Partial characterization of a soluble ATPase from pea cotyledon mitochondria.

A partially purified soluble ATPase (ATP phosphohydrolase, EC 3.6.1.3) from pea cotyledon mitochondria was characterized. Inhibition patterns with azide, NaF, and cold, and a stimulation by 2,4-dinitrophenol were typical of F1-ATPases from mammalian mitochondria. The enzyme hydrolysed GTP, ITP, and ATP, but not CTP, UTP, ADP, or IDP. ATPase and ITPase activities were strongly inhibited by ADP and to a lesser extent by IDP. Distinctive properties of the pea mitochondrial enzyme were activation by high concentrations of CaCl2 and stimulation by NaCl.     

Regulation by alcohols and anions of soluble ATPase activity in chloroplasts and mitochondria

The alcohols and anions present at the medium before the beginning of ATPase reaction increased or decreased the lag-time of ATPase hydrolysis by chloroplast coupled factor according to the extent of their inhibitory or stimulating action. The effect of alcohols and anions on the ATPase activity of coupled factor of chloroplasts and mitochondria develops less than 2 s in case of their introduction during the ATP hydrolysis.     

Properties of a soluble ATPase from castor bean endosperm mitochondria

An ATPase was extracted and purified from castor bean endospermmitochondria. The enzyme is stable at 60°C only in the presenceof ATP in the incubation medium. It is less stable at 0°Cthan at 30°C but is stabilized by ammonium sulfate or glycerol.Activity is dependent on the presence of Mg⁺⁺, and in the presenceof Mg⁺⁺ is enhanced by 2,4-dinitrophenol, but is not inhibitedby oligomycin. The enzyme hydrolyzes ITP in addition to ATP,but ITPase activity is hardly enhanced by 2,4-dinitrophenol.This preparation has many properties in common with the ATPase(coupling factor 1) from beef heart mitochondria. (Received November 8, 1969; )     

Changes in respiration of mitochondria isolated from cotyledons of ethylene-treated pea seedlings

Treatment of intact, germinating pea (Pisum sativum L. cv. Homesteader) seedlings with ethylene enhanced the cyanide-resistant respiration of mitochondria isolated from the cotyledons. The level of enhancement depended on the concentration of ethylene. Thus, exposure to 0.9 l·l^-1 of ethylene in air for days 4–6 of germination had little effect on cyanide-resistant respiration, while exposure to 130 l·l^-1 increased it from 10 to 50 nmol O₂·min^-1·(mg protein)^-1. The length of exposure to ethylene also affected the degree of enhancement. According to some literature data, lipoxygenase (EC 1.13.11.12) activity can be mistaken for cyanide-resistant respiration, but in our preparations of purified pea mitochondria ethylene had no effect on lipoxygenase activity, nor did the gas disrupt the outer mitochondrial membrane. Bahr and Bonner plots of respiration in the presence of salicylhydroxamic acid (SHAM) indicated that ethylene did not affect respiration proceeding via the cytochrome pathway. Thus, increases in total respiration in mitochondria from cotyledons of ethylene-treated pea seedlings reflect increases in cyanide-resistant respiration.Abbreviations Cyt c cytochrome c - SHAM salicylhydroxamic acid     

Action of alkyl cations and the natural ATPase inhibitor from mitochondria on soluble mitochondrial ATPase

The effect of the natural ATPase inhibitor and octylguanidine on the ATPase activity of soluble oligomycin-insensitive mitochondrial F₁ were compared. Both compounds induced a maximal inhibition of 60–80% in various preparations of F₁ studied. The inhibition was of the uncompetitive type with respect to MgATP, and the action of the compounds was partially additive. The data suggest that octylguanidine reproduces the action of the natural ATPase inhibitor. Alkylammonium salts also affect the ATPase activity in a similar form. F₁ bound to Sepharose-hexylammonium is largely inactive, whilst free hexylammonium at higher concentrations induces only a partial inhibition of the activity. This suggests that the degree of immobilization of F₁ is related to the magnitude of inhibition of ATPase activity induced by alkyl cations. The binding of F₁ to Sepharose-hexylammonium is prevented by high concentrations of Na⁺ or K⁺.     

