Molecular Weight and Quaternary Structure of Yeast <scp>l</scp>‐Lactate Dehydrogenase (Cytochrome <i>b</i><sub>2</sub>)

Pajot, Patrick; Groudinsky, Olga

doi:10.1111/j.1432-1033.1970.tb00833.x

Cited by 108 publications

(54 citation statements)

References 21 publications

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“…Standard methods for growth of Escherichia coli, plasmid purification, DNA manipulation and transformation were used as described previously [9]. Enzymes Wild-type and mutant flavocytochromes b2 expressed in E. coli were isolated from cells, which had been stored at -20°C, using a previously reported purification procedure [8] haem separated on a Sephadex G-25 gel-filtration column (1.5 cm x 15 cm; Sigma), equilibrated and eluted with Caps (3-cyclohexylamino-1-propanesulphonic acid) buffer (Sigma), pH 11, I0.10 mol/l at 4°C in darkness, using their known visible absorption maxima [11,12]. The Caps buffer consisted of 0.01 M NaOH titrated against Caps solution to pH 11 and adjusted to I 0.10 mol/l using NaCl.…”

Section: Dna Manipulation Strains and Growthmentioning

confidence: 99%

Strategic manipulation of the substrate specificity of Saccharomyces cerevisiae flavocytochrome b2

Daff

Manson

Reid

et al. 1994

Biochemical Journal

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Flavocytochrome b2 from Saccharomyces cerevisiae acts physiologically as an L-lactate dehydrogenase. Although L-lactate is its primary substrate, the enzyme is also able to utilize a variety of other (S)-2-hydroxy acids. Structural studies and sequence comparisons with several related flavoenzymes have identified the key active-site residues required for catalysis. However, the residues Ala-198 and Leu-230, found in the X-ray-crystal structure to be in contact with the substrate methyl group, are not well conserved. We propose that the interaction between these residues and a prospective substrate molecule has a significant effect on the substrate specificity of the enzyme. In an attempt to modify the specificity in favour oflarger substrates, three mutant enzymes

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Section: Dna Manipulation Strains and Growthmentioning

confidence: 99%

Strategic manipulation of the substrate specificity of Saccharomyces cerevisiae flavocytochrome b2

Daff

Manson

Reid

et al. 1994

Biochemical Journal

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“…L'hydroxyapatite employite pour les chromatographies est prkparee selon [16] On precipite alors l'ensemble des proteines par addition de sulfate d'ammonium B pH constant jusqu'k goo/, de la saturation. On redissout ce pritcipite dans un faible volume de phosphate 50 mM, pH 6 Chromatographie sur hydroxyapatite. Selon les preparations, il peut &re nitcessaire de chromatographier une troisibme fois la protitine dans les mBmes conditions que ci-dessus mais sur une colonne de dimensions reduites (1,5 x 20 em) avec un volume de gradient de 300 ml (2 x 150 ml).…”

Section: Matgriel Et Mgthodesunclassified

“…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.…”

Section: Inhibition De La Cristallisation Par Le Fluorure De Ph6nylmcunclassified

Sur les deux formes moleculaires du cytochrome b2 de Saccharomyces cerevisiae

Jacq

Lederer

1972

Eur J Biochem

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Several authors showed that a modification of the enzyme must occur immediately before crystallization [ll, 121.We show here that in the presence of phenylmethylsulfonyl fluoride crystallization does not take place. We describe a new method for purifying the protein, using phenylmethylsulfonylfluoride a t all steps. The cytochrome b, purified in this manner shows kinetic properties identical with those described for the enzyme present in crude extracts (the so-called physiological cytochrome b, [ill), and significantly different from those of the crystalline enzyme.We further show that an essential structural difference between the two purified forms of the enzyme lies in their tetrameric structure : whereas the protomer of crystallized cytochrome b, is constituted by two polypeptide chains of unequal size (36000 and 21 000 daltons [8,9]), the protomer of physiological cytochrome 6 , is constituted by only one polypeptide chain of Mr 57000 daltons. The transformation of the latter form into the former must result from a proteolytic cleavage, which enables the less soluble, modified form to crystallize.I n the light of our results, it seems that physiological cytochrome b, is a tetramer composed of four polypeptide chains carrying one heme and one FMN each. It is tempting to postulate that each chain is folded into two globules linked by an exposed segment of polypeptide.Finally, our results cast doubt as to the significance of most results concerning cytochrome b,, which were obtained with the crystallized, artifactual form.Le cytochrome b,, ou L-lactic0 cytochrome c

