hydrolysis of pyrophosphates (ATP, ADP, inorganic pyrophosphate, etc.), aromatic monophosphate esters (phenyl phosphate, p-nitrophenyl phosphate, etc.), and certain other phosphate compounds such as phosphorylenol pyruvate, acetyl phosphate, and phosphoamide. The purified enzyme has little activity toward ordinary phosphomonoesters. For example, sugar phosphates, glycerol phosphate, serine phosphate, and small serine phosphate peptides are poor substrates. Activators. Enzyme activity is considerably enhanced by the presence of reducing agents. Ascorbic acid and thioglycolic acid at the optimal concentration of 5 X 10-'~ M activate the enzyme about tenfold. At a concentration of 10-2M, thioglycerol, cysteine, or 2-mercaptoethanol increases enzyme activity about sixfold. Glutathione is not an effective activator. Effect of Metals. Ferrous iron completely replaces the reducing agents and gives maximal stimulation at the optimal concentration of 10-3M. Higher concentrations of ferrous iron inhibit the enzyme. Ca ++, Mg ÷÷, Mn ++, Cu ++, Co ++, Ni ++, Zn +÷, and Fe +++, tested in the range of 10-4 to 10-2M, did not stimulate phosphoprotein phosphatase activity and caused about 50% inhibition at 10-3 M. Inhibitors. Molybdate is the most effective agent, giving 50% inhibition at 4 X10-cM and complete inhibition at 10-4 M. Some metal chelators are potent inhibitors; 1,10-o-phenanthroline and a,a-dipyridyl inhibit the enzyme 50% at 10-5 M and 4 X 10-2 M, respectively. In contrast to molybdate, however, neither chelating agent inhibits the enzyme completely. Less effective inhibitors include p-chloromercuribenzoate (40% at 10-~ M), fluoride (60% at 4 >( 10-3 M), iodoacetate (30% at l0 -2 M), and phosphate (40% at 10-2 M). Azide, cyanide, and ethylenediaminetetraacetate are without effect at 10-2 M. Stability. The purified enzyme is stable at --18 ° for several months. Enzyme activity is preserved after exposure to temperatures up to 7 0 ° for prolonged periods of time, but the enzyme is rapidly inactivated at 37 ° in the presence of ascorbic acid or thioglycolic acid. Ferrous ions as well as substrate protect the enzyme against this inactivation. Kin. The Km for casein is about 6 X 10-4 M for the enzyme preparation described.