Oxidative and phosphorylative activity of mitochondria from pea cotyledons during maturation of the seed

Summary The respiration rate and the activity of some mitochondrial enzymes from pea cotyledons have been followed during the final phases of seed development, when the relative water content of the cotyledons dropped from 65 to 13%. Succinate, malate and -ketoglutarate oxidase activity, and succinate and malate dehydrogenase activity per cotyledon increased when the relative water content dropped from 65 to about 55%. A further drop of the relative water content was accompanied by a strong decrease of the activity of the succinate and malate oxidase system, but only a slight decrease of succinate and malate dehydrogenase activity. Mitochondrial fractions from air-dry, mature cotyledons showed a low activity of the succinate and malate oxidase system but their dehydrogenase activity was relatively high. The phosphorylation efficiency and respiratory control gradually decreased during maturation. These results indicate that during maturation of the pea seed certain mitochondrial enzymes partly lose their activity.     

Action of alkyl cations and the natural ATPase inhibitor from mitochondria on soluble mitochondrial ATPase.

The effect of the natural ATPase inhibitor and octylguanidine on the ATPase activity of soluble oligomycin-insensitive mitochondrial F1 were compared. Both compounds induced a maximal inhibition of 60-80% in various preparation of F1 studied. The inhibition was of the uncompetitive type with respect to MgATP, and the action of the compounds was partially additive. The data suggest that octylguanidine reproduces the action of the natural ATPase inhibitor. Alkylammonium salts also affect the ATPase activity in a similar form. F1 bound to Sepharose-hexylammonium is largely inactive, whilst free hexylammonium at higher concentrations induces only a partial inhibition of the activity. This suggests that the degree of immobilization of F1 is related to the magnitude of inhibition of ATPase activity induced by alkyl cations. The binding of F1 to Sepharose-hexylammonium is prevented by high concentrations of Na+ or K+.     

Solubilization and partial purification of n,n'-dicyclohexylcarbodiimide-sensitive ATPase from pea cotyledon mitochondria

The N,N′-dicyclohexylcarbodiimide (DCCD)-sensitive ATPase of pea (Pisum sativum L.) cotyledon mitochondria was solubilized from submitochondrial particle membranes with sodium cholate and ammonium sulfate. Ammonium sulfate precipitation of the enzyme resulted in an increase in specific activity. At between 38% and 45% saturated ammonium sulfate, 20% of the ATPase activity was precipitated, with a specific activity 4 to 5 times higher than that of the crude enzyme. The precipitate was highly sensitive to DCCD.

The properties of the ammonium sulfate preparation were investigated. It contained levels of cytochrome and NADH dehydrogenase contamination comparable to those of the highly purified F₀F₁ preparations from animal tissue. The high degree of purification was corroborated by sodium dodecyl sulfate electrophoresis.

     

Purification of a soluble ATPase from rat liver mitochondria by AMP-Sepharose affinity chromatography.

ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity was shown in the soluble fraction of rat liver micochondria. Two molecular forms (ATPase 1 and 2) were isolated. ATPase 1 has already been studied. The present paper deals with the purification method of ATPase 2 which was achieved by the following steps: (NH4)2SO4 precipitation. DEAE-cellulose chromatography, hydroxyapatite chromatography, Sephadex G100 filtration and AMP-Sepharose affinity chromatography. The purified protein was characterized by bidimensional polyacrylamide gel electrophoresis. Molecular weight evaluated by SDS-polyacrylamide gel electrophoresis and Sephadex G100 gel filtration was found to be 61 500 +/- 3000.     

Inhibition of soluble yeast mitochondria ATPase by ethidium-bromide.

Surface galactosyl residues of alive embryonal carcinoma cells were tritium-labeled by the galactose oxidase NaB(3H)4 method. The labeled glycopeptides isolated from Pronase digests of the cells, were found to be similar to those prepared from endogeneously labeled embryonal carcinoma cells. They consisted mostly of high molecular weight material, and contained receptors for peanut agglutinin. It is concluded that at least a fraction of the high molecular weight glycopeptides characteristic of early embryonic cells, is displayed on the cell suface.     

Mechanism of glycerol and methanol action on soluble ATPase of mitochondria

The effects of methanol, butanol, glycerol, glucose, sucrose and inorganic anions on the activity of soluble ATPase from mouse liver mitochondria were studied. Glycerol inhibited, while methanol stimulated the enzyme activity uncompetitively with respect to ATP and competitively with respect to each other. Glycerol-induced inhibition of ATPase was competitive with respect to sulphite; methanol competed with thiocyanate for the enzyme activity. The Arrhenius plots for ATPase revealed bends at 20 degrees and 30 degrees C in the presence of sulphite, chlorine, thiocyanate, glycerol and methanol. It was assumed that all the compounds tested influenced soluble ATPase by changing the nucleophilic activity of H2O.     

