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Carlsberg Research Communications

, Volume 47, Issue 2, pp 119–141 | Cite as

Partial separation of individual enzyme activities of an ACP-dependent fatty acid synthetase from barley chloroplasts

  • Peter Bordier Høj
  • Jørn Dalgaard Mikkelsen
Article

Abstract

An acyl carrier protein (ACP) dependent fatty acid synthetase (fas) from barley chloroplast stroma was purified five-fold by ammonium sulphate precipitation and gel filtration on Sephacryl S-300. The β-ketoacyl-ACP reductase, β-ketoacyl-ACP synthetase, acetyl-CoA:ACP transacylase and malonyl-CoA:ACP transacylase activities were resolved on the Sephacryl S-300 column with apparent molecular weights of respectively 125, 92, 82 and 41 kilodalton. The fas activity exhibited an apparent molecular weight of 87 kilodalton resulting from the overlapping portions of the component activities. A fifth component of the active fas, ACP, was separated completely from the other four individual enzyme activities by the ammonium sulphate precipitation. When the fas purified by gel filtration was applied to a Mātrex Gel Blue B column, the component activities were separated into two groups. A bound fraction contained all the malonyl-CoA:ACP transacylase whereas the β-ketoacyl synthetase activity was exclusively present in the non-bound fraction. Neither the bound nor the non-bound fraction showed any fas activity alone, but complete reconstitution of fas activity was obtained when both protein fractions were combined. The barley chloroplast fas is therefore not a multifunctional protein but consists of at least five separable components. Characterization with respect to cofactor requirements was also performed. Variation of certain cofactor concentrations markedly altered the pattern of fatty acid synthesis.

Keywords

Affinity chromatography gel filtration radio-gas liquid chromatography fatty acids arsenite Coenzyme A derivatives multienzyme complex monofunctional polypeptide multifunctional polypeptide 

Abbreviations

ACP

acyl carrier protein

DTNB

5′,5-dithiobis(2-nitro benzoic acid)

DTT

1,4-dithiotreitol

Hepes

N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid

HPLC

high pressure liquid chromatography

kD

kilodalton

Tricine

N-(Tris(hydroxy-methyl)-methyl)-glycin

SDS

sodium dodecyl sulphate

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Copyright information

© Carlsberg Laboratory 1982

Authors and Affiliations

  • Peter Bordier Høj
    • 1
    • 2
  • Jørn Dalgaard Mikkelsen
    • 1
    • 2
  1. 1.Department of PhysiologyCarlsberg LaboratoryCopenhagen Valby
  2. 2.Institute of GeneticsUniversity of CopenhagenCopenhagen K

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