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Biomedical subjects

P K Stumpf

Publications and source records attributed to P K Stumpf.

At least 37 records · Page 2Linked to original sources
PubMed1983

Regulation of Plant Acetyl-CoA Carboxylase by Adenylate Nucleotides.

The assay of acetyl-CoA carboxylase (EC 6.4.1.2) does not follow ideal zero-order kinetics when assayed in a crude extract from wheat (Triticum aestivum L.) germ. Our results show that the lack of ideality is the consequence of contamination by ATPase and adenylate kinase. These enzyme activities generate significant amounts of ADP and AMP in the assay mixture, thus limiting the availability of ATP for the carboxylase reaction. Moreover, ADP and AMP are competitive inhibitors, with respect to ATP, of acetyl-CoA carboxylase. Similar relationships between adenylate nucleotides and acetyl-CoA carboxylase are found in isolated chloroplasts. There is no evidence that acetyl-CoA carboxylase activity in the extracts of the plant systems examined is altered by covalent modification, such as a phosphorylation-dephosphorylation cycle. A scheme is presented that illustrates the dependency of acetyl-CoA carboxylase and fatty acid synthesis on the energy demands of the chloroplasts in vivo.

Journal Article↗
PubMed1983

Reduction of N-acetyl methionine sulfoxide in plants.

An enzymic activity which catalyzes the reduction of N-acetyl-methionine sulfoxide to l-N-acetyl-methionine has been observed in a wide variety of plant tissues. Its activity depended on the presence of dithiotreithol in the incubation medium. l-Methionine-sulfoxide was essentially inactive as a substrate. Of all the physiological reductants tested, only thioredoxin partially replaced dithiothreithol. When fractions obtained by gradient centrifugation of gently disrupted barley protoplasts were assayed for the reductase, the activity was largely associated with chloroplasts although approximately 15% was found in the cytosolic compartment. The enzyme, isolated from spinach chloroplasts, had a broad pH optima between 7.0 and 8.0, and its K(m) for N-acetyl methionine sulfoxide is 0.4 millimolar. The possible participation of this ubiquitous enzyme in enzyme regulation is discussed.

Journal Article↗
PubMed1983

Some Enzymic Activities in the Germinating Oil Palm (Elaeis guineensis) Seedling.

In germinating oil palm (Elaeis guineensis var D x P) seedling, an active lipase was present in the shoot but absent from both the kernel and the haustorium. It has an optimum pH of 6.2 and a smaller peak at pH 8.6. The shoot lipase was active against a number of mono-, di-, and triacylglycerols as well as the endogenous lipids present in the shoot, haustorium, and kernel. Activity against related substrates were in the order: trilaurin > dilaurin > monolaurin but monopalmitin > dipalmitin > tripalmitin. The level of the enzyme in the seedling was highest at a relatively early stage of growth (18-21 days) and also higher in dark-grown seedlings. Glyoxylate bypass enzymes (malate synthetase and isocitrate lyase), glutamate-oxaloacetate transaminase, phosphoenolpyruvate carboxykinase and lauroyl-coenzyme A oxidase were located in the haustorium. The levels of the enzymes paralleled seedling development and were slightly higher in light-grown seedlings. Fatty acyl-coenzyme A synthetase activity was very low and was found in both the shoot and haustorium.

Journal Article↗
PubMed1983

The Metabolism of the Germinating Oil Palm (Elaeis guineensis) Seedling : In Vivo Studies.

