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.     

Solubilization and partial purification of ATPase from a rose cell plasma membrane fraction

Some isolates of the fungus Nectria haematococca Berk. and Br. can demethylate pisatin, a phytoalexin from pea (Pisum sativum L.). Pisatin demethylation appears to be necessary for tolerance to pisatin and virulence on pea, and is catalyzed by a microsomal cytochrome P-450. We now report solubilization of this enzyme from N. haematococca microsomes. Pisatin demethylase activity was obtained in the high speed supernatant of detergent treated microsomes, if detergent was removed before assay. The CO-binding spectrum of the soluble enzyme preparation indicated the presence of cytochrome P-450. Cholic acids were the most effective of the detergents tested for solubilizing enzyme activity. Loss of enzyme activity during solubilization was reduced by certain protease inhibitors, but not by substrate, reducing agents, antioxidants, or phospholipids. The most effective solubilization medium tested was 1% sodium cholate, 100 millimolar potassium phosphate, 500 millimolar sucrose, 1 millimolar phenylmethylsulfonyl fluoride, pH 7.5, which yielded approximately 30% of the pisatin demethylase and over 95% of the NADPH-cytochrome c reductase in the soluble fraction. Demethylase activity was lost when the reductase was removed by adsorption on 2′,5′-ADP-agarose. The demethylase activity of reductase-free fractions could be restored by adding a reductase preparation purified approximately 100-fold from microsomes of N. haematococca isolate 74-8-1, which does not demethylate pisatin. We conclude that pisatin demethylase requires NADPH-cytochrome c reductase for activity. The inability of some isolates to demethylate pisatin appears to be due to the absence of a suitable cytochrome P-450, rather than to a lack of functional reductase.     

Carnitine acetyltransferase in pea cotyledon mitochondria

Purified pea cotyledon mitochondria did not oxidise acetyl-CoA in the presence of carnitine. However, acetylcarnitine was oxidised. It was concluded that acetylcarnitine passed through the mitochondrial membrane barrier but acetyl-CoA did not. Only a sensitive radioactive assay detected carnitine acetyltransferase in intact mitochondrion or intact mitoplast preparations. When the mitochondria or mitoplasts were burst, acetyl-CoA substrate was available to the matrix carnitine acetyltransferase and a high activity of the enzyme was measured. The inner mitochondrial membrane is there-fore the membrane barrier to acetyl-CoA but acetylcarnitine is suggested to be transported through this membrane via an integral carnitine: acylcarnitine translocator. Evidence is presented to indicate that when the cotyledons from 48-h-grown peas are oxidising pyruvate, acetylcarnitine formed in the mitochondrial matrix by the action of matrix carnitine acetyltransferase may be transported to extra-mitochondrial sites via the membrane translocator.     

Solubilization and partial purification of aromatase from chicken ovary

Aromatase, the cytochrome P-450 that converts androgen to estrogen, has been solubilized from chicken ovarian microsomes with the nonionic detergent Emulgen 913. Following chromatography on gel filtration, anion exchange, dye affinity, and hydrophobic media, ovarian aromatase is purified up to 27-fold with 10-15% recovery. Separation of the cytochrome P-450 aromatase from NADPH cytochrome P-450 reductase is achieved during the purification. The partially purified enzyme is stable for as long as 6 months when frozen in liquid nitrogen in buffer containing dithiothreitol, glycerol, Emulgen and 150 mM KCl.     

Restoration of ATP synthesis in urea-treated membranes prepared from pea cotyledon mitochondria

Submitochondrial particles were prepared from pea cotyledon mitochondria by sonication in a medium containing 5 mM MgCl₂. The resulting particles (Mg²⁺-submitochondrial particles) catalyzed oxidative phosphorylation at the rate of 100–200 nmol ATP formed / min per mg protein. Treatment of Mg²⁺-submitochondrial particles with 3.0 M urea resulted in a preparation of highly resolved particles with low ATPase activity and no capacity for oxidative phosphorylation. However, the resulting membranes were not capable of reconstitution of oxidative posphorylation with the purified mitochondrial F₁-ATPase. Urea particles capable of reconstitution of oxidative phosphorylation could be prepared by extracting Mg²⁺-submitochondrial particles with concentrations of urea ranging from 1.7 to 2.0 M. We have used 1.9 M urea for large-scale preparation of urea particles that could be stored in liquid nitrogen without any loss of reconstitution capacity. The residual oxidative phosphorylation rate of these particles was 6–8 nmol ATP / min per mg protein and this rate could increase to 60–70 nmol ATP / min per mg protein on incubation with saturating amounts of purified mitochondrial F₁-ATPase. In contrast to the mitochondrial F₁, purified activated pea chloroplast CF₁ was unable to stimulate ATP synthesis in 1.9 M urea particles.     

Solubilization and partial purification of GABAB receptor from bovine brain

gamma-Aminobutyric acid (GABA)B receptor has been solubilized and partially purified by an affinity column chromatography. GABAB receptor was solubilized by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in the presence of asolectin. The solubilized GABAB receptor was adsorbed on baclofen-coupled epoxy-activated Sepharose 6B. The affinity matrix adsorbed 80% of the solubilized [3H]GABA binding activity to GABAB receptor, and approximately 75% of the adsorbed activity could be eluted with 1 M KC1. GABAB receptor binding in the fraction eluted from affinity column was displaced by GABA, baclofen and 2-hydroxy saclofen in a dose-dependent manner. Furthermore, the purified GABAB receptor showed approximately 2800-fold purification as compared with the original solubilized fraction and possessed the specific binding activity of 17.68 p mol/mg of protein. This binding consisted of a single binding site with a dissociation constant of 64.4 nM. The present results indicate that affinity column chromatographic procedures using baclofen-coupled epoxy-activated Sepharose 6B are suitable for the partial purification of GABAB receptor from cerebral tissues.     

