Microsomal membrane lipids from pea shoots and Ranunculus sceleratus petioles

Microsomal membranes, relatively free of chloroplasts and mitochondria, were prepared from etiolated pea stems and Ranunculus sceleratus petioles by differential centrifugation. Relative quantitative analyses were made of the phospholipid, glycolipid, sterol and fatty acid contents.     

Trypsin inhibitor from bambara pea

An antitryptic activity has been identified in the flour of dry non-germinated seed of an African leguminous plant, the bambara pea (Voandzeia subterranea). This inhibitor has been purified by trichloracetic acid and ammonium sulphate precipitations followed by gel filtration on Sephadex G25, ion exchange chromatography and gel filtration on Sephadex G75. Antitryptic activity increased 50-fold. Its purity has been verified by electrophoresis on polyacrylamide gel and gel chromatography. Its MW is 13 200 in the denatured and reduced forms and 26 300 in the native form. It is resistant to thermal denaturation and appears to be in monomeric form when entirely denatured.     

Characterization and localization of an ATP diphosphohydrolase activity (EC 3.6.1.5) in sarcolemmal membrane from rat heart

In the present report we describe an ATP diphosphohydrolase (apyrase EC 3.6.1.5) in rat cardiac sarcolemma. It is Ca2+ dependent and is insensitive to ouabain, orthovanadate, N-ethylmaleimide (NEM), lanthanum, and oligomycin that are classical ATPase inhibitors. Sodium azide that is a mitochondrial inhibitor at low concentrations, did not affect the enzyme activity at 5.0 mM or below. In contrast, at high concentrations (> 10 mM) sodium azide inhibited the enzyme. Levamisole, a specific inhibitor of alkaline phosphatase and P1, P5-di(adenosine 5-)pentaphosphate (Ap5A), a specific inhibitor of adenylate kinase did not inhibit the enzyme. Mercury chloride showed a parallel inhibition of the hydrolysis of both substrates of apyrase. Similar inhibition profiles are powerful evidence for a common catalytic site for the hydrolysis of both substrates. The enzyme has an optimum pH range of 7.5–8.0 and catalyzes the hydrolysis of triphospho- and diphosphonucleosides other than ATP or ADP. The apparent Km (Michaelis constant) and Vmax (maximal velocity) are 62.1 ± 5.2 M and 1255.7 ± 178 mol inorganic phosphate liberated/min/mg with ATP and 59.4 ± 4.3 M and 269.2 ± 39 mol inorganic phosphate liberated/min/mg with ADP. Enzyme markers indicated that this apyrase is associated with the plasma membrane. A deposition of lead phosphate granules on the outer surface of the sarcolemmal vesicles was observed by electron microscopy in the presence of either ATP or ADP as substrate. It is suggested that the ATP diphosphohydrolase could regulate the concentration of extracellular adenosine, and thus is important in the control of vascular tone and coronary flow.     

Dye-sensitized photo-oxidation of acid ribonuclease from pea cotyledons

In crude extracts, pea cotyledon acid ribonuclease is not inactivated by photo-oxidation, but after 150-fold purification, it is markedly inactivated when illuminated in the presence of methylene blue at pH 7.2. It is, however, still resistant to methylene blue-sensitized photo-oxidation at pH 5.4. These data suggest that photo-oxidation of methionine, cysteine and tryptophan has no effect on enzyme activity, whereas photo-oxidation of histidine markedly reduces catalytic acitivity. It is thus likely that the mode of action of acid ribonuclease from pea cotyledons is similar to that of pancreatic ribonuclease.     

Recombinant protein purification from pea

To assess the suitability of transgenic peas as a host for protein production from the perspective of ease of recovery, a strain containing recombinant beta-glucuronidase with poly(histidine) tail (GUSH6) was evaluated for solubility of the target protein in relation to native components (proteins, carbohydrates, and phenolics). Recovery of the recombinant GUSH6 from aqueous extracts by immobilized metal affinity chromatography with coupled Co(2+) yielded a nearly pure product with IDA (enrichment factor (EF) = 260) or NTA (EF = 200) resin. Single-step recoveries were also possible by isoelectric precipitation (EF = 4), polyelectrolyte precipitation (EF = 1.5), and anion-exchange chromatography (EF = 3.1), but enrichment factors were low.     

