Molecular Weight and Quaternary Structure of Yeast l‐Lactate Dehydrogenase (Cytochrome b₂)

Abstract

Amino acid analyses of L-lactate dehydrogenase from baker's yeast show that the minimum molecular weight (53000 daltons) of the protein is much lower than found in the literature (80000). This result, combined with those reported in the following papers, leads to a revision of the dimeric model generally accepted for cytochrome b,. Following the physicochemical studies of Morton et al. [5], the enzymically active L-lactate dehydrogenase has been considered t o have two haems and two flavins for a weight of 160000-1180000, although, as pointed out by Armstrong [9], some discrepancy existed between the results obtained by the various techniques. It was further shown by Baudras and Labeyrie [6] that cytochrome b, can be dissociated into subunits by urea and guanidinium chloride.Amino acid analyses carried out in our laboratory on type I1 cytochrome b, gave results inconsistent with the previously accepted dimeric structure of the protein. We report here the results of our work, which, taken together with those presented in the following papers [7--81, would lead to a tetrameric structure for L-lactate dehydrogenase and to a revision of its molecular weight. Amino Acid Analyses Type I1 cytochrome b, prepared as described above was used for all amino acid determinations. MATERIALS AND METHODS PreparationAn aliquot of the stock solution was diluted in 0.1 M phosphate buffer pH 7.2 containing 20 p. M EDTA, and the concentration determined by measuring the absorbance of the Soret band at 424nm in the presence of sodium dithionite. The spectrometer used was either a Cary 15 or a Cary 14; with a 1 om pathlength the absorbance a t the maximum of the reduced Soret band was on the order of 0.6 to 1. Carefully measured aliquots of that same dilution (usually 0.2 to 0.4 ml) were then pipetted into hydrolysis tubes. A known amount of norleucine and &-amino p-guanido propionic acid as reference standards was added and after thorough mixing the samples were lyophilized.

Molecular Weight and Quaternary Structure of Yeast l‐Lactate Dehydrogenase (Cytochrome b2)

“…This figure is somewhat higher than 52500 & 2000 estimated in the preceding paper [17]. The standard deviation of our determinations is 1100; but the results may be also affected by a systematic error (0-5O/,) due to water trapped in the "dry" protein material; nevertheless the agreement between the dry weight and the protein weight estimated by aminoacid analyses carried out by Jacq and Lederer on the same sample [I71 makes us confident in our results.…”

Sur les deux formes moleculaires du cytochrome b2 de Saccharomyces cerevisiae

“…On sait que le cytochrome b, cristallisi? possede comme groupements prosthktiques 4 hemes et 4 FMN [5,6] pour une molecule qui est probablement t6tra-merique [7]. Compte tenu de ce que nous avons dit plus haut, il en est vraisemblablement de m6me pour Ie cytochrome 6, physiologique.…”

Flavocytochrome b2: Reactivity of Its Flavin with Molecular Oxygen

“…It catalyzes the oxidation of l -lactate to pyruvate at the expense of cytochrome c in the mitochondrial intermembrane space (). Each subunit of the tetrameric enzyme is composed of two domains: a heme-binding domain (residues 1−99) and an FMN-binding domain (residues 100−511) ( , ). In the crystallographic asymmetric unit (a dimer), one heme domain out of two is invisible, due to its mobility.…”