NADH-Monodehydroascorbate oxidoreductase is one of the redox enzymes in spinach leaf plasma membranes

Abstract

Amino acid analysis of internal sequences of purified NADH-hexacyanoferrate(III) oxidoreductase (NFORase), obtained from highly purified plasma membranes (PM) of spinach (Spinacia oleracea L.) leaves, showed 90 to 100% homology to internal amino acid sequences of monodehydroascorbate (MDA) reductases (EC 1.6.5.4) from three different plant species. Specificity, kinetics, inhibitor sensitivity, and cross-reactivity with anti-MDA reductase antibodies were all consistent with this identification. The right-side-out PM vesicles were subjected to consecutive salt washing and detergent (polyoxyethylene 20 dodecylether and 3-[(3-cholamido-propyl)-dimethylammonio]-1-propane sulfonate [CHAPS]) treatments, and the fractions were analyzed for NFORase and MDA reductase activities. Similar results were obtained when the 300 mm sucrose in the homogenization buffer and in all steps of the salt-washing and detergent treatments had been replaced by 150 mm KCl to mimic the conditions in the cytoplasm. We conclude that (a) MDA reductase is strongly associated with the inner (cytoplasmic) surface of the PM under in vivo conditions and requires washing with 1.0 m KCl or CHAPS treatment for removal, (b) the PM-bound MDA reductase activity is responsible for the majority of PM NFORase activity, and (c) there is another redox enzyme(s) in the spinach leaf PM that cannot be released from the PM by salt-washing and/or CHAPS treatment. The PM-associated MDA reductase may have a role in reduction of ascorbate in both the cytosol and the apoplast.

Figure 1

Scheme of the fractionation protocol.

Figure 2

Internal amino acid (aa) sequences of the NFORase purified from spinach leaf PM vesicles. Frozen PM vesicles were thawed and diluted with buffer with low ionic strength and low osmotic potential and then pelleted by centrifugation. Enzyme 1 was purified from the pellet after CHAPS solubilization, and enzyme 2 was purified from the supernatant containing proteins released by the freezing-thawing procedure and hypo-osmotic shock. Search for homologous internal sequences in the Swiss-Prot databank identified highly homologous sequences in monodehydroascorbate radical reductases in three different species. Numbers on the right are the amino acid positions of the highly homologous sequences in the known MDA reductases. Bold letters indicate identity.

Figure 3

Redox activity of the NFORase (enzyme 1) when the concentration of MDA (▪), NADH (•), and NADPH (▴) was varied and the concentrations of NADH and MDA, respectively, were held constant. Results presented are from one enzyme preparation; two other preparations gave similar results (for K_m values, see Results).

Figure 4

Immunoblotting of the PM and supernatant fractions of the MDA reductase activity fractionation experiment using anti-MDA reductase antibodies. Lanes A to F, Fractions from PM preparation with Suc in the homogenization buffer; lanes J to O, fractions from PM preparation with KCl in the homogenization buffer. A and J, PM; B and K, S1 fraction; C and L, S2 fraction; D and M, S3 fraction; E and N, S4 fraction; F and O, S5 fraction; G and H, Mono-Q fraction Q27 and affinity fraction A40 (Bérczi et al., 1995); and I, standard proteins. About the same total MDA reductase activity (approximately 5 nmol min⁻¹) was applied in each lane (except lane I). The positions of standard proteins of known molecular mass are shown on the left. All fractions were from the same experiment. Three independent fractionation experiments gave similar results. The high-molecular-mass band in lanes F and O is an artifact probably caused by CHAPS.

Figure 5

Schematic representation of the location of PM-bound MDA reductase and of the functional interaction between the trans-PM b-type Cyt and the PM-bound MDA reductase.