Purification and Properties of Mesophyll and Bundle Sheath Cell α-Glucan Phosphorylases from Zea mays L.

Abstract

Two major a-glucan phosphorylases (I and II) from leaves of the C4 plant corn (Zea mays L.) were previously shown to be compartmented in mesophyll and bundle sheath cells, respectively (C Mateyka, C Schnarrenberger 1984 Plant Sci Lett 36: 119-123 a-Glucan phosphorylases in leaves ofthe two C3 plants spinach and pea have previously been shown to have a very distinct compartmentation: one is located in the cytosol and one or two in the chloroplasts (13,19,25). They are part of two sets of isoenzymes in glycolysis, gluconeogenesis and the oxidative pentose phosphate cycle in the cytosol and in the plastids of plant cells, respectively (20). The cytosol and plastid phosphorylases of C3 plant leaves have many properties in common; however, they differ greatly in their substrate affinity for glycogen, in their effect on some maltodextrins (17,22,28), and in their immunological crossreactivity (5, 29).Similar to C3 plants, multiple forms of phosphorylase can also be isolated from leaves of the C4 plant corn (11). It could be ' Part of this work was supported by Deutsche Forschungsgemeinschaft.shown that one of the two major phosphorylases in corn was restricted to mesophyll cells and the other to bundle sheath cells. These findings explain previous (2) and recent (23) reports on phosphorylase activity for mesophyll and bundle sheath cells in corn leaves. Since it may argued that the cytosol and plastid phosphorylases of C3 plants are homologous with the mesophyll and bundle sheath cell phosphorylases of C4 plants we have looked for, and report here on properties of corn leaf phosphorylases in order to gain evidence for such a conclusion. MATERIALS AND METHODSEnzyme Sources. Corn (Zea mays L.), type Inrafruh, was grown from seeds in greenhouses. For enzyme isolation 600 g of leaf material without midribs was cut with scissors into small pieces, homogenized in 2.4 L of 100 mM imidazol/HCl (pH 7.0), 0.5% polyvinylpyrrolidone, 20 mm 2-ME,2 and 0.1 mm PMSF for 1 min, first with a Waring Blendor (Waring Products, New Hartfort, CT) and subsequently with an Ultra-Turrax (Janke & Kunkel, Staufen/FRG) at full speed at 0°C. The suspension was squeezed through two layers of cheesecloth and centrifuged at 24,000g for 20 min at 4°C. The pellet was discarded.Correspondingly, 400 g ofderibbed spinach (Spinacia oleracea L.) was homogenized in 1 L of the same buffer for 1 min with a Waring Blendor. The suspension was processed in the same way as with corn.Enzyme Purification. All purification steps were performed at 4°C. The buffer used for all subsequent operations was 0.01 M Tris/HCl (pH 7.2), 20 mM 2-ME, and 0.1 mM PMSF.Enzymes of the crude extracts were fractionated by adding solid (NH4)2SO4 to a final concentration of 35% saturation at pH 7.2. After centrifugation at 24,000g in a GSA-rotor of a Sorvall RCIIB centrifuge, (NH4)2SO4 was added to the supernatant to give 60% saturation. Precipitated proteins were dissolved in buffer, dialyzed overnight, and applied to a DEAE-cellulose (Whatman, Springfield Mill, Springfield, Mai...

Two Distinct Aldolases of Class II Type in the Cyanoplasts and in the Cytosol of the Alga Cyanophora paradoxa

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Two Phosphorylases from Suspension-cultured Soybeans

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