Changes in the properties of L-lactate dehydrogenase (cytochrome beta-2) from yeast during preparation of the crystalline enzyme.

Abstract

1. 1. Cytochrome b2, l-lactate dehydrogenase (l-lactate:cytochrome c oxidoreductase, EC 1.1.2.3) was present in the 10 000 × g supernatant of yeast cells broken in a Nossal shaker and to a lesser extent (30–40% of the total activity) in particles sedimenting between 1000 × g and 10 000 × g. The particulate enzyme became soluble on treatment with butanol-lactate. Lactate dehydrogenase from both sources behaved identically on electrophoresis in starch gel and migrated as a narrow anionic zone at pH 8.3. 2. 2. On storage of lactate dehydrogenase at 4° after partial purification by fractionation with acetone or polyethylene glycol, the Michaelis constant for l-lactate with ferricyanide as acceptor increased from 0.6 mM to 1.7 mM and the constant with ferricytochrome c as acceptor increased from 0.08 mM to 0.5 mM. The mean mobility of the enzyme also increased and multiple bands of l-lactate dehydrogenase activity were found on electrophoresis of the stored enzyme. 3. 3. It has not been possible to obtain crystalline enzyme from partly purified yeast extracts without prior storage or dialysis for at least 24 h at 4°. After crystallization, yeast l-lactate dehydrogenase was found to have the higher Km, greater electrophoretic mobility and multiple electrophoretic bands characteristic of the stored enzyme. Saturation of partly purified enzyme solutions with butanol prevented these changes and also prevented crystallization. 4. 4. Recrystallization of l-lactate dehydrogenase did not require prolonged storage or dialysis and the preferential crystallization of previously crystallized 35S-labelled lactate dehydrogenase from a solution of non-radioactive uncrystallized enzyme indicated that modification must precede crystallization in the conditions used here.