Carnitine content of pea seedling cotyledons

Using a radioactive assay for the determination of carnitine, the amount in pea cotyledons was shown to vary with age.     

Purification of ctp: cholinephosphate cytidylyl-transferase from pea stems

CTP:cholinephosphate cytidylyltransferase (EC 2.7.7.15) was purified from pea (Pisum sativum) stems. The purification involved ammonium sulphate fractionation, ion exchange chromatography, removal of proteases with α₂-macroglobulin and gel filtration. The purified enzyme had K_m values for phosphorylcholine and CTP of 2.1 mM and 0.55 mM respectively. It was found to have a pH optimum of 7.5, a requirement for Mg²⁺ and an M_r of 56000. It could not utilize phosphorylethanolamine and its activity was not stimulated by added phospholipids.     

Purification and properties of diamine oxidase from pea epicotyls

Diamine oxidase (DAO) (EC 1.4.3.6) was purified from pea epicotyls to homogeneity by the criterion of polyacrylamide gel electrophoresis (PAGE). The pu     

Kinetin effect on ATP synthesis and on adenylate kinase activity in pea seeds

Pea seed powder incubated in the presence of AMP and phosphoenolpyruvate (PEP) accumulated relatively large amounts of ATP. The rate of accumulation in     

Isolation and properties of lactate dehydrogenase from germinating pea plants

Lactate dehydrogenase (LDH) was isolated from pea seedlings by means of protamine sulphate and (NH₄)₂SO₄ fractionation and chromatography on DEAE-cellulose and Sephadex G-150. The enzyme had a MW of ca 145 500. The kinetic properties studied were the lactate oxidation pH optimum (9·1) and the pyruvate reduction pH optimum (7·1). K_m values were determined for four natural substrates (Lactate, pyruvate, NAD⁺ and NADH) and for other acids (glycollate, α-ketoglutarate and glyoxylate). The K_i value was determined for p-chloromercuribenzoate (PCMB) which is a noncompetitive inhibitor of LDH from pea plants, and the course of irreversible inhibition of the enzyme by iodoacetamide (IA) and n-ethylmaleimide (NEMI) was studied. Preincubation of LDH with the coenzyme protects against PCMB inhibition, indicating the important role of the sulfhydryl group in the active site.     

Characterization of a proton pump from pea stem microsomes

Abstract The present work deals with the characterization of an ATP-dependent proton translocation monitored by the ΔpH probe acridine orange. The ATP-dependent proton translocation has an optimum activity at pH 6.5 and is substrate specific for ATP. It is stimulated by Cl⁻, HCO₃⁻ and Br⁻, but is insensitive to several monovalent cations. Divalent cations (Mg²⁺ or Mn²⁺) are required for proton translocation, while in the presence of Ca²⁺ no uptake is observed. NO₃⁻, NO₂⁻ and citrate strongly inhibit proton uptake. On the contrary, F⁻, SO₄²⁻, malate, pyruvate, succinate, oxalate and acetate have no inhibitory effect. Proton uptake is stimulated by valinomycin and unaffected by molybdate. Two thiols, dithioerythritol and dithiothreitol, are able partially to prevent the FCCP-abolished proton uptake or partially restore the ATP-dependent proton translocation in FCCP-collapsed vesicles. It is suggested that pea stem microsomes possess an electrogenic ATPase, acting as a proton pump, which, on the basis of its characteristics, can be tentatively associated with membranes of tonoplast origin.     

