Download inhibition of very long chain fatty acid synthesis in barley and wild

Biochemical Society Transactions (1999) 27
119
A C Y G C O A: G L Y C E R O G f P H O S P H A T E
ACYLTRANSFERASE F R O M OIL PALM (Elaeis
guineensis) TISSUES
121
A1 23
INHIBITION OF VERY LONG CHAEV FATTY ACID
SYNTHESIS IN BARLEY AND WILD OATS BY
THIOCARBAMATE HERBICIDES.
Arif M. Manaf and John L. Harwood
Adam Baldwin, Hilary Rogers, Dennis Francis & John Harwood
Cardiff School of Biosciences, Cardiff University, PO Box 911,
Cardiff CFl 3US, UK.
Cardiff School of Biosciences, Cardiff University, PO Box 91 I ,
Cardiff CFI 3US, U.K.
Oil palm (Elueis pineensis) membrane-bound acyl-CoA :
glycerol-3-phosphate acyltransferases (GPAT, EC 2.3.15), which
are localised in the endoplasmic reticulum, were studied using
high-speed particulate fractions from tissue cultures or mesocarp
acetone powders. The fractions were assayed with [‘4C]glycerol
3-phosphate and acyl-CoAs and incorporation of radioactivity
was optimised. The optimal conditions were similar between the
two preparations. Experiments with different acyl-CoA substrates
showed that the GPAT in both preparations had a significant
preference for palmitate, in keeping with the high percentage of
this fatty acid in palm oil. GPAT was solubilised from both
preparations with optimal solubilisation being achieved at 0.5%
(w/v) CHAPS concentrations. Solubilised GPATs were purified
about 70-fold using DE52 ion-exchange chromatography and
Sephadex G-l 00 molecular exclusion chromatography.
Comparison of SDS-PAGE gel patterns for the purified GPATs
suggested that the enzymes have molecular masses in the range of
56-67 m a . In comparison, the chloroplast GPATs, which use
acyl-ACPs, have molecular masses of 42-43 kDa (pea, spinach)
or of 30-40 kDa (squash).
Thiocarbamates are long-established herbicides known to
interfere with surface wax formation. We have shown that they
have a major effect on the synthesis of very long chain fatty acidswhich are precursors for surface waxes. However, the inhibitory
characteristics are such that a metabolite, probably the
sulphoxide, rather than the parent compound is the active reagent.
In order to probe the molecular mechanism of inhibition, as well
as the reason for safener action we have carried out further
experiments with pebulate and its sulphoxide.
We have used Horderim vrrlgure (barley) and the
pernicious weed, Avena hrdoviciur?u(wild oat). Pebulate and its
sulphoxide were both effective at inhibiting, selectively, fatty acid
elongation in these plants, consistent with data obtained
previously with peas and wheat. However, the sulphoxide was
more effective especially when irr vi/ro assays were made of
arachidonyl-CoA or behenoyl-CoA elongation. These plants
were also sensitive to pebulate or its sulphoxide in vivo.
One potential safener, dichlormid, known to induce
glutathione S-transferases (GST’s) has been utilised in our
experiments, to enhance the target plants’ ability for the
detoxification of the sulphoxide. An increased synthesis of very
long chain fatty acids occured also in response to treatment with
dichlormid and various other safeners.
122
The analysis by electrospray ionisation mass spectrometry
of phosphatidylcholine synthesis from [I3C] choline by
cultured cells.
Anthony D Postle, Sarah M Wright and Alan N Hunt
Child Health, University of Southampton, SO16 6YD
120
Substrate specificity of rat heart Phosphatidate Phosphohydrolase
Reghu Venkatesan and David Saggerson
Department of Biochemisty
University College London
Gower Street
London WC 1E 6BT
Type 2 PAPS are magnesium-dependent enzymes which hydrolyze
a variety of lipid substrates, namely phosphatidate(PA),
lysophatidate,
ceramide-I-phosphate
and
sphingosine-lphosphate, in a variety of tissues. We have shown that a variety of
PA substrates of defined acyl-chain composition have different
magnesium dependencies. The results indicate that rat heart
membrane PAP2 may show a preference for shodmedium
saturated (e.g C12:O) or longer unsaturated (e.g C18:l) acyl chain
lengths.
Measurement of the rate of phosphatidylcholine (PC) synthesis has
typically involved the analysis by thin layer chromatography of the
incorporation of a radiolabelled substrate. More detailed analysis of
the synthetic rates of individual molecular species of PC has required
extensive use of laborious HPLC procedures. We describe a novel
approach to quantify rates of synthesis of PC molecular species, using
electrospray ionisation mass spectroscopy (ESI-MS). Cultured cells,
both IMR-32 neuroblastoma and U937 promyeloid leukaemia cells,
were transferred into choline-deficient medium and incubated with
[I,2-”CC]-choline (98 mole %) for up to 24 hours. Cells were
harvested at specified times and their total lipids extracted with
chlorofordmethanol. Using BondElut NH2 cartridges, PC was
isolated from the organic phase and water-soluble metabolites from
the aqueous phase. PC compositions were determined by tandem
MS/MS in a Quattro 11 triple quadrapole ESI-MS using a nanoflow
interface. Selective fragmentation of sodiated PC species generated a
diagnostic ion at m/z=147, which comprised a five membered cyclic
alkane phosphate. As this fragment contained carbons I and 2 from
the choline moiety, PC species synthesised from [ I ,2-’3C]-choline
generated a corresponding fragmentation ion at m/z=149.
Consequently, parent scan analysis of m/z=147 and m/z=149
distinguished clearly between endogenous and newly synthesised PC
species.
Correction for the ”C enrichment in substrate
phosphorylcholine enabled absolute rates of synthesis of individual
molecular species of PC to be calculated. This methodology is rapid,
sensitive, and has considerable potential for studies in vivo as well as
in cultured cells.