Solubilization and partial purification of hyaluronate synthetase from oligodendroglioma cells

Hyaluronate synthetase was solubilized with digitonin from crude membranes of mouse oligodendroglioma cells. Detergent extraction was carried out in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered saline with an optimal digitonin to protein ratio (w/w) of 0.7-0.8. The solubilized synthetase was partially purified approximately 230-fold by gel filtration and ion-exchange chromatography. The solubilized enzyme displayed similar properties to membrane-bound enzyme: (a) it synthesized high molecular weight hyaluronate which eluted in the void volume of a Sepharose CL-2B column; (b) the apparent Km values obtained for UDP-GlcUA and UDP-GlcNAc were 50 and 100 microM, respectively; and (c) treatment of intact cells with hyaluronidase prior to extraction with digitonin resulted in a 3-fold increase in solubilized synthetase activity. Furthermore, gel filtration chromatography of the solubilized hyaluronidase-treated synthetase complex showed that it was smaller than the solubilized untreated synthetase complex, due to shorter nascent-bound hyaluronate. The solubilized synthetase was shown to be associated with hyaluronate in the form of a complex. Both hyaluronidase-treated and -untreated synthetase-hyaluronate complexes after solubilization were adsorbed by an affinity matrix using the hyaluronate binding domain of rat chondrosarcoma proteoglycan as ligand. This solubilized active enzyme preparation should allow the identification and characterization of the components of the hyaluronate-synthetase complex.     

The effect of rotenone on respiration in pea cotyledon mitochondria

Respiration utilizing NAD-linked substrates in mitochondria isolated from cotyledons of etiolated peas (Pisum sativum L. var. Homesteader) by sucrose density gradient centrifugation exhibited resistance to rotenone. The inhibited rate of α-ketoglutarate oxidation was equivalent to the recovered rate of malate oxidation. (The recovered rate is the rate following the transient inhibition by rotenone.) The inhibitory effect of rotenone on malate oxidation increased with increasing respiratory control ratios as the mitochondria developed. The cyanide-resistant and rotenone-resistant pathways followed different courses of development as cotyledons aged. The rotenone-resistant pathway transferred reducing equivalents to the cyanide-sensitive pathway. Malic enzyme was found to be inhibited competitively with respect to NAD by rotenone concentrations as low as 1.67 micromolar. In pea cotyledon mitochondria, rotenone was transformed into elliptone. This reduced its inhibitory effect on intact mitochondria. Malate dehydrogenase was not affected by rotenone or elliptone. However, elliptone inhibited malic enzyme to the same extent that rotenone did when NAD was the cofactor. The products of malate oxidation reflected the interaction between malic enzyme and malate dehydrogenase. Rotenone also inhibited the NADH dehydrogenase associated with malate dehydrogenase. Thus, rotenone seemed to exert its inhibitory effect on two enzymes of the electron transport chain of pea cotyledon mitochondria.     

Inhibition of cyanide-resistant respiration in pea cotyledon mitochondria by chloroquine

The action on mitochondrial respiration of a ubiquinone analog, chloroquine, has been studied using purified mitochondria from the cotyledons of germinating peas (Pisum sativum L. var. Homesteader). Chloroquine at 3 millimolar did not inhibit malate or succinate oxidation at pH 7.2, but it did inhibit malate (but not succinate) oxidation at pH 8.2. Cyanide-resistant respiration was also inhibited.     

Solubilization, partial purification and properties of N-methylglutamate dehydrogenase from Pseudomonas aminovorans.

1. Extracts of amine-grown Pseudomonas aminovorans contained a particle-bound N-methylglutamate dehydrogenase (EC 1.5.99.5). The enzyme was not present in succinate-grown cells, and activity appeared before growth began in succinate-grown cells which had been transferred to methylamine growth medium. 2. Membrane-containing preparations from methylamine-grown cells catalysed an N-methylglutamate-dependent uptake of O2 or reduction of cytochrome c, which was sensitive to inhibitors of the electron-transport chain. 3. N-Methylglutamate dehydrogenase activity with phenazine methosulphate or 2,6-dichlorophenol-indophenol as electron acceptor could be solubilized with 1% (w/v) Triton X-100. The solubilized enzyme was much less active with cytochrome c as electron acceptor and did not sediment in 1 h at 150000g. Solubilization was accompanied by a change in the pH optimum for activity. 4. The solubilized enzyme was partially purified by Sepharose 4B and hydroxyapatite chromatograpy to yield a preparation 22-fold increased in specific activity over the crude extract. 5. The partially-purified enzyme was active with sarcosine, N-methylalanine and N-methylaspartate as well as with N-methylglutamate. Evidence suggesting activity with N-methyl D-amino acids as well as with the L-forms was obtained. 6. The enzyme was inhibited by p-chloromercuribenzoate, iodoacetamide and by both ionic and non-