Characterization of an ATP diphosphohydrolase (EC 3.6.1.5) in synaptosomes from cerebral cortex of adult rats

Data from the literature have demonstrated that synaptosomal preparations from various sources can hydrolyze externally added ATP. Various authors characterized this activity as an ecto-ATPase. In the present report, we demonstrate that synaptosomal preparations obtained from the cerebral cortex of rats show ATPase activity that could not be dissociated from ADPase activity, suggesting that an ATP-diphosphohydrolase is involved in ATP and ADP hydrolysis. Furthermore, the ATP and ADP hydrolysis could not be attributed to associations of enzymes that could mimic an ATP-diphosphohydrolase because none of the following activities were detected in our assay conditions inorganic pyrophosphatase, adenylate kinase, or nonspecific phosphatases. A possible association between an ATPase and an ADPase was excluded on the basis of both the kinetics and much additional data on inhibitors, ion dependence, pH, etc. The present results demonstrate that in synaptosomal preparations from cerebral cortex an ATP-diphosphohydrolase is involved, at least in part, in ATP and ADP hydrolysis.Abbreviations DCCD dicyclohexylcarboiimide - EDTA ethylenediaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Pi inorganic phosphate Enzymes ATP diphosphohydrolase, Apyrase (EC 3.6.1.5) - ATPase ATP phosphohydrolase (EC 3.6.1.3) 5-nucleotidase (EC 3.1.3.5) Hexokinase (EC 2.7.1.1) Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) Adenylate kinase (EC 2.7.4.3) Inorganic pyrophosphatase (EC 3.6.1.1) - ATP pyrophosphohydrolase (EC 3.6.1.8) - LDH lactate dehydrogenase (EC 1.1.1.27) - SDH succinate dehydrogenase (EC 1.3.1.6) - ACHE acethylcholinesterase (EC 3.1.1.7) - G-6-Pase glucose-6-phosphatase (EC 3.1.3.9) - NADPH cytoehrome c oxidoreductase (NCR) (EC 1.6.2.4)     

Glycolipids and glycoproteins formed from UDP-galactose by pea membranes

Pea membranes were incubated with UDP-[¹⁴C]galactose and sequentially extracted with lipid solvents and 2% sodium dodecyl sulfate (SDS). At least three-quarters of the products were SDS-soluble. All fractions contained some [¹⁴C]glucose, indicating the presence of an active epimerase which, however, could be inhibited by ADP-ribose. The chloroform-methanol extract contained mainly neutral galactosyl lipids and a small amount of dolicyl monophosphoryl glucose. The chloroform-methanol-water extract contained trace amounts of lipid-linked galactosyl oligosaccharide with properties comparable to polyisoprenyl pyrophosphoryl derivatives. Polyacrylamide gel electrophoresis of SDS-soluble products indicated the formation of both immobile and mobile components with similar size distribution (Sepharose CL-6B). The mobile component only was susceptible to hydrolysis by protease. Periodate oxidation analysis of SDS-soluble and -insoluble products indicated that they were composed mainly of 1 → 6 galactosyl residues, i.e. as in many arabinogalactan proteins and arabinogalactans.     

Isolation and preliminary characterization of cryptic satellite DNA in pea

Optimum conditions have been established for isolation of ‘cryptic’ satellite DNA from the genome of pea (Pisum sativum), using gradients of CS₂SO₄ containing silver ions. At an Ag⁺ :DNA-P ratio (R) of 0.1, and at alkaline pH, four fractions are obtained: mainband (buoyant density 1.437 g cm³; 67% of total DNA), satellite I (buoyant density 1.582 g/cm³; 7% of total DNA), satellite II (buoyant density 1.520 g/cm³, 11% of total) and satellite III (buoyant density variable between 1.45 and 1.51 g/cm³; 15% of total). The reiterated DNA content of these four fractions has been investigated by reassociation experiments conducted over a Cot range of 1 × 10^?5 to 2.0. All four fractions contain at least two kinetic components; each fraction, including the mainband, consists at least partly of highly reiterated DNA. Ribosomal RNA hybridizes only to the mainband.     

