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P K Stumpf

Publications and source records attributed to P K Stumpf.

At least 55 records · Page 3Linked to original sources
PubMed1981

Synthesis of Long-Chain Acyl-CoA in Chloroplast Envelope Membranes.

The chloroplast envelope is the site of a very active long-chain acylcoenzyme A (CoA) synthetase. Furthermore, we have recently shown that an acyl CoA thioesterase is also associated with envelope membrane (Joyard J, PK Stumpf 1980 Plant Physiol 65: 1039-1043). To clarify the interacting roles of both the acyl-CoA thioesterase and the acyl-CoA synthetase, the formation of acyl-CoA in envelope membranes was examined with different techniques which permitted the measurement of the actual rates of acyl-CoA formation. Using [(14)C]ATP or [(14)C]oleic acid as labeled substrates, it can be shown that the envelope acyl-CoA synthetase required both Mg(2+) and dithiothreitol. Triton X-100 slightly stimulated the activity. The specificity of the acyl-CoA synthetase was determined either with [(14)C]ATP or with [(3)H]CoA as substrates. The results obtained in both cases were similar, that is, as substrates, the unsaturated fatty acids were more effective than saturated fatty acids, the velocity of the reaction increased from lauric acid to palmitic acid, and the maximum velocity was obtained with unsaturated C(18) fatty acids.The results obtained suggest that the acyl-CoA thioesterase associated with envelope membranes could be an ultimate control to prevent the transport (outside of the chloroplast) or the insertion (into chloroplast lipids) of fatty acids with chains shorter than C(16).

Journal Article↗
PubMed1981

Recent studies of the enzymic synthesis of ricinoleic Acid by developing castor beans.

Oleate Delta(12)-hydroxylase activity was measured in extracts of developing castor bean seeds. Most of the hydroxylase activity is associated with microsomes. However, when microsomes are washed, the activity is completely lost. Some (50%) of the activity can be restored by addition of the 100,000g supernatant to the washed microsomes. Supernatant extracts (100,000g) of developing safflower seeds are able to restore all (100%) of the hydroxylase activity to the washed castor bean microsomes. In addition, purified mammalian catalase can restore some (25%) of the activity to the microsomes but is not as effective as either castor bean or safflower 100,000g supernatants. The K(m) of the hydroxylase for oxygen is 4 micromolar. Inasmuch as the activity was not inhibited by high concentrations of either carbon monoxide or cyanide, neither the involvement of cytochrome P450 nor other cytochrome systems is suggested. The enzyme system was not saturated by oleoyl-CoA, even at concentrations as high as 200 micromolar. When [(14)C]oleoyl-CoA is supplied as a substrate, the acyl component is rapidly transferred to phosphatidylcholine (PC). Hydroxylation may occur on PC or on a lipid which receives its acyl component from PC. However, exogeneously added 2-[1-(14)C]oleoyl-PC was hydroxylated at a much lower rate than was [1-(14)C]oleoyl-CoA added as the primary substrate.

Journal Article↗
PubMed1980

Desaturation of Oleic and Linoleic Acids by Leaves of Dark- and Light-grown Maize Seedlings.

Oleate and linoleate desaturation in leaves of maize seedlings was largely independent of previous light treatment of the seedlings; there was no evidence of light-induced desaturase activities. These results are in sharp contrast to those observed with developing cucumber cotyledons in which pronounced increase in desaturation occurs after exposure of tissue to light. The rates of desaturation of oleate were about four times those of linoleate in both etiolated and 16-hour greened maize leaves. In both etiolated and greened tissues, about two-thirds of the label from oleate was esterified after 4 hours, half of which was in phosphatidylcholine. Phosphatidylcholine and diglyceride contained large proportions of [(14)C]linoleate formed from [(14)C]oleate but not [(14)C]linolenate. In monogalactolipid, about two-thirds of the labeled fatty acids were linolenate. In vivo desaturase activity was present in tissue of widely different levels of differentiation and chlorophyll content obtained from light-grown maize seedlings.

Journal Article↗
PubMed1980

Characterization of an acyl-coenzyme a thioesterase associated with the envelope of spinach chloroplasts.

