Immunological detection of NADH-specific enoyl-ACP reductase from rape seed (Brassica napus)--induction, relationship of alpha and beta polypeptides, mRNA translation and interaction with ACP.
An antibody has been raised against rape seed enoyl-ACP reductase. This recognizes both the alpha and beta polypeptides of the enzyme. Immunoblotting of fresh seed demonstrates that beta is not present in seed material, and that it is produced by proteolysis during isolation. It is thus deduced that rape seed enoyl reductase is an alpha 4 homotetramer. Leaf material from both rape and Arabidopsis have an enoyl reductase with a similar electrophoretic mobility to the rape seed enzyme when analyzed on SDS-PAGE. Quantitative immunoassay has demonstrated that the enzyme continually increases during lipid deposition, indicating that an increase in this enzyme is required to sustain high levels of lipid biosynthesis. In vitro translation experiments show that the enzyme is nuclear coded and synthesized as a precursor form. Immunogold electron microscopy has demonstrated that enoyl reductase is located in plastids. It is shown that ACP-Sepharose may be used as a matrix in the purification of enoyl-ACP reductase.
Slabas
A R
AR
Unilever Research, Sharnbrook, U.K.
Cottingham
I R
IR
Austin
A
A
Hellyer
A
A
Safford
R
R
Smith
C G
CG
D000213:Acyl Carrier Protein / Q000378:metabolism*; D000918:Antibody Specificity; D015153:Blotting, Western; D001937:Brassica / Q000201:enzymology* / Q000235:genetics; D002846:Chromatography, Affinity; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); D004790:Enzyme Induction; D007124:Immunoenzyme Techniques; D007150:Immunohistochemistry; D046911:Macromolecular Substances; D009243:NAD / Q000378:metabolism; D010088:Oxidoreductases / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D011863:Radioimmunoassay; D012639:Seeds / Q000201:enzymology; D013379:Substrate Specificity
D000213:Acyl Carrier Protein; D046911:Macromolecular Substances; D012333:RNA, Messenger; D009243:NAD; D010088:Oxidoreductases; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)