Christopher Sidebottom

Induction, purification and characterization of NADH-specific enoyl acyl carrier protein reductase from developing seeds of oil seed rape (Brassica napus)

The induction kinetics of enoyl reductase have been studied in the developing rape seed. Enzyme activity rapidly increases prior to the onset of lipid accumulation and remains high even after lipid synthesis has ceased. Enoyl reductase has been purified from developing seed by a purification procedure consisting of conventional blue Sepharose chromatography combined with HPLC ion-exchange and hydrophobic chromatography. The final preparation when analysed by polyacrylamide gel electrophoresis was homogeneous on gradient gels but on linear gels shows a doublet with components α and β of molecular weight of 34 800 and 33600, respectively. The enzyme has a native molecular weight of 140000 by gel filtration. The amino acid composition of the enoyl reductase has been determined and is remarkably similar to that reported for the spinach enzyme. Enoyl reductase is active over a wide pH range and has an absolute specificity for NADH; the apparent Km for NADH is 7.6 μM. The apparent Km values for crotonyl-CoA and crotonyl acyl carrier protein were 178 μM and less than 1 μM, respectively. Enoyl reductase activity is sensitive to inhibition by p-chloromercuribenzoate and N-ethylmaleimide but insensitive to iodoacetamide.

Evidence That a 77-Kilodalton Protein from the Starch of Pea Embryos Is an Isoform of Starch Synthase That Is Both Soluble and Granule Bound

In this paper we provide further evidence about the nature of a 77-kD starch synthase (SSII) that is both soluble and bound to the starch granules in developing pea (Pisum sativum L.) embryos. Mature SSII gives rise to starch synthase activity when expressed in a strain of Escherichia coli lacking glycogen synthase. In transgenic potatoes (Solanum tuberosum L.) expressing SSII, the protein is both soluble and bound to the starch granules. These results confirm that SSII is a starch synthase and indicate that partitioning between the soluble and granule-bound fraction of storage organs is an intrinsic property of the protein. A 60-kD isoform of starch synthase found both in the soluble and granule-bound fraction of the pea embryos is probably derived by the processing of SSII and is different gene product from GBSSI, the exclusively granule-bound 59-kD isoform of starch synthase that is similar to starch synthases encoded by the waxy genes of cereals and the amf gene of potatoes. Consistent with this, expression in E. coli of an N-terminally truncated version of SSII gives rise to starch synthase activity.

3-Oxoacyl-[ACP] reductase from oilseed rape (Brassica napus)

3-Oxoacyl-[ACP] reductase (E.C. 1.1.1.100, alternatively known as beta-ketoacyl-[ACP] reductase), a component of fatty acid synthetase has been purified from seeds of rape by ammonium sulphate fractionation, Procion Red H-E3B chromatography, FPLC gel filtration and high performance hydroxyapatite chromatography. The purified enzyme appears on SDS-PAGE as a number of 20-30 kDa components and has a strong tendency to exist in a dimeric form, particularly when dithiothreitol is not present to reduce disulphide bonds. Cleveland mapping and cross-reactivity with antiserum raised against avocado 3-oxoacyl-[ACP] reductase both indicate that the multiple components have similar primary structures. On gel filtration the enzyme appears to have a molecular mass of 120 kDa suggesting that the native structure is tetrameric. The enzyme has a strong preference for the acetoacetyl ester of acyl carrier protein (Km = 3 microM) over the corresponding esters of the model substrates N-acetyl cysteamine (Km = 35 mM) and CoA (Km = 261 microM). It is inactivated by dilution but this can be partly prevented by the inclusion of NADPH. Using an antiserum prepared against avocado 3-oxoacyl-[ACP] reductase, the enzyme has been visualised inside the plastids of rape embryo and leaf tissues by immunoelectron microscopy. Amino acid sequencing of two peptides prepared by digestion of the purified enzyme with trypsin showed strong similarities with 3-oxoacyl-[ACP] reductase from avocado pear and the Nod G gene product from Rhizobium meliloti.