The enzymic hydrolysis of acyl-coenzyme A occurs in intact and purified chloroplasts. The different components of spinach chloroplasts were separated after a slight osmotic shock and the purified envelope membranes were shown to be the site of very active acyl-CoA thioesterase activity (EC 3.1.2.2.). The enzyme, which had a pH optimum of 9.0, was not affected by sulfhydryl reagents or by serine esterase inhibitors. However, the acyl-CoA thioesterase was strongly inhibited by unsaturated fatty acids, especially oleic acid, at concentrations above 100 micromolar. In marked contrast, saturated fatty acids had only a slight effect on the thioesterase activity. Substrate specificities showed that the velocity of the reaction increased with the chain length of the substrate from decanoyl-CoA to myristoyl-CoA and then decreased with the chain length from myristoyl-CoA to stearoyl-CoA. Interestingly, oleoyl-CoA was only slowly hydrolyzed. These results suggest that the envelope acyl-CoA thioesterase coupled with an envelope acyl-CoA synthetase may be involved in a switching system which indirectly allows acyl transfer from acyl carrier protein derivatives to unsaturated acyl-CoA derivatives and ensures the predominance of unsaturated 18 carbon fatty acids in plants. Furthermore, the position of both acyl-CoA thioesterase and synthetase in the envelope membranes suggest that these two enzymes may be involved in the transport of oleic acid from the stroma phase to the cytosol compartment of the leaf cell.

Journal Article↗
PubMed1980

Long Chain (C(20) and C(22)) Fatty Acid Biosynthesis in Developing Seeds of Tropaeolum majus: AN IN VIVO STUDY.

The storage triacylglycerols of nasturtium (Tropaeolum majus) seeds are composed principally of cis-11-eicosenoate and cis-13-docosenoate. To investigate the biosynthesis of these C(20) and C(22) fatty acids, developing seed tissue was incubated with various (14)C-labeled precursors. Incubation with [1-(14)C]acetate produced primarily cis-11-[1-(14)C]eicosenoate and cis-13-[1,3-(14)C]docosenoate in the triacylglycerol fraction, the odd-carbon [U-(14)C]oleate also formed from [(14)C] acetate was in the polar lipid fraction. Kinetic data showed that this oleate was not channeled into cis-11-eicosenoate nor cis-13-docosenoate over a 24-hour period. Under suitable conditions, nasturtium seed could also produce [(14)C]stearate, [(14)C]eicosenoate, and [(14)C]docosenoate from [1-(14)C]acetate. The results are discussed in terms of the number of pathways producing fatty acids. From pool size and other considerations, the results can be rationalized only in terms of different de novo systems for oleate biosythesis, one supplying oleate for incorporation into phospholipids and the other supplying oleate for chain elongation and subsequent esterification into triacylglycerols. Because of the probable heterogeneous nature of the seed tissue, it is not known if these two systems are operating in different cell types, in the same cell type at different stages of development, or in the same cell type concurrently.

Journal Article↗
PubMed1980

Biosynthesis of C(20) and C(22) Fatty Acids by Developing Seeds of Limnanthes alba: CHAIN ELONGATION AND Delta5 DESATURATION.

The storage triacylglycerols of meadowfoam (Limnanthes alba) seeds are composed essentially of C(20) and C(22) fatty acids, which contain an unusual Delta5 double bond. When [1-(14)C]acetate was incubated with developing seed slices, (14)C-labeled fatty acids were synthesized with a distribution similar to the endogenous fatty acid profile. The major labeled product was cis-5-eicosenoate, with smaller amounts of palmitate, stearate, oleate, cis-5-octadecenoate, eicosanoate, cis-11-eicosenoate, docosanoate, cis-5-docosenoate, cis-13-docosenoate, and cis-5,cis-13-docosadienoate. The label from [(14)C]acetate and [(14)C]malonate was used preferentially for the elongation of endogenous oleate to produce cis-[(14)C]11-eicosenoate, cis-13-[(14)C]docosenoate, and cis-5,cis-13-[(14)C]docosadienoate and for the elongation of endogenous palmitate to produce the remaining C(20) and C(22) acyl species. The Delta5 desaturation of the preformed acyl chain and chain elongation of oleate and palmitate were demonstrated in vivo by incubation of the appropriate 1-(14)C-labeled free fatty acids. Using [1-(14)C]acyl-CoA thioesters as substrates, these enzyme activities were also demonstrated in vitro with a cell-free homogenate.

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PubMed1980

Polyunsaturated Fatty Acid Biosynthesis in Cotyledons from Germinating and Developing Cucumis sativus L. Seedlings.

Etiolated Cucumis sativus L. cotyledons preferentially catabolized exogenous [1-(14)C]oleic acid and [1-(14)C]linoleic acid with relatively little incorporation into complex lipids or desaturation of the (14)C-labeled fatty acids. Following a 16-hour exposure to light, the greening cotyledons efficiently desaturated the exogenous (14)C-labeled fatty acids. A small amount of oleate desaturation to linoleate was observed in etiolated tissue, but hardly any linoleate desaturation to alpha-linolenate was detected. Both oleate and linoleate desaturation showed diurnal variations with maxima at the end of light periods and minima at the end of dark periods. Illumination of etiolated tissue by flashing light, as opposed to continuous light, failed to stimulate either chlorophyll or alpha-linolenic acid biosynthesis, and both processes could be halted or reversed by 10 micrograms per milliliter cycloheximide. Production of polyunsaturated fatty acids from [1-(14)C]acetate, [1-(14)C]oleic acid, and [1-(14)C]linoleic acid, by greening cucumber cotyledons, was markedly affected by tissue integrity with finely chopped cotyledons having very little capacity for their synthesis and intact seedlings showing the highest rates.

