Lactic dehydrogenase and cytochrome b2 of baker's yeast. The deoxyribose polynucleotide component and the physicochemical properties of the crystalline enzyme

Abstract

Sedimentation of crystalline cytochrome b2 (lactic dehydrogenase) of baker''s yeast in an ultracentrifuge revealed only 1 component, having a sedimentation coefficient (S20,w) of 7.7 s. Electrophoresis at pH 6.65, 7.05 and 8.8 showed partial dissociation of a polynucleotide component of the enzyme. Separation of nucleotide-depleted enzyme from intact enzyme was detected after electrophoresis for 269 minutes at pH 8.8. There was no separation of a flavoprotein from a haemoprotein. The results indicate that crystalline cytochrome b2 contains only 1 major protein component. There are approximately 15 (polymerized) deoxy- ribonucleotide residues/mole of haem in the crystalline enzyme. The base composition (adenine, 6; thymine, 5; guanine, 2; cytosine, 2 moles) of the polynucleotide differs considerably from that of yeast deoxy-ribonucleic acid. Recrystallized cytochrome b2 contains no detectable ribose or uracil, indicating the absence of ribonucleotides. Dissociation of the deoxyribopolynucleotide of cytochrome b2 by 0.7-saturated ammonium sulphate or by digestion with pancreatic deoxyribonuclease prevented crystallization of the enzyme by anaerobic dialysis at low ionic strength. An amorphous precipitate was formed on recombination of the polynucleotide and nucleotidedepleted enzyme. The nucleotide is not essential for dehydrogenase activity. No uptake or exchange of inorganic phosphate during oxidation of lactate catalyzed by the crystalline enzyme could be demonstrated. The possible function of the polynucleotide is discussed. There is approximately 0.56% P, 0.74% S and 15.2% N in the crystalline enzyme, and approximately 14.2% of N in the nucleotide-free enzyme. The properties of cytochrome b2, a crystalline deoxyribonucleoprotein, are compared with those of other nucleoproteins.