Alternative respiration of fungus Phycomyces blakesleeanus.

Respiratory characteristics of germinating spores, developing mycelium and mitochondria of the fungus Phycomyces blakesleeanus were investigated by means of oxygen Clark-type electrode. The effects of respiratory inhibitors and metabolic compounds on oxygen consumption were tested. It was demonstrated that P. blakesleeanus apart of cyanide-sensitive respiration, CSR, possess alternative respiration, (cyanide-resistant respiration, CRR) which is constitutive and whose capacity decreases during development. Maximum is observed for activated spores where CRR capacity is significantly greater than CSR. After treatment with antimycin A, a third type of respiration insensitive to antimycin A and low concentration of SHAM (sufficient for inhibition of CRR), but sensitive to cyanide and high concentration of SHAM, has been expressed.

Characterization of NAD-dependent malate dehydrogenases from spinach leaves.

Spinach leaves were used to extract isoforms of NAD-dependent malate dehydrogenase (NAD-MDH) (EC 1.1.1.37), either soluble or bound to microsomal, plasma, or chloroplast envelope membranes. All fractions were subjected to isoelectric focusing analysis, which showed that purified chloroplast envelopes contain an NAD-MDH isoform tightly bound to the membranes, since treatment with 0.5 or 1% Triton X-100 was not able to release the enzyme from the envelopes. In contrast, plasma membranes released an isoform with a pI of 3.5 following treatment with 0.5% Triton X-100. The most abundant soluble leaf isoform had a pI of 9, while the chloroplast stroma contained an isoform with a pI of 5.3. Kinetic analysis of oxaloacetate (OAA)-dependent NADH oxidation in different fractions gave different Km values for both substrates, the envelope- and plasma membrane-bound NAD-MDH exhibiting the highest affinities for OAA. Leaf plasma membrane-bound MDH exhibited a high capacity for both reaction directions (malate oxidation and OAA reduction), while the two chloroplast isoforms (stromal and envelope-bound) preferentially reduced OAA. Our results indicate that the chloroplast envelope contains a specifically attached NAD-MDH isoform that could provide direct coupling between chloroplast and cytosol adenylate pools.