Derek Herbert

Oxygen induction of a novel fatty acid n-6 desaturase in the soil protozoon, Acanthamoeba castellanii.

Induction of fatty acid desaturation is very important for the temperature adaptation of poikilotherms. However, in oxygen-limited late-exponential-phase Acanthamoeba castellanii cultures, oxygen alone was able to induce increased activity of a fatty acid desaturase that converts oleate into linoleate and which has been implicated in the temperature adaptation of this organism. Experiments with Delta(10)-nonadecenoate showed that the enzyme is an n -6 desaturase rather than a Delta(12)-desaturase. It also used preferentially 1-acyl-2-oleoyl-phosphatidylcholine as substrate and NAD(P)H as electron donor. The involvement of cytochrome b (5) as an intermediate electron carrier was shown by difference spectra measurements and anti-(cytochrome b (5)) antibody experiments. Of the three protein components of the desaturase complex, oxygen only increased the activity of the terminal (cyanide-sensitive) protein during n -6 desaturase induction. The induction of this terminal protein paralleled well the increase in overall oleate n -6 desaturation. The ability of oxygen to induce oleate desaturase independently of temperature in this lower eukaryotic animal model is of novel intrinsic interest, as well as being important for the design of future experiments to determine the molecular mechanism of temperature adaptation in poikilotherms.

Use of plant cell cultures to study graminicide effects on lipid metabolism

Graminicides belonging to the cyclohexanedione and aryloxyphenoxypropionate classes are well established to act by disrupting acyl lipid biosynthesis via specific inhibition of acetyl-CoA carboxylase. Species of grass inherently resistant to such herbicides, or biotypes of grassy weed species which display acquired resistance to recommended rates of graminicide application, are known to possess an altered plastidic multifunctional acetyl-CoA carboxylase showing reduced sensitivity to these herbicides in vitro. Studies reported here demonstrate that cell suspension cultures of maize, a graminicide-sensitive species and Poa annua, a graminicide-insensitive species, display a similar differential sensitivity of acyl lipid biosynthesis as tissue from corresponding intact plants. Acyl lipid biosynthesis in P. annua can be inhibited if sufficiently high concentrations of graminicide are used. The major plastidic form and the minor cytosolic forms of acetyl-CoA carboxylase were successfully purified from maize cell suspensions, were compared to those from leaf tissue and were shown to be differentially inhibited by graminicides in a similar manner to their counterparts from leaf tissue. These studies demonstrate that cell suspensions are useful for studying the mode of action of graminicides, especially in view of the limited amount of material obtainable from many grassy species which are very fine-growing.

Graminicide-binding by acetyl-CoA carboxylase from Poa annua leaves

Abstract A partially purified acetyl-CoA carboxylase (ACCase) preparation from leaves of a graminicide-resistant grass, Poa annua (annual meadow grass), was weakly inhibited by an aryloxyphenoxypropionate graminicide, quizalofop. The I 50 value of 10 μM was ca 300-fold higher than that previously determined for an ACCase from a susceptible grass species, maize. Poa annua ACCase exhibited negative cooperativity in binding quizalofop (Hill coefficient = 0.54±0.02). Inhibition was approximately equal in sensitivity to the concentrations of acetyl-CoA or ATP in the assay medium and appeared close to being non-competitive for these substrates. A double-reciprocal plot of initial velocity with the substrate pair acetyl-CoA plus ATP produced a parallel line pattern which was the expected pattern for a Ping-Pong reaction mechanism. The results from these kinetic measurements are discussed in relation to possible reasons for the insensitivity of P. annua ACCase to graminicides. It is concluded that cooperativity of herbicide-binding may be a possible indicator of such insensitivity.

Enzyme Studies on Isoforms of Acetyl-CoA Carboxylase

Acetyl-CoA carboxylase is recognised generally as being a key enzyme in acyl lipid formation. In plants it has been shown to be important as a regulatory enzyme in light-driven lipid synthesis [1] and has a high flux control coefficient under such conditions [2]. Recently, characterisation of different isoforms of acetyl-CoA carboxylase from plants has been made. In Poaceae such as maize there are two multifunctional proteins [3]. By contrast, the dicotyledon pea contains a multienzyme complex form of acetyl-CoA carboxylase in the mesophyll chloroplasts but a multifunctional protein isoform in the cytosol of epidermal cells [4].