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PubMed1980

In Vivo Pathway of Oleate and Linoleate Desaturation in Developing Cotyledons of Cucumis sativus L. Seedlings.

Exogenous [1-(14)C]oleic acid and [1-(14)C]linoleic acid were taken up and esterified to complex lipids by greening cucumber (Cucumis sativus L.) cotyledons. Both (14)C-labeled fatty acids were initially esterified to phosphatidylcholine prior to eventual accumulation in triacylglycerols and galactolipids. Kinetic data suggest that esterification occurs prior to desaturation and that phosphatidylcholine is the initial site of both [(14)C]-oleate and [1-(14)C]linoleate esterification and of [1-(14)C]oleate desaturation to [1-(14)C]linoleate. [1-(14)C]Linoleic acid was esterified more rapidly than [(14)C]oleic acid and its desaturation product, [1-(14)C]alpha-linolenate, occurred mainly on monogalactosyl diacylglycerol, although some was also observed on the other major acyl lipids, including phosphatidylcholine.

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PubMed1979

Subcellular localization of acyl carrier protein in leaf protoplasts of Spinacia oleracea.

This communication demonstrates that all de novo fatty acid biosynthesis in spinach leaf cells requires acyl carrier protein (ACP) and occurs specifically in the chloroplasts. Antibodies raised to purified spinach ACP inhibited at least 98% of malonyl CoA-dependent fatty acid synthesis by spinach leaf homogenates. Therefore, the presence of ACP in a compartment of the spinach leaf cell would serve as a marker for de novo fatty acid biosynthesis. A radioimmunoassay capable of detecting 10(15) mol (10(-11) g) of spinach ACP was developed to measure the levels of ACP in leaf cell components isolated by sucrose gradient centrifugation of a gentle lysate of spinach leaf protoplasts. All of the ACP of the leaf cell could be attributed to the chloroplast. Less than 1% of the ACP associated with chloroplasts resulted from binding of free ACP to chloroplasts. Of interest, ACP from Escherichia coli, soybean, and sunflower showed only partial crossreactivity with spinach ACP by the radioimmunoassay. These results strongly suggest that, in the leaf cell, chloroplasts are the sole site for the de novo synthesis of C16 and C18 fatty acids. These fatty acids are then transported into the cytoplasm for further modification and are either inserted into extrachloroplastic membrane lipids or returned to the chloroplast for insertion into lamellar membrane lipids.

Acyl Carrier Protein↗
PubMed1979

Light-dependent Induction of Polyunsaturated Fatty Acid Biosynthesis in Greening Cucumber Cotyledons.

Greening cucumber (Cucumis sativus L.) cotyledons exhibited dramatic increases in the ability to desaturate exogenously added [1-(14)C]oleic acid and [1-(14)C]linoleic acid within 2 to 3 hours of illumination. These increases were effectively inhibited by 10 micrograms per milliliter cycloheximide. Oleate desaturation remained at a high level in constant light for 5 to 6 days after induction and then declined by about 50%; when returned to the dark, the tissue showed a sharp decrease in conversion of [(14)C]oleate to [(14)C]linoleate. Linoleate desaturation reached a maximum about 15 hours after induction and declined immediately thereafter while the tissue still was in the light; after induction had peaked return of the tissue to the dark showed a dramatic fall of linoleate desaturation. The changes in desaturation were correlated with the conversion of the principal fatty acid in the etiolated cotyledons, linoleate, to alpha-linolenate, and with the assembly of the chlorophyll-containing photosynthetic membranes. The incorporation of [1-(14)C]acetate into lipids showed no significant light stimulation. The role of light in the regulation of certain aspects of plant metabolism during development is discussed.

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PubMed1979

Elongation Pathway for alpha-Linolenic Acid Synthesis in Spinach Leaves: A Reexamination.

Leaf slices from spinach exhibited considerable variation in their incorporation of [(14)C]bicarbonate and [1-(14)C]acetate into fatty acids. Reductive ozonolysis studies indicated that all the (14)C-labeled fatty acids were synthesized de novo; no in vivo evidence was found for the previously proposed elongation of hexadecatrienoate to alpha-linolenate.

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