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“…This operation was repeated twice. After the second centrifugation, the pellet was dissolved in a minimum amount of buffer B (buffer A without ammonium sulfate) and the solution was filtrated through a Sephadex G-25 column (d = 2 cm, h = 10 cm) equilibrated in the same buffer.H-flavocytochrome b, concentrations were determined with a Perkin Elmer 555 spectrophotometer using the absorption coefficient E~~~~~ = 183 m M -l cm-' for the dithionitereduced form [16]. …”

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confidence: 99%

Proteolytic Cleavage of Hansenula anomala Flavocytochrome b₂ into Its Two Functional Domains

Gervais¹,

Risler²,

Corazzin³

1983

European Journal of Biochemistry

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In a previous work, we have described the tryptic cleavage of yeast flavocytochrome b, into its two functional domains: a cytochrome h, core and a flavodehydrogenase. The lactate dehydrogenase efficiency of the latter was, however, dramatically low, only about 1 :< that of intact flavocytochrome b,. Our present study concerns a new flavodehydrogenase derivative of Hansenula anomala flavocytochrome 6 , which spontaneously dissociates from the cytochrome domain when the polypeptide bridge connecting thein is cleaved by Staphylococcus aureus V 8 protease I. This flavodehydrogenase was purified and some of its functional and structural properties were studied. It presents an exceptionally high lactate dehydrogenase activity, about 80 "/, that of flavocytochrome b,. This result clearly demonstrates that the cytochrome domain is not necessary for the lactate dehydrogenase function and suggests an autonomous folding for both domains.Our results are discussed in terms of 'gene fusion'.Each chain of yeast flavocytochrome b, has been shown to be folded into three globules: n, E and / I ' joined together in the 'intact' enzyme by two protease-sensitive bridges aa' and cd [I -41 (cf. These two functional domains, flavodehydrogenase and cytochrome, which spontaneously dissociate from each other when their connecting bridge cd is cleaved by trypsin, were isolated as stable, pure, molecular entities, termed FDH, and nT respectively [2, 3, 91. Unfortunately, this polypeptide bridge cd is not the part of the native chain which is most sensitive towards trypsin: the aa' zone is cleaved about 20-times faster [lo], so that FDHT was always found altered by this undesirable inner cleavage, which was achieved with a loss of about 20 amino acid residues [3].From a functional point of view, the first cleavage in aa' results in a 96 %loss of activity [I I]; the isolated 'nicked' FDH, entity itself only retains about 1 % of the ferricyanide reductase activity of native flavocytochrome b2 [lo]. These results did not elucidate whether such a low activity was due to the aa' cleavdge itself or to the release of cytochrome b2 core resulting from the second cleavage at cd.This study presents information concerning this question. Despite the hypersensitivity of the aa' zone towards a number of proteases [I2 -141, we succeeded in obtaining a cleavage in the cd zone faster than in the aa' zone. This was achieved with Hansenula anomala flavocytochrome b2 and the highly specific Staphylococcus aureus V 8 protease 1. We describe a method for separating the so-formed 'intact' flavodehydrogenase part which we currently term 'FDH,,', from the other proteolysis products. Steady-state catalytic parameters of this new flavocytochrome h, derivative have been determined and compared with those of the native flavocytochrome b, and its tryptic derivatives. MATERIALS A N D METHODS Hansenula anomala Flavocytochrome b,This was prepared according to [I51 with the minor modifications described in [I I]. It was stored as an ammonium sulfate precipitate (50 % satur...

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

Cited by 108 publications

References 21 publications

Strategic manipulation of the substrate specificity of Saccharomyces cerevisiae flavocytochrome b2

Strategic manipulation of the substrate specificity of Saccharomyces cerevisiae flavocytochrome b2

Sur les deux formes moleculaires du cytochrome b2 de Saccharomyces cerevisiae

Proteolytic Cleavage of Hansenula anomala Flavocytochrome b₂ into Its Two Functional Domains

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

Cited by 108 publications

References 21 publications

Strategic manipulation of the substrate specificity of Saccharomyces cerevisiae flavocytochrome b2

Strategic manipulation of the substrate specificity of Saccharomyces cerevisiae flavocytochrome b2

Sur les deux formes moleculaires du cytochrome b2 de Saccharomyces cerevisiae

Proteolytic Cleavage of Hansenula anomala Flavocytochrome b2 into Its Two Functional Domains

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

Proteolytic Cleavage of Hansenula anomala Flavocytochrome b₂ into Its Two Functional Domains