Preparation of Escherichia coli pyruvate oxidase utilizing a thiamine pyrophosphate affinity column

Thomas A. O'Brien; Herbert L. Schrock; Patricia Russell; Robert Blake; Robert B. Gennis

doi:10.1016/0005-2744(76)90054-1

Preparation of Escherichia coli pyruvate oxidase utilizing a thiamine pyrophosphate affinity column

Thomas A. O'Brien, Herbert L. Schrock, Patricia Russell, Robert Blake, Robert B. Gennis

Biochemistry

Research output: Contribution to journal › Article › peer-review

Abstract

An improved procedure is reported for the purification of Escherichia coli pyruvate oxidase (pyruvate:ferricytochrome b₁ oxidoreductase, EC 1.2.2.2), a peripheral membrane flavo-enzyme, which is much more reproducible and requires considerably less time than the original purification scheme. The key element in this protocol is a new Sepharose-based affinity resin designed for the isolation of thiamine pyrophosphate-requiring enzymes. The synthesis, partial characterization, and use of two such affinity resins is described. Pyruvate oxidase is a pure, homogeneous protein as it is eluted from the affinity resin. The enzyme is a tetramer with a subunit molecular weight of approx. 60 000. The subunits appear to be identical. The isoelectric point of pyruvate oxidase is 5.6.

Original language	English (US)
Pages (from-to)	13-29
Number of pages	17
Journal	BBA - Enzymology
Volume	452
Issue number	1
DOIs	https://doi.org/10.1016/0005-2744(76)90054-1
State	Published - Nov 8 1976

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General Medicine

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10.1016/0005-2744(76)90054-1

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title = "Preparation of Escherichia coli pyruvate oxidase utilizing a thiamine pyrophosphate affinity column",

abstract = "An improved procedure is reported for the purification of Escherichia coli pyruvate oxidase (pyruvate:ferricytochrome b1 oxidoreductase, EC 1.2.2.2), a peripheral membrane flavo-enzyme, which is much more reproducible and requires considerably less time than the original purification scheme. The key element in this protocol is a new Sepharose-based affinity resin designed for the isolation of thiamine pyrophosphate-requiring enzymes. The synthesis, partial characterization, and use of two such affinity resins is described. Pyruvate oxidase is a pure, homogeneous protein as it is eluted from the affinity resin. The enzyme is a tetramer with a subunit molecular weight of approx. 60 000. The subunits appear to be identical. The isoelectric point of pyruvate oxidase is 5.6.",

author = "O'Brien, {Thomas A.} and Schrock, {Herbert L.} and Patricia Russell and Robert Blake and Gennis, {Robert B.}",

note = "Funding Information: phate affinity resin. We would especially like to acknowledge the aid of Dr. Lowell P. Hager whose experience with this system proved most valuable. This research was supported by grants from the National Institutes of Health, HL16101 (R.B.G.) and GM 07768 to Lowell Hager. T.A.O'B., H.L.S. and P.R. were Trainees of the Biophysical Chemistry Training Program at the University of Illinois and supported by PHS GM 00722. R.B.G. is grateful for support from PHS Career Development Award K04 HL00040.",

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doi = "10.1016/0005-2744(76)90054-1",

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T1 - Preparation of Escherichia coli pyruvate oxidase utilizing a thiamine pyrophosphate affinity column

AU - O'Brien, Thomas A.

AU - Schrock, Herbert L.

AU - Russell, Patricia

AU - Blake, Robert

AU - Gennis, Robert B.

N1 - Funding Information: phate affinity resin. We would especially like to acknowledge the aid of Dr. Lowell P. Hager whose experience with this system proved most valuable. This research was supported by grants from the National Institutes of Health, HL16101 (R.B.G.) and GM 07768 to Lowell Hager. T.A.O'B., H.L.S. and P.R. were Trainees of the Biophysical Chemistry Training Program at the University of Illinois and supported by PHS GM 00722. R.B.G. is grateful for support from PHS Career Development Award K04 HL00040.

PY - 1976/11/8

Y1 - 1976/11/8

N2 - An improved procedure is reported for the purification of Escherichia coli pyruvate oxidase (pyruvate:ferricytochrome b1 oxidoreductase, EC 1.2.2.2), a peripheral membrane flavo-enzyme, which is much more reproducible and requires considerably less time than the original purification scheme. The key element in this protocol is a new Sepharose-based affinity resin designed for the isolation of thiamine pyrophosphate-requiring enzymes. The synthesis, partial characterization, and use of two such affinity resins is described. Pyruvate oxidase is a pure, homogeneous protein as it is eluted from the affinity resin. The enzyme is a tetramer with a subunit molecular weight of approx. 60 000. The subunits appear to be identical. The isoelectric point of pyruvate oxidase is 5.6.

AB - An improved procedure is reported for the purification of Escherichia coli pyruvate oxidase (pyruvate:ferricytochrome b1 oxidoreductase, EC 1.2.2.2), a peripheral membrane flavo-enzyme, which is much more reproducible and requires considerably less time than the original purification scheme. The key element in this protocol is a new Sepharose-based affinity resin designed for the isolation of thiamine pyrophosphate-requiring enzymes. The synthesis, partial characterization, and use of two such affinity resins is described. Pyruvate oxidase is a pure, homogeneous protein as it is eluted from the affinity resin. The enzyme is a tetramer with a subunit molecular weight of approx. 60 000. The subunits appear to be identical. The isoelectric point of pyruvate oxidase is 5.6.

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Preparation of Escherichia coli pyruvate oxidase utilizing a thiamine pyrophosphate affinity column

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