The metabolism of (14)C-labeled fatty acids and triacylglycerols was followed in intact germinating oil palm seedlings as well as in tissue slices. In the germinating seedling, the shoot contained a normal pattern of membrane fatty acids (mainly C(16), C(18:1), C(18:2)) but the kernel contained about 68% C(12) and C(14) fatty acids. Haustorium fatty acids were intermediate between the two. [(14)C]Acetate was actively metabolized by shoot and haustorium slices but not so actively by the kernel. Approximately 9% to 17% was converted to water-soluble substances, 4% to 6% to CO(2), and 0.5% to 5.9% to lipids. The fatty acids synthesized in the shoot and haustorium were mainly C(16), C(18), and C(18:1) fatty acids but in the kernel about 18% to 32% of the (14)C-fatty acids were C(12) fatty acids.[(14)C]Lauric acid was absorbed and metabolized by haustorium slices and by the haustorium in intact seedlings; it was partly esterified to triacylglycerols and also converted to water-soluble substances and insoluble tissue material. In contrast, tri-[(14)C]laurin was absorbed but not metabolized. The haustorium also absorbed other fatty acids but the longer chain (C(16) and C(18)) fatty acids were not esterified or metabolized further. Preincubation of the haustorium with plant hormones or in the presence of kernel tissue did not alter its inactivity towards tri-[(14)C]laurin.When tri-[(14)C]laurin or [(14)C]lauric acid were injected into the seed or the shoot, there was no movement or radioactivity to other parts of the seedling. When injected into the shoot, but not into the seed, tri-[(14)C] laurin was hydrolyzed and partly metabolized to water-soluble substances.

Journal Article↗
PubMed1982

Purification and characterization of the stearoyl-acyl carrier protein desaturase and the acyl-acyl carrier protein thioesterase from maturing seeds of safflower.

Two enzymes involved in oleic acid biosynthesis have been purified from immature safflower seed. The stearoyl-acyl carrier protein (ACP) desaturase which catalyzes the formation of the double bond of oleate has been purified 200-fold and is a dimer with a molecular weight of 68,000. The enzyme shows strong preference for stearoyl-ACP as substrate; by comparison of its activity with stearoyl-CoA and palmitoyl-ACP as substrates, it appears that the ACP moiety is primarily important for binding of substrate and the chain length is important for catalytic activity. The desaturase requires 56 microM oxygen for half-maximal activity, 400 microM oxygen for maximal activity, and is stimulated severalfold by catalase. The acyl-ACP thioesterase has been purified 700-fold and is also a dimer of molecular weight 74,000. It shows a 5-fold preference for oleoyl-ACP versus stearoyl-ACP and is relatively inactive with corresponding acyl-CoAs.

Acyl Carrier Protein↗
PubMed1982

Isolation and function of spinach leaf beta-ketoacyl-[acyl-carrier-protein] synthases.

Crude spinach leaf extract readily forms the stearoyl derivative of acyl-carrier-protein (ACP) when acetyl-ACP and malonyl-ACP are incubated together. Palmitoyl-ACP is also elongated by malonyl-ACP to stearoyl-ACP. When beta-ketoacyl-ACP synthase {3-oxoacyl-[ACP] synthase; acyl-[ACP]:malonyl-[ACP] C-acyltransferase (decarboxylating), EC 2.3.1.41} is purified with decanoyl-ACP as the assay substrate, palmitoyl-ACP elongation activity is lost. When palmitoyl-ACP is the assay substrate, another protein is isolated that specifically elongates palmitoyl-ACP to beta-ketostearoyl-ACP but has no activity towards decanoyl-ACP. The first protein is designated beta-ketoacyl-ACP synthase I and participates in the conversion of acetyl-ACP to palmitoyl-ACP, whereas the second protein is designated beta-ketoacyl-ACP synthase II, and its substrate specificity is highly restricted to myristoyl-ACP and palmitoyl-ACP. The purification of synthase II is described, and its activity is compared to synthase I. Reconstitution experiments with the highly purified nonassociated enzymes in fatty acid synthesis plus synthases I and II clearly demonstrate the roles of these two proteins in fatty acid synthesis.

Journal Article↗
PubMed1982

Origin of acetate in spinach leaf cell.

