Activation Kinetics of NAD-Dependent Malic Enzyme of Cauliflower

NAD-dependent malic enzyme (EC 1.1.1.39) was obtained from isolated mitochondria of cauliflower buds (Brassica okracca L., var. botrytis). The NAD-linked activty is accompanied by a minor NADP-linked activity. Some contaminant NADP-malc enzyme from the supernatant and the plasma membrane is usually present in crude mitochondrial preparations. NAD-dependent malic enzyme has been purified 38-fold by ammonium sulfate fractionation and gel permeation chromatography, to a specific activity up to 2 micromoles per minute per millgram. The nature of the activating effect of coenzyme A and dithiothreitol has been investigated. Both compounds act by decreasing the apparent Michaelis constants for i-Imalate and NAD' (and NADP ), V,,,,= remini approximately constant. However, enzyme fully activated by dithiothreitol can still be stimulated up to 2.4-fold by coenzyme A treatment. Velocity versus substrate responses show hyperbolic kinetics under present assay conditions (pH 7.5, 2 millolar Mn2+), but biphasic kinetics have been observed with enzyme purife in the presence of 10 millmar dithiothreitol, suggesting enzyme heterogeneity with respect to an activated state. This condition is reverted to linearity by treatment with coenzyme A. K. values do not vary with changing concentrations of the second substrate. Enzyme molecular weight is 400,000 in the completely activated state and 200,000 in the 'inactivated' state; intermediate forms are also found. All coenzyme A derivatives tested are effective activators, showing activation constants lower than for coenzyme A itself. The concentration dependence of the activation is sigmoidal. The presence of an NAD-dependent malic enzyme in plant mitochondria was recognized by Macrae and Moorhouse (12). The enzyme required an activator such as CoA, acetyl-Co A, fructose-1,6-bP, sulfate, or a reductant for full activity (2-4, 9, 11), although these compounds do not exert appreciable effects on the activity of intact mitochondria (2-4). The role of malic enzyme in mitochondrial substrate oxidation and respiration has been widely investigated (3, 5, 6, 12, 14, 18). Recent work (2, 5, 6) indicates a localization in the mitochondrial matrix, similar to the homologous mammalian protein (13) and at variance with other views (3, 14). Davies and Patil (4) have interpreted the regulation ofNADmalic enzyme as connected to the control of intracellular proton activity (pH-stat), involving also PEP carboxylase. This possibility has recently been discussed (5, 17). The enzyme activity is particularly high in several plants possessing either the C4 or the CAM pathway of photosynthesis (2, 5, 7, 9). In these species, malate decarboxylation represents a major source of CO2 for photosynthetic carbon assimilation. 1 Supported by grants from the Consiglio Nazionale delle Ricerche (Rome) for 1978 and 1979. The involvement of this enzyme in such diverse metabolic functions renders its regulatory mechanism (2-4, 9, 11) an attractive object of investigation. In this paper we are presenting some results on the nature of the activation by CoA and DTT, and new data on the accompanying NADP-dependent activity. MATERIALS AND METHODS Enzyme Purification. About 150 g of cauliflower buds were homogenized in a chilled medium consisting of 40 mm Tris-HCl (pH 7.5), 3 mm 2-mercaptoethanol, and 350 mm sucrose on ice. The filtered homogenate was centrifuged 10 min at 1,500g (2°C). The mitochondria were sedimented by centrifuging the supernatant at 10,000g for 20 min. The precipitate was suspended to 25 ml with the aid of a Potter-Elvehjem homogenizer in 40 mM TrisHCI (pH 7.5) and 1 mm 2-mercaptoethanol, then the mitochondria were washed by a similar centrifugation and resuspended in this buffer. Crystalline ammonium sulfate was now dissolved to 35% saturation, and the precipitate (20,000g, 30 min) was discarded. The supernatant fluid was brought to 55% ammonium sulfate saturation and centrifuged. The partially purified enzyme (referred to in the following as ammonium sulfate fraction) was resuspended in the same buffer containing 10 mm DTT and 0.1 mM CoA, incubated 1 h at room temperature, clarified (40 min, 100,000g), and applied to a column (2.6 x 60 cm) packed with Sepharose 6B, and equilibrated with 20 mM Tris-HCl (pH 7.5), 10 mM DL-malate (pH 7), 5 mm DTT, 1 mm MnCl2, 0.1 mM NAD+, and 100 mM NaCl. This medium permits complete recovery of the activity applied. Fractions of 100 drops were collected and assayed for activity and A2w. The enzyme was usually stored as ammonium sulfate precipitate (55% saturation) at 18°C. When necessary, aliquots were thawed and rapidly desalted through a Sephadex G-25 column (1 x 20 cm) equilibrated with 20 mm Tris-HCl (pH 7.5), 1 mim EDTA, and 0.1 mm 2-mercaptoethanol. The enzyme in this or in similar buffers (containing only traces of reductants) will be defined as 'inactivated. ' Assays. NAD-dependent malic enzyme was assayed spectrophotometrically at 340 nm in a mixture containing 100 ,mol TrisHC1 (pH 7.50), 1.5 ,umol NAD+, 10 ,umol sodium L-malate, 2 ,umol MnCl2, and Triton X-100 (0.25%) in a volume of 1.0 ml at 25°C. Triton was omitted in experiments with purified protein. CCO2 and CCR activities were determined as described elsewhere (16). Protein concentration was measured by a Coomassie Blue G-250 method (1) with minor modifications. Isopycnic Centrifugation. A crude mitochondrial sediment was washed in the homogenization medium (containing 2 mM EDTA), and applied on top of a sucrose density gradient in an SW 27 tube. All solutions contained 20 mm Tris-HCl (pH 7.5), 1 mM 2Abbreviations: CCO, cytochrome c oxidase; CCR, NADH-cytochrome c reductase. 1191 D ow naded rom http/academ ic.p.com /plphys/art8/5/1191/6076684 by gest on 18 M arch 2022