Partial purification and characterization of the maize mitochondrial pyruvate dehydrogenase complex

Abstract

The pyruvate dehydrogenase complex was partially purified and characterized from etiolated maize (Zea mays L.) shoot mitochondria. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed proteins of 40, 43, 52 to 53, and 62 to 63 kD. Immunoblot analyses identified these proteins as the E1beta-, E1alpha-, E2-, and E3-subunits, respectively. The molecular mass of maize E2 is considerably smaller than that of other plant E2 subunits (76 kD). The activity of the maize mitochondrial complex has a pH optimum of 7.5 and a divalent cation requirement best satisfied by Mg2+. Michaelis constants for the substrates were 47, 3, 77, and 1 &mgr;m for pyruvate, coenzyme A (CoA), NAD+, and thiamine pyrophosphate, respectively. The products NADH and acetyl-CoA were competitive inhibitors with respect to NAD+ and CoA, and the inhibition constants were 15 and 47 &mgr;m, respectively. The complex was inactivated by phosphorylation and was reactivated after the removal of ATP and the addition of Mg2+.

Figure 1

A, Fractionation profile for a typical rate-zonal glycerol gradient. Approximately 3 mg of 400K enzyme was loaded onto this gradient. Fractions 19 through 30 were pooled and concentrated for the SDS gel shown in Figure 2. B, Coomassie blue-stained SDS-PAGE of odd-numbered glycerol-gradient fractions 7 to 33. Positions of protein standards are indicated on the left (in kilodaltons). The position of the 110-kD protein is indicated on the right with an arrowhead. U, Units.

Figure 2

SDS-PAGE and corresponding immunoblots of lysed mitochondria (total mito), the supernatant from the 100,000g centrifugation (100K super), the 400K enzyme (400K pellet), and the pooled mtPDC activity fraction from the glycerol-gradient fractionation (glycerol). A, Coomassie blue-stained SDS-PAGE gel loaded with 5 μg of protein per lane. Positions of protein standards are indicated on the left and calculated molecular mass values of the predominant bands in the glycerol fraction are indicated on the right (in kilodaltons). B, Four replica protein blots of the fractions in A were probed with anti-subunit antibodies. One microgram of protein was loaded per lane. The molecular masses of the protein bands are indicated on the left (in kilodaltons).

Figure 3

Two-dimensional gel electrophoresis of glycerol-gradient-enriched maize mtPDC and corresponding immunoblots. A, Ten micrograms of glycerol-gradient-enriched mtPDC was resolved by IEF in the first dimension followed by SDS-PAGE. B and C, One microgram of protein was resolved as in A, transferred to nitrocellulose, and probed with antibodies (Ab) to the E1α- and E1β-subunits. The molecular masses of the protein bands are indicated on the left (in kilodaltons).

Figure 4

Amino acid alignment of N-terminal amino acid sequences for maize 52- and 53-kD proteins and deduced amino acid sequences for yeast, Arabidopsis, and human E2-subunits. The number of amino acid residues (aa) before the homologous region is indicated to the left of the sequences. Shading indicates an identical amino acid. X indicates a cycle of Edman degradation for which no determination was made.

Figure 5

Divalent cation requirement for the 400K enzyme. The 400K enzyme was dialyzed for 2 h in 2 L of 2 mm EDTA and 2 mm EGTA to remove endogenous cations, and then dialyzed twice in 2 L of 20 mm Tes, pH 7.5, and 0.5 mm DTT to remove the chelators. Enzyme was added to an assay vessel that contained divalent cations and necessary components. Rates are expressed as relative percentages of the maximum rate (0.093 μmol min⁻¹ mg⁻¹).

Figure 6

A, ATP-dependent inactivation of the 400K enzyme. Equimolar amounts of Mg²⁺ and ATP were added to PDC preparations to the final concentrations indicated. The control did not have any MgATP added. One-hundred-microgram samples of enzyme were removed at various time intervals and assayed for activity. Activity is expressed as a percentage of the control (0.16 μmol NADH formed min⁻¹ mg⁻¹ protein) at time 0. B, The effect of Mg²⁺ on reactivation of P-PDC. The 400K enzyme was incubated with 200 μm MgATP until the inactivation of mtPDC ceased. Free ATP was then removed with 2.5 units of hexokinase (HK) and 2 mm Glc at 30 min. The 400K enzyme was then divided into four aliquots to which EDTA, MgCl₂, or buffer (control) was added to the final concentrations indicated.