Purification and Properties of Acid Pyrophosphatase from the Potato Yasuko TANEMURA, Hiroshi WADA*, Takashi ITO*, Haruhito TSUGE* and Kazuji OHASHI* Department of Home Economics, Shotoku Gakuen Women's Junior College, Gifu 500 *Department of Agricultural Chemistry, Gifu University, 1-1 Yanagido, Gifu 501-11 Some properties of acid pyrophosphatase (EC 3. 6. 1. 1) purified from potato tubers were inves tigated by combining water extraction, acid treatment, fractionation with ammonium sulfate and acetone, cellulose chromatography and column chromatographies on P-cellulose and DEAE-Sephadex A-50. Two fractions (I and II) were obtained on DEAE-Sephadex A-50. Fraction II was purified to 2500-fold with a 4.7% yield. Pyrophosphate was the most active substrate for the purified enzyme (fraction I and II). However, the enzyme also acted on tripoly- and trimeta-phosphate, and these activities could not be separated from each other. The purified enzyme (fraction II) had an optimum ph of 5.0 `6.0 and an optimum temperature of 50 Ž. The difference in substrate specificity between fractions I and II was remarkable. Frac tion I was inactive toward monoesters as ƒà-glycerophosphate and nucleoside monophosphates, while fraction II was a non-specific enzyme. Fraction II was markedly inhibited by Hg2+ and F-, and the Km value of fraction II determined with pyrophosphate was 12.2 ~10-3 M. (Received April 21, 1983)
Fig. 1. Cellulose Column Chromatography of Potato Acid Pyrophosphatase. The enzyme solution from stage 4 (cf. Table I) was brought to 80% saturation with ammonium sulfate. The precipitate collected by centrifugation was mix ed with cellulose pre-equilibrated with 80% saturated ammonium sulfate solution. The mixture was applied to a cellulose column (2. 7 ~ 5cm) and then cellulose was layered over it. The column was eluted with a linear gradient of 80 `0% saturated ammonium sulufate solu tion in 0. 1 M acetate buffer, ph 4. 6. Fractions of 10g were collected. They were dialyzed overnight against 0.01m acetate buffer, ph 4.6 and then pyrophosphatase activity was measured in a 2. 5ml reaction mixture containing 20 mm sodium pyrophosphate, 5 mm EDTA, 72 rum ace tate buffer, ph 4. 6 and 0. 1ml enzyme solution. After incubation at 37 Ž for 30 min, 1. 25ml ice-cold 10% trichloroacetic acid was added and the mixture was filtrated. In order to prevent spontaneous hydrolysis of pyrophosphate, the filtrate was assayed for Pi by a modified Fiske - Sabbarow method(17). After addition of amino naphtholsulfonic acid solution, the mixture was left at 20 Ž for 15 min exactly, and the absor bance was read at 720 nm. One enzyme unit was defined as the amount of enzyme which liberated 1.0 umol Pi/min. - œ-, pyrophospha tase activity ; - œ-, protein. Fig. 2. P-Cellulose Column Chromatography of Potato Acid Pyrophosphatase. The concentrated enzyme solution from stage 5 (cf. Fig. 1) was applied to a P-cellulose column (2. 7 ~10cm) pre-equilibrated with 0.01M acetate buffer, ph 4. 6. After washing with 500ml of 0. 15M acetate buffer, ph 5.6, the column was eluted with a linear gradient of 0 `0.6m NaCl in 0.2M acetate buffer, ph 5.6. - œ-, pyrophosphatase activity; - œ-, protein ; NaCl con centration.
Fig. 4. Polyacrylamide Gel Electrophoresis of Purified Enzyme. Electrophoreses of 100,ƒÊg sample (stage 4 and stage 7 (I)) and 4 `5,ƒÊg Fig. 3. DEAE-Sephadex A-50 Column Chroma tography of Potato Acid Pyrophosphatase. The pyrophosphatase fraction from stage 6 (cf. Fig.2) was concentrated to a small volume(2 ` sample (stage 7 (II)) were carried out in tris glycine buffer, ph 9.4, as the running buffer for 2 hr at 2 ma/tube. The gel was stained with Amido black 10B. 3 ml) with collodion bag and applied to a DEAE - Sephadex A-50 column (1. 5 ~ 68cm) pre-equilibrated with 0.01m acetate buffer, ph 5.6. Elution was carried out with a linear gradient of 0 `0.3M NaCl in 0.01M acetate buffer, ph 4.6. - œ-, pyrophosphatase activity; - œ-, pro tein ; -, NaCl concentration.
Table II. Substrate Specificity of Acid Pyrophosphatase Fig. 5. Effect of ph on the Activity of Purified Enzyme. Assay conditions were as described in Fig. 1, except that 0. 1 M acetate buffer, ph 4.6 `6.0, 0. 1 M tris-maleate buffer, ph 7.0 ` 8. 0 and 0. 1 M glycine-naoh buffer, ph 9.0, were used. The final substrate concentrations were 20 mm for PPi ( œ), 10 mm for TPi ( ) and TM ( ). Enzyme activity was measured at ph 5. 6 with fraction I or II. Final substrate concentration was 10 mm. The enzyme activities were cal culated from Pi liberated from various sub strates. Relative activity was expressed as the percent of those for PPi. Table III. Effect of Various Substances on Activity of Acid Pyrophosphatase (Fraction II) Fig. 6. Effect of Temperature on the Activity of Acid Pyrophosphatase. Assay conditions were as described in Fig. 1, except for 0. 1 M acetate buffer, ph 5.6 and temperature used. The enzyme activity was measured at ph 5.6 in the presence of various substances.