A lactate oxygenase from Mycobacterium phlei: III. Evidence for the subunit structure With an appendix electron microscopic studies—“Morphology”

Abstract

Crystalline lactate oxygenase from Mycobacterium phlei with a molecular weight of 350,000 has been shown to be composed of six subunits of similar size. The bonds responsible for holding these subunits together appear to be strong, and dissociation of subunits does not occur in the presence of 8 m urea and 100 m m 2-mercaptoethanol. However, a complete dissociation of the enzyme is achieved by treatment with 5 m guanidine hydrochloride and 100 m m 2-mercaptoethanol. As determined by sedimentation and diffusion, the subunit molecular weight is 54,000–57,000, corresponding to the minimum molecular weight obtained from flavin content. A similar value is estimated by gel electrophoresis in the presence of sodium dodecyl sulfate. Quantitative amino-terminal amino acid analysis by the dinitrofluorobenzene method shows the presence of 1 mole serine/56,000 g of the enzyme protein. Digestion with carboxypeptidase B releases 1 mole arginine residue/56,000 g of the enzyme protein. Selective carboxyl-terminal tritiation also indicates that the arginine is the sole carboxyl-terminal amino acid. Further examination of negatively stained electron microscopic images of this enzyme (Appendix) leads us to the deduction of a plausible structural model of the enzyme molecule. Appearances of the molecule, crystals, and their transient chain or plane segment structures are explained in terms of the proposed model.