Showing papers by "John L. Harwood published in 1980"

Desaturation of linoleic acid from exogenous lipids by isolated chloroplasts.

Abstract: When [14C]diacylgalactosylglycerol was added to isolated pea or lettuce chloroplasts linolenate synthesis was seen. The desaturation of [14C]linoleate in diacylgalactosylglycerol to [14C]linolenate was stimulated by the addition of a soluble protein fraction containing lipid-exchange activity. Other [14C]acyl lipids were ineffective, except that [14C]phosphatidylcholine in the presence of UDP-galactose and sn-glycerol 3-phosphate could also supply [14C]linoleate for desaturation. These results are consistent with a role of diacylgalactosylglycerol in linolenate synthesis, as indirectly suggested by labelling experiments.

Fatty acid elongation by a particulate fraction from germinating pea

Abstract: The synthesis of fatty acids from [14C]malonyl-CoA was studied with a high-speed particulate fraction from germinating pea (Pisum sativum). The variety used (Feltham First) produced mainly saturated fatty acids with palmitate (30--40%) and stearate (40--60%) predominating. Several palmitate-containing lipids stimulated overall synthesis and, in addition, increased the percentage of label in stearate. The production of stearate was severely inhibited by preincubation of the microsomal fraction with snake venom phospholipase A2 or by incubation with Rhizopus arrhizus lipase. Addition of a series of di-saturated phosphatidylcholines, with different acyl constituents, resulted in stimulation of overall fatty acid synthesis as well as an increase in the radiolabelling of the fatty acid two carbon atoms longer than the acyl chain added. This chain lengthening of fatty acids donated from phosphatidylcholine was due to the action of both fatty acid synthetase and palmitate elongase. The latter would utilize dipalmitoyl phosphatidylcholine and was sensitive to arsenite whereas fatty acid synthetase would use dilauroyl phosphatidylcholine and was sensitive to cerulenin. The results are discussed in relation to previous data obtained in vivo on plant fatty acid synthesis and current suggestions for the role of phosphatidylcholine in this process.

Inhibition of fatty acid biosynthesis by metronidazole

Abstract: A number of inhibitors have been used as probes in characterizing details of plant fatty acid synthesis. These include such molecules as arsenite, cerulenin, cyanide, fluoride and thiocarbamate herbicides (cf. Harwood, 1979). In order to increase our knowledge of reaction details, we began work with metronidazole (2-methylS-nitroimidazole1-ethanol), a drug widely used clinically for the treatment of diseases caused by anaerobic bacteria and protozoa (Gruneberg & Titsworth, 1973). This compound has been suggested to be a rather specific inhibitor of reactions using reduced ferredoxin and, accordingly, it was expected to inhibit desaturation of stearate to oleate (a reaction apparently requiring ferredoxin as the usual source of reduced equivalents; Jacobsen et al., 1974). Initially, metronidazole was tested in two plant systems in vivo-germinating pea (Pisum satiuum) and saillower (Carthamus tinctorius) seeds. When allowed to imbibe [\"Clacetate, these two kinds of seeds synthesized rather different patterns of fatty acids in the first 24 h of germination. Saillower synthesized 34% palmitate and 5 1% oleate of the total 14C-labelled fatty acids, whereas pea synthesized only saturated fatty acids (33% palmitate, 67% stearate). As expected, metronidazole strongly inhibited the formation of I1'Cloleate by saillower, but, surprisingly, also altered the pattern of I4Clabelled fatty acids made by pea. Thus, for example, the ratio of [14C]palmitate/[14C]stearate increased from 0.5 to 1.0 with 20 mM metronidazole in pea without the total incorporation of radioactivity into fatty acids being appreciably affected. A third system tested in vivo was the cucumber (Cucumis sativus) cotyledon. When incubated with ''C-labelled fatty acids, this tissue exhibits high rates of oleate and linoleate desaturation (Murphy et al., 1980). Metronidazole was found to inhibit linoleate, but not oleate, desaturation in this tissue. Because of the unexpected inhibition of palmitate elongation by metronidazole, the present study was expanded to include a number of systems in vitro. A direct test of metronidazole against the ferredoxin-requiring spinach chloroplast stearoyl(acyl carrier protein) (ACP) desaturase showed a high amount of inhibition (Table 1). This inhibition was also seen with the alternative electron donor, flavodoxin. When chloroplasts were prepared from cucumber cotyledons and incubated with [I4Clacetate, palmitate and oleate were essentially the only fatty acids synthesized. In the presence of metronidazole, oleate synthesis was inhibited but, instead of [14Clstearate building up, [14Clpalmitate was increased (Table 1). This indicated an inhibition of palmitate elongase such as had been observed in the germinating-pea system. The inhibition of palmitate elongase by metronidazole was further examined in the soluble fraction from germinating pea (Bolton & Harwood, 1977). The malonyl-CoA-dependent synthesis of stearate in this fraction was inhibited virtually completely by 2 mM-metronidazole (Table 1). At this concentration of inhibitor the total incorporation of radioactivity from [14Clmalonyl-CoA was still at 80% of the control value, indicating that fatty acid synthetase was unaffected. Since there was no evidence that ferredoxin was involved in palmitate elongation, it seemed possible that metronidazole was inhibiting the reaction by an oxidoreduction interaction with another component. Theoretically, the redox potential of NADH and NADPH should make them capable of reducing metronidazole, although a direct test could not show this (Chen & Blanchard, 1979). We tested a number of NADH or NADPH-requiring enzymes (e.g. lactate dehydrogenase, triose phosphate dehydrogenase, malate dehydrogenase, alcohol dehydrogenase) for metronidazole inhibition, but failed to record any noticeable effect. However, since the effect of metronidazole is not specific for ferredoxin, but will also occur with flavodoxin or the hydrogenase from Clostridium pasteurianum (Chen & Blanchard, 1979), we presume that the inhibition of palmitate elongase was due to the involvement of reduced equivalents of appropriate electronegativity in the reaction. Further studies are required to determine the nature of these reduced equivalents and the prevalence of metronidazole inhibition of different fatty acid elongations or desaturations.