Mitochondria were isolated from spinach (Spinacia oleracea L.) leaves using a Percoll gradient step. The high purity of the organelle fraction is demonstrated by electron microscopy and biochemical parameters. In the matrix space of these mitochondria, a short-chain acyl-coenzyme A hydrolase is present that converts acetyl-coenzyme A to acetate and coenzyme A with reasonable rates (K(m), 150 micromolar; V(max), 140 nanomoles acetate formed milligram(1) protein hour(-1)). The enzyme is product inhibited by coenzyme A-sulfhydryl, other thiols are ineffective; however, the disulfides 5,5'-dithio-bis-(2-nitrobenzoate) and cystamine stimulate the hydrolysis. The possible role of this mitochondrial enzyme as a means of generating free acetate from pyruvate via acetyl-coenzyme A in the mitochondria of mature spinach leaves is discussed. It is suggested that free acetate moves rapidly from the mitochondrion to the chloroplast where acetyl-coenzyme A synthetase, solely localized in this organelle, converts the metabolically inert free acetate to the highly active acetyl-coenzyme A.

Journal Article↗
PubMed1982

Fatty Acid Synthetase of Spinacia oleracea Leaves.

The molecular organization of fatty acid synthetase system in spinach (Spinacia oleracea L. var. Viroflay) leaves was examined by a procedure similar to that employed for the safflower system (Carthamus tinctorius var. UC-1). The crude extract contained all the component activities (acetyl-CoA:ACP transacylase, malonyl-CoA:ACP transacylase, beta-ketoacyl-ACP synthetase, beta-ketoacyl-ACP reductase, beta-hydroxyacyl-ACP dehydrase, and enoyl-ACP reductase [I]) involved in the synthesis of fatty acids, but enoyl-ACP reductase (II) present in safflower seeds extract could not be detected spectrophotometrically. By polyethylene glycol fractionation followed by several chromatographic procedures, i.e. Sephadex G-200, hydroxyapatite, and blue-agarose, the component enzymes were clearly separated from one another. Properties of beta-ketoacyl-ACP reductase, beta-hydroxyacyl-ACP dehydrase, and enoyl-ACP reductase (I) from spinach were compared with the same enzymes in safflower seeds and Escherichia coli.From these results, it was concluded that the fatty acid synthetase system of spinach leaves, as well as that of safflower seeds, was nonassociated and similar to the Escherichia coli system.

Journal Article↗
PubMed1982

Solubilization and Characterization of an Acyl-Coenzyme A : O-LYSOPHOSPHOLIPID ACYLTRANSFERASE FROM THE MICROSOMES OF DEVELOPING SAFFLOWER SEEDS.

Acyl-CoA:O-lysophospholipid acyltransferase activity was measured in extracts of developing safflower (Carthamus tinctorius var. UC-1) seeds. Acyltransferase activity was present in all subcellular fractions. The microsomal acyltransferase activity was solubilized with either 30 mm-n-octylglucoside, 1% deoxycholate, or 100 mum lysophosphatidylcholine. The pH optimum of the lysophosphatidylcholine-dependent acyltransferase activity was at 9.0. Among the various acyl-CoA's tested, oleoyl-CoA was the best substrate and with it the enzyme had a K(m) of 9.5 mum. Among the various acyl acceptors tested, lysophosphatidylcholine (oleoyl) gave the highest activity and the optimal concentration was 30 mum. Acyltransferase activity was stimulated by ethanol and inhibited by bovine serum albumin and divalent cations.

Journal Article↗
PubMed1981

Inhibition of plant fatty acid synthesis by nitroimidazoles.

1. The effect of the addition of a number of nitroimidazoles was tested on fatty acid synthesis by germinating pea seeds, isolated lettuce chloroplasts and a soluble fraction from pea seeds. 2. All the compounds tested had a marked inhibition on stearate desaturation by lettuce chloroplasts and on the synthesis of very-long-chain fatty acids by pea seeds. 3. In contrast, the effect of the drugs on total fatty acid synthesis from [14C]acetate in chloroplasts was related to the compound's electron reduction potentials. 4. Of the compounds used, only metronidazole had a marked inhibition on palmitate elongation in the systems tested. 5. The mechanism of inhibition of plant fatty acid synthesis by nitroimidazoles is discussed and the possible relevance of these findings to their neurotoxicity is suggested.

Acetates↗