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.     

Identification of a glycine transporter from pea leaf mitochondria

Mitochondria isolated from pea leaves possess a glycine transporter that is capable of moving glycine from the cytosol into the matrix, the site of glycine decar?ylase. The carrier was inhibited by mersalyl, p-chloromercuribenzoate, and the glycine analogues, glycine hydroxamate and aminoacetonitrile. Glycine uptake was dependent on the transmembrane pH gradient and was inhibited by uncouplers and electron transport inhibitors. Glycine transport was not, however, inhibited by the glycine decar?ylase inhibitor, arsenite. This transporter is responsible for the movement of glycine into the mitochondria and provides an important step in photorespiration.     

Effects of medium osmolarity on the release of amino acids from isolated cotyledons of developing pea seeds

The release of endogenous amino acids from isolated, immature pea (Pisum sativum L. cv. Marzia) cotyledons was investigated in relation to their developmental stage and the osmolarity of the bathing medium. The water potential of the cotyledons was about-1.1 MPa from which it could be inferred that the osmolarity of their apoplastic fluids will be approximately 450 mosmol·l^?1. The time course of amino-acid release conformed to an exponential function. Rate constants of the release were in the range 0.3 to 0.9 · h ^?1. No indication was found for increased permeability of the plasmamembrane for amino acids at low medium osmolarity. Rate constants were even 1.5-fold lower in 0 mM mannitol than in medium with 400 mM mannitol. This effect could be ascribed to reduced protein synthesis in hypotonic media. In the presence of 400 mM mannitol the release was nearly proportional to the total amino-acid pool of the cotyledons and ranged from 12% to 8% for the various developmental stages. Amino-acid release was stimulated by incubation in a hypotonic medium (< 400 mM mannitol), up to fourfold in a medium without mannitol where as much as 45% of the cotyledonary amino-acid content could be released. The extra aminoacid release induced by the hypotonic condition declined during development and eventually vanished completely. Release of amino acids into a medium with 400 mM mannitol was more selective than into a medium without mannitol. For instance, arginine was one of the main constituents of the cotyledonary amino-acid pool (19%) as well as of the released amino-acid mixture when the medium contained no mannitol (10%), whereas it was virtually absent when the medium contained 400 mM mannitol. As an overall interpretation of these results, it is proposed that the hypotonic condition greatly enhances the permeability of the tonoplast (not that of the plasmalemma) for amino acids so that the otherwise well-sequestered amino acids in the vacuole become available for release into the bathing medium.     

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.     

Properties of a DNA-dependent ATPase from rat mitochondria.

A DNA-dependent ATPase has been highly purified from rat liver mitochondria and characterized. The enzyme catalyzes the hydrolysis of ATP or dATP in the presence of single-stranded DNA cofactor and a divalent cation. The Km values for ATP and dATP are 0.15 mM and 0.35 mM, respectively. The enzyme activity is highly sensitive to N-ethylmaleimide. The sedimentation coefficient of the enzyme is 8.3 S in a glycerol gradient. From this and data on Sephadex G-200 gel filtration, the molecular weight of the native enzyme was calculated to be about 190,000. All the natural single-stranded DNAs tested were equally effective for the ATPase activity, but synthetic deoxyhomopolymer poly(dC) was found to be more effective than natural single-stranded DNAs. Synthetic and natural RNAs had no effect on the activity.     

Biosynthesis of Fatty acids by a soluble extract from developing soybean cotyledons

Fractionation of developing soybean cotyledons into cellular components demonstrates that most of the activity necessary to incorporate acetate-1-¹⁴C into lipid remains in the supernatant from a 198,000g spin for 1 hr. The system studied is dependent upon ATP, CoA, and CO₂. Concentrations of ATP greater than 4 × 10⁻³m are inhibitory, while 1 × 10⁻⁴m CoA is needed for optimal activity. Avidin inhibition of acetate incorporation into lipid could be reversed by biotin. Studies indicated that NADPH is a better source of reducing power than NADH. The system studied is inhibited by p-chloromercuribenzoic acid and this inhibition can be reversed by an excess of GSH. The system studied shows maximum activity in tris buffer at pH 8.6 or in glycine buffer, pH 9.4.