Aldrin epoxidase from pea roots

The membrane system which epoxidizes aldrin to dieldrin in pea roots was separated from polyphenolic inhibitors and soluble peroxidase by rapid anaerobic gel filtration. Epoxidase activity was strongly inhibited by cytochrome-c, cyanide, electron acceptors and polyphenols and was enhanced by chelating and solubilizing agents which inhibit cytochrome P450 systems.     

Effect of phenylalanine and its metabolites on ATP diphosphohydrolase activity in synaptosomes from rat cerebral cortex

The in vitro effects of phenylalanine and some of its metabolites on ATP diphosphohydrolase (apyrase, EC 3.6.1.5) activity in synaptosomes from rat cerebral cortex were investigated. The enzyme activity in synaptosomes from rats subjected to experimental hyperphenylalaninemia (-methylphenylalanine plus phenylalanine) was also studied. In the in vitro studies, a biphasic effect of phenylalanine on both enzyme substrates (ATP and ADP) was observed, with maximal inhibition at 2.0 mM and maximal activation at 5.0 mM. Inhibition of the enzyme activity was not due to calcium chelation. Moreover, phenylpyruvate, when compared with phenylalanine showed opposite effects on the enzyme activity, suggesting that phenylalanine and phenylpyruvate bind to two different sites on the enzyme. The other tested phenylalanine metabolites (phenyllactate, phenylacetate and phenylethylamine) had no effect on ATP diphosphohydrolase activity. In addition, we found that ATP diphosphohydrolase activity in synaptosomes from cerebral cortex of rats with chemically induced hyperphenylalaninemia was significantly enhanced by acute or chronic treatment. Since it is conceivable that ATPase-ADPase activities play an important role in neurotransmitter (ATP) metabolism, it is tempting to speculate that our results on the deleterious effects of phenylalanine and phenylpyruvate on ATP diphosphohydrolase activity may be related to the neurological dysfunction characteristics of naturally and chemically induced hyperphenylalaninemia.     

Effect of pisatin inducing fungi on pea polyamines

The spermine and spermidine content of pea pod tissue is not significantly altered by inoculation with the pisatin-inducing fungi, Fusarium solani. Although these polyamines induce pisatin, it appears that they do not accumulate in levels sufficient to serve as internal mediators of pisatin production in infected tissues.     

Kinetic properties of two starch phosphorylases from pea seeds

Both of the starch phosphorylase fractions from Victory Freezer pea seeds, that can be separated by DEAE—cellulose chromatography and purified by Sepharose 4B-starch affinity chromatography, contain pyridoxal 5′-phosphate. The addition of further quantities of pyridoxal 5′-phosphate causes inactivation. Both enzymes showed similar bi-substrate kinetics with d-Glc-1-P and varying amounts of amylopectin and also with P_i and varying amounts of amylopectin. In the direction of glucan sythesis the K_m for amylopectin with phosphorylase II was much higher than with phosphorylase I. However, the two enzymes differed in their behaviour on glucan degradation at varying concentrations of P_i. With phosphorylase II the K_m for amylopectin was dependent on the concentration of P_i but that for phosphorylase I was constant. Phosphorylase II was strongly inhibited by ADPG in the direction of glucan degradation but only slightly in the direction of glucan synthesis by both ADPG and UDPG. Phosphorylase I was only slightly inhibited by ADPG in both directions and by UDPG in synthesis. UDPG inhibited both enzymes moderately in glucan degradation,     

Characteristics of starch in developing pea seeds

Changes in the physical and chemical properties of pea starch in developing seeds have been investigated. Increase in the iodine-binding capacity of st     

The effect of light on lipoxygenase activity in dwarf pea seedlings

Continuous illumination of 10-day-old etiolated dwarf pea seedlings caused an increase in lipoxygenase activity. At the same time the activity in both stem and leaf tissue decreased. The lipoxygenase isoenzymes of the whole seedling and separated leaf and stem tissue were affected differently by light. It is concluded that lipoxygenase is not involved directly in photosynthesis or chloroplast development.