Characterization from Mature Kernels and Anaerobically Treated Roots

Pyruvate decarboxylase (PDC) was purified from mature, dry maize kernels and from roots of anaerobically treated maize seedlings and partially characterzed. PDC was purified to a specific activity of 96 units per milligram protein from kernels and to 41 units per milligram protein from root. The subunit molecular masses were estimated to be 61,000 and 60,000 for kernel PDC and 59,000 and 58,000 for root PDC. The pH optimum for each enzyme was 5.8. Since the pH optimum is nearly one pH unit below the value reported for the cytoplasm of anaerobically metabolizing maize roots (pH 6.7 1 0.2), we investigated the effects of pH 5.8 and 6.6 on the cooperative kinetics observed for PDC from each source. The maximum Hill coefficients (nH) were much greater at each pH for the kernel PDC (pH 5.8, nH = 2.5 and pH 6.6, nH = 3.2) than for the root PDC (pH 5.8, nH = 14A and pH 6.6, nH = 1.8). The cooperative kinetics observed with respect to pyruvate were asymmetric. Potassium inhibited maize PDC and was competitive with pyruvate (root PDC K,= 16 millimolar and kernel PDC KJ= 10 millimolar). Pyruvate decarboxylase, PDC3 (EC 4.1.1.1) catalyzes the decarboxylation of pyruvate to acetaldehyde; ADH (EC 1.1.1.1) then catalyzes the reduction ofthe acetaldehydeto ethanol. These two enzymes thus catalyze a pathway in which NAD+ is regenerated under anaerobic conditions. Pyruvate, the first substrate of this pathway, occurs at a major branch point in glycolysis between anaerobic and aerobic metabolism, and the regulation between these two metabolic routes is likely to occur both at the level of PDC synthesis and of PDC activity. The syntheses of PDC and ADH are specifically enhanced under anaerobic conditions (9, 18), and significant increases in the activities of these enzymes are observed (9, 18). One functional mechanism proposed to control this anaerobic pathway is the regulating effect of cytoplasmic pH on PDC activity (7). PDC is inactive at the pH ofaerobically metabolizing tissues. However, the cytoplasmic ' Supported by United States Department of Agriculture CRG 83CRCR-1-1201, the University of Wisconsin, and United States Department of Energy. The investigations reported were included in the dissertation submitted by T. C. L. to the Graduate College, University of Wisconsin, Madison, in partial fulfillment of the requirements for the Ph.D. degree. 2Present address: INC4, MS C345, Los Alamos National Laboratory, Los Alamos, NM 87545. 3Abbreviations:PDC, pyruvate decarboxylase (EC 4.1.1.1); ADH, alcohol dehydrogenase (EC 1.1.1.1); EDTA-Na, ethylenediaminetetraacetic acid, sodium salt; TPP, thiamine pyrophosphate. pH of anaerobically metabolizing tissues is significantly lower than the pH of aerobically functioning tissues and this decrease allows PDC to function. The PDC's from sweet potato root (14, 15), wheat (7, 22), and brewer's yeast (3, 24) bind pyruvate cooperatively; thus, pyruvate levels have also been proposed as a regulatory mechanism. The effect(s) that changing the pH may have on these cooperative kinetics has not been investigated using a plant PDC. An additional mechanism to regulate this anaerobic pathway in plants is the inhibition of PDC by phosphate, a mechanism similar to the phosphate inhibition of yeast PDC (3). A complete study of the possible effects of phosphate salts on plant PDC activity has not been done. Since maize seedlings must carry out anaerobic metabolism to survive growth in anaerobic conditions such as in water-logged soils, the regulation ofPDC is important. Therefore, we purified and partially characterized PDC from anaerobically treated maize roots and from maize kernels. The effects of pH and of pyruvate concentration on the cooperative kinetics were investigated for the enzyme from each of these two sources. The possible inhibition of maize PDC by phosphate and potassium was also studied. MATERIALS AND METHODS Reagents and Buffers. Sepharose 6B was purchased from Pharmacia Inc.4 The mol wt standards and biochemical reagents were from Sigma. All other chemicals were of reagent grade. Buffer A contained 100 mM K-phosphate (pH 6.5), 50 mm TPP, 5 mm MgCl2, and 5 mm 2-mercaptoethanol. Buffer B contained 25 mM imidazole-HCI (pH 6.8), 5 mM MgC92, 50 zlM TPP, and 5 mm 2mercaptoethanol. Purification of Pyruvate Decarboxylase from Kernels. Mature, dry maize (Zea mays L., W438 x A632) kernels (500 g) were ground and the flour was homogenized with 1 L of buffer A in a Waring Blendor (medium speed for 1 min). All purification steps were at 4C. The homogenate was filtered through cheesecloth and centrifuged (10,000g, 25 min). Protamine sulfate (2% w/v) was added to the supernatant with approximately 1 ml of protamine sulfate solution added per 20 ml of supernatant. The solution was stirred for 10 min, clarified by centrifugation (15,000g, 20 min) and pH adjusted to 7.5 with I M K2HPO4. The solution was brought to 25% saturation with solid (NH4L)SO4, sfirred for 45 min, and centrifuged (20,000g, 20 min). The supernatant was brought to 37% saturation with (NH4)2SO4, stirred for 45 min, and centrifuged (30,000g, 20 4The mention of firm names or trade products does not imply that they are endorsed or recommended by the United States Department of Agriculture or by the University ofWisconsin over other firms or similar products not mentioned.