POTATO LECTIN : A THREE-DOMAIN GLYCOPROTEIN WITH NOVEL HYDROXYPROLINE-CONTAINING SEQUENCES AND SEQUENCE SIMILARITIES TO WHEAT-GERM AGGLUTININ

Potato (Solanum tuberosum) tuber lectin is a chitin-binding, hydroxyproline-rich glycoprotein, which may be involved in the defence mechanism of the plant. We had previously obtained evidence that it consists of at least two very dissimilar domains. The aim was to use a combination of accurate determinations of molecular weight and protein sequencing to gain more accurate information on the domains. Accurate determinations of the molecular weight of the lectin by a MALDI mass spectrometer have shown that the subunit molecular weight is 65,500 (+/- 1100) and that of a totally deglycosylated sample is 31,250 (+/- 30). This means that the lectin is 52.3 (+/- 1)% carbohydrate with a considerable number of glycoforms being present. Partial sequences and other analyses are consistent with the existence of three distinct domains. These are: (1) an N-terminal region which is rich in proline but poor in hydroxyproline; (2) a glycosylated region with a glycosylated molecular weight of 45,300 (+/- 1100) and a deglycosylated molecular weight of 11,050 (+/- 50) which is extremely rich in glycosylated hydroxyproline residues with a similar sequence to extensins; and (3) a cystine-rich domain which has the sugar binding site shows partial conservation of a repeated motif common to many chitin-binding proteins of the hevin family including wheat-germ agglutinin. The closest similarity seems to be to the sequence of potato basic chitinase.

Purified enoy-[acyl-carrier-protein] reductase from rape seed (Brassica napus) contains two closely related polypeptides which differ by a six-amino-acid N-terminal extension

Abstract An improved method for the preparation of rape seed enoyl reductase is described. The result is a preparation with fewer impurities, as judged by SDS-polyacrylamide gel electrophoresis, which is obtained more quickly and in greater yield. However, in common with the previous protocol (Slabas, A.R., Sidebottom, C.M., Hellyer, A., Kessell, R.M.J. and Tombs, M.P. (1986) Biochim. Biophys. Acta 877, 271–280) the purified enzyme contains two polypeptides which only differ in molecular mass by about 1000 Da. The larger of these was separated from a mixture of both, by reverse-phase chromatography on a C-4 column, and sequenced from the N-terminus. The sequence of nineteen amino acids thus obtained was used to unravel the mixed sequence from the native preparation. This shows that the two polypeptides in such preparations differ by a six-amino-acid N-terminal extension. Furthermore, no notable differences were detected between the amino-acid compositions of the single and the mixed polypeptides, indicating that a comparable degree of similarity is probably maintained throughout the remainder of the two polypeptides.

Amino acid sequence analysis of rape seed (Brassica napus) NADH-enoyl ACP reductase

The biosynthesis of fatty acids in plants is catalysed by a type II, dissociable fatty acid synthetase (for review see [ 15]. This enzyme system contains at least seven catalytic domains and a central acyl carrier protein (ACP). The individual catalytic reactions are performed on ACP substrates [6] to which the acyl groups are attached via the 4phosphopantetheine group of the protein. Much is known about the structure of ACP in plants. cDNA [8, 9] and genomic clones have been isolated [3, 5, 7] and the tissue-specific and temporal regulation of the gene has been reported [3]. However, comparatively little is known concerning the structure of catalytic components of the plant fatty acid synthetase complex. Two reductive steps occur in core fatty acid biosynthesis, which are catalysed by enoyl ACP reductase and fl-ketoacyl ACP reductase. Enoyl ACP reductase catalyses the second reductive step in fatty acid biosynthesis and there are two forms of this enzyme, a NADH and a N A D P H enzyme. These are termed type I and type II respectively, and the activities have been separated in Safflower seed [10]. Whilst the type I and type II enzymes are present in seed material, only type I is present in leaf material [ 11 ]. The activity of the type I enoyl ACP reductase has been measured during seed development in oil seed rape. The activity rises prior to major storage lipid synthesis and the shape of the activity profile closely resembles that of lipid deposition [12]. Western blotting has demonstrated that the protein is continually synthesized during lipid deposition, presumably to meet the high demand for fatty acid synthesis in that part of seed development [ 14]. The enzyme is tetrameric and consists of four identical subunits of 33.6 kDa [ 14] . The rape enzyme has two arginine residues which are covalently modified by phenylyglyoxal, following which the enzyme is inactivated. Such inactivation can be protected by pre-incubation with ACP or coenzyme A, indicating that the arginine residues are either at the active site of the enzyme or that substrate binding results in a conformational change which affords protection [2]. This interaction with ACP has been demonstrated further by affinity chromatography of enoyl ACP reductase on an ACP-sepharose column. In this report, we present extensive amino acid sequence data derived from homogeneous NADH-specific enoyl ACP reductase from oil seed rape. Homogeneous 0c-4 NADH-specific enoyl ACP