John B. Ohlrogge

The primary structure of spinach acyl carrier protein

Acyl carrier protein (ACP) from spinach leaves has been purified to homogeneity by high-performance liquid chromatography with an anion-exchange column. The amino acid sequence of one major ACP in spinach leaves, ACP-I, has been determined by automated Edman degradation. It consists of the following 82 amino acids: (sequence in text). Sequencing of the intact polypeptide provided data for the first 57 residues. Cleavage of the succinylated ACP with CNBr at Met-46, followed by sequencing of the fragment mixture, provided data for the final 36 residues. The C-terminal alanine was confirmed by carboxypeptidase Y digestion. The spinach ACP has 40, 70, and 25% homology with Escherichia coli, barley, and rabbit ACPs, respectively. The results not only provide the first complete sequence of a plant ACP, but also provide insight into the structural and evolutionary relationships among plant, animal, and bacterial ACPs.

Partial purification and characterization of two forms of malonyl-coenzyme A:Acyl carrier protein transacylase from soybean leaf tissue

Investigation of malonyl-CoA:acyl carrier protein transacylase from soybeans has shown that this fatty acid biosynthetic enzyme occurs in at least two isozymic forms. Both forms exist as soluble, low-molecular-mass polypeptides (approx 43 kDa) which catalyze one of the first committed steps in the synthesis of C16 and C18 fatty acids. We have partially purified the two forms of this enzyme from soybean leaf tissue 1200- and 3900-fold respectively. Isozyme 1 does not adhere to ion-exchange or blue dye affinity chromatographic supports and elutes from a polybuffer exchanger column at a pH of 7.3. Isozyme 2 requires salt to be eluted from ion-exchange and affinity matrices and elutes from a polybuffer exchanger column at a pH of 5.3. The two forms of malonyl-CoA:acyl carrier protein transacylase also differ in their sensitivity to catalytic inhibitors, heat treatment, and inhibition by acyl-CoA ester substrates. Both forms utilize malonyl-CoA as the preferred substrate, and polyacrylamide gel electrophoresis of reaction products indicated that malonyl-acyl carrier protein was the major product formed. Analysis of developing soybean seeds indicates that only one form (isozyme 1) is predominant, whereas leaf tissue possesses both isozymes.

Acylation of plant acyl carrier proteins by acyl-acyl carrier protein synthetase from Escherichia coli☆

The acyl-acyl carrier protein synthetase from Escherichia coli has been examined for its ability to specifically acylate acyl carrier protein (ACP) from higher plants in order to develop an assay for plant ACP, and to prepare labeled acyl-ACP of plant origin. It was found that the E. coli enzyme was able to acylate ACP from spinach, soybean, avocado, corn, and several other plants. The acylation was very specific because, in crude extracts of spinach leaves where ACP represented approximately 0.1% of the total soluble protein, ACP was shown to be the only protein acylated. In contrast to other E. coli enzymes that display 2- to 10-fold lower rates with plant versus bacterial ACP, the kinetic constants (Km and Vmax) for acyl-ACP synthetase were found to be essentially identical for spinach and E. coli ACP when acylated with palmitic acid. Palmitic, myristic, lauric, stearic, and oleic acid could all be esterified to both spinach and E. coli ACP with similar specificity. Procedures are described that allow the assay of ACP in plant extracts at the nanogram level.

A novel, general radioimmunoassay for acyl carrier proteins

A radioimmunoassay (RIA) for acyl carrier proteins (ACP) is described that is based on the competitive binding between [3H]acyl-ACP and unlabeled ACP of the same species. The radiolabeled antigen, [3H]palmitoyl-ACP, is enzymatically synthesized by Escherichia coli acyl-ACP synthetase. Because acyl-ACP synthetase can specifically radiolabel ACP in crude extracts from several plant sources, the use of this enzyme to prepare [3H]acyl-ACP obviates the need for pure preparations of each ACP. Preparation of [3H]acyl-ACP with a specific activity of 15 Ci/mmol allows RIA detection of total ACP in crude plant extracts at the nanogram level. Because antibodies against spinach ACP partially crossreact with ACP from many plant sources, RIAs for other plant species can be constructed using only one preparation of antibody. ACP preparations from safflower, soybean, avocado, corn, and E. coli show a decreasing order of partial immunocross-reactivity with spinach ACP-specific antiserum, as examined by RIA using spinach [3H]palmitoyl-ACP.

Toxicity of activated oxygen: Lack of dependence on membrane unsaturated fatty acid composition

Membrane unsaturated fatty acid oxidation has been suggested as a mechanism of toxicity for a variety of activated oxygen species. We have tested this hypothesis by manipulating the fatty acid composition of an Escherichia coli mutant that is unable to synthesize unsaturated fatty acids. To provide a wide range of susceptibility to membrane oxidation we have replaced the naturally occurring monoenoic acyl chains with cyclopropanes to greatly reduce the unsaturation level and with linoleate to increase the membrane unsaturation. These cultures were treated with ozone, hydrogen peroxide, singlet oxygen and paraquat. In no case was there substantial protection from toxicity afforded by cyclopropanes nor was there enhancement of toxicity to cells with the polyunsaturated membranes. We suggest, therefore, that oxidation of membrane unsaturated fatty acids is not an essential component of the toxicity to E. coli of active oxygen species.

A Possible Differential Role for Plant Acyl Carrier Protein Isoforms in Higher Plants

Acyl carrier protein (ACP) dependent fatty acid synthesis (FAS) in plant leaf tissues is exclusively localized in the chloroplast (1). Two forms of ACP have been purified from spinach and barley leaf, but, only one ACP isoform predominates in spinach seed tissue (2, 3). Oleic acid is the major product of fatty acid synthesis by isolated chloroplasts (4). The regulation of plant lipid metabolism is believed to involve the export of oleic acid from its site of synthesis (plastid) to sites of complex lipid metabolism (eg. ER). This pathway requires the initial release of oleic acid from ACP by oleoyl-ACP thioesterase (5). In addition, the oleoyl moiety may also enter glycerolipid synthesis within the plastid through the action of acyl-ACP acyl-transferase (6). The preferred substrate for both the thioesterase and glycerol-3-phosphate acyl transferase reactions is 18: 1-ACP (6, 7).

A Preliminary Characterization of Plant Holo-Acyl Carrier Protein Synthase

Acyl Carrier Protein (ACP) is a central cofactor in the biosynthesis of fatty acids and complex acyl lipids (1). ACP and CoA have a common prosthetic group, 4′-phosphopantetheine, to which fatty acids and intermediates are attached as thioesters. Using an E. coli auxotroph, Alberts and Vagelos (2) interpreted their in vivo experimental findings as suggesting that during de novo synthesis of ACP the prosthetic group is donated by CoA.