Inhibition of Glycolipid Biosynthesis in Chloroplasts by Ozone and Sulfhydryl Reagents

Abstract

The metabolism of uridine 5'-pyrophosphate-galactose by spinach (Spinacia oleracea) chloroplast preparations was inhibited by ozone. The formation of digalactosyl diglyceride and trigalactosyl diglyceride was inhibited much more than the formation of monogalactosyl diglyceride, steryl glycoside, and acylated steryl glycoside. Essentially identical results were obtained when glycolipid synthesis was inhibited by N-ethyl maleimide, p-hydroxymercuribenzoate, and CdCL. lodoacetate and iodoacetamide affected neither the total incorporation of sugar from uridine 5'-pyrophosphate-galactose nor distribution of the incorporated sugar in the various glycolipids.Ozone reacted with model membrane systems prepared with egg lecithin. In the absence of reduced glutathione, products included malonaldehyde and hydrogen peroxide. In the presence of glutathione, malonaldehyde was still produced, but the glutathione was oxidized and no peroxide was detected. When these studies were extended to chloroplast preparations, it was also found that malonaldehyde was produced and glutathione was oxidized.It was concluded that ozone inhibits glycolipid biosynthesis in chloroplast preparations by way of oxidation of enzyme sulfhydryl groups but that this reaction may be a secondary effect of oxidation of unsaturated fatty acids.The synthesis of galactosyl diglycerides by cell-free preparations from leaves of higher plants was first reported by Neufeld and Hall (18). Their results have been substantiated and extended by subsequent work. This work has shown that in addition to the predominant naturally occurring monogalactosyl diglyceride and digalactosyl diglyceride, there is at least one additional compound, trigalactosyl diglyceride, which becomes labeled during the incubation of chloroplast preparations with 4C-UDP galactose (11,17,20).The ratio of monogalactosyl diglyceride, digalactosyl diglyceride, and trigalactosyl diglyceride, synthesized by cell-free preparations, can be changed by variations in pH and temperature (17 MATERIALS AND METHODS Chloroplasts. Chloroplasts were prepared from spinach (Spinacia oleracea) purchased at local markets. The leaves were washed in distilled water and the petioles and midribs were removed. The leaves (75 g) were ground in a Waring Blendor (70 v, 5 sec) with an equal weight of ice-cold homogenizing medium (0.5 M sucrose, 10 mm phosphate, pH 7.5). The homogenate was filtered through cheesecloth and the filtrate was centrifuged at 200g for 2 min. The pellet was discarded and the supernatant was centrifuged at 1 OOg for 7 min. The supernatant was discarded and the pellet was resuspended in 0.1 M phosphate, pH 7.5. Chlorophyll concentration was determined by the method of Arnon (2), and the chloroplast suspension was diluted so that addition to the reaction mixtures would give 1 mg of chlorophyll per vessel.Reaction Mixtures. Reaction mixtures for the study of glycolipid biosynthesis consisted of 100 ,umoles of tris-Cl, pH 7.5, or phosphate, pH 7.5; 0.4 ml of the broken chloroplast suspension con...

Effects of Ozone and Peroxyacetyl Nitrate on Polar Lipids and Fatty Acids in Leaves of Morning Glory and Kidney Bean

“…Peters and Mudd (21) In kidney bean plants exposed to ozone, the decreases in phospholipid and MGDG content may be due to either the inhibition of lipid synthesis, the increase of hydrolysis, or a combination of these. It is well known that ozone inhibits lipid biosynthesis in spinach chloroplast suspensions or mitochondrial or microsomal fractions from rat lung by way of oxidation of sulfhydryl enzymes (12,13,21). Moreover, Mudd et al (13) reported that glycolipid biosynthesis was inhibited in chloroplasts treated with ozone and the formation of DGDG was more strongly inhibited than the formation of MGDG.…”

“…It is well known that ozone inhibits lipid biosynthesis in spinach chloroplast suspensions or mitochondrial or microsomal fractions from rat lung by way of oxidation of sulfhydryl enzymes (12,13,21). Moreover, Mudd et al (13) reported that glycolipid biosynthesis was inhibited in chloroplasts treated with ozone and the formation of DGDG was more strongly inhibited than the formation of MGDG. However, the present study showed that MGDG was more sensitive to ozone than was DGDG.…”

“…Ozone not only destroys the double bond in unsaturated fatty acids, but also affects lipid biosynthesis (12,13). Lipid synthesis from acetate and acetyl-CoA (12) and the incorporation of galactose from UDP-galactose into galactolipids (13) were inhibited by bubbling ozone through a chloroplast suspension. PAN in vitro also inhibited the synthesis of fatty acids from acetate (11).…”

Exposure of spring wheat (Triticum aestivum) to ozone in open‐top chambers. Effects on acyl lipid composition and chlorophyll content of flag leaves

“…The ducmg malondialdehyde (Heath, 1978). Malondimechanisms underlying the effects of ozone on crop aldehyde was also formed when ozone reacted with yield are not fully understood, although ozone-phosphatidylcholine (PC) in model membrane induced reductions in net photosynthesis have been systems, again demonstrating an ozone effect on observed in many plant species, including wheat unsaturated fatty acids (Mudd et al, 1971). Ozone has also been reported to affect membrane lipids in situ.…”

Free Fatty Acids Regulate Two Galactosyltransferases in Chloroplast Envelope Membranes Isolated from Spinach Leaves

“…7), Mudd et al ( 18) reported that incorporation of UDP-Gal into oligogalactolipids, GGGT activity, was inhibited by ozone bubbling into the suspension of spinach chloroplasts and suggested that hydrogen peroxide produced during the oxidation of fatty acids by ozone was responsible for GGGT inhibition. Thus, the lack of an effect of ozone on GGGT activity in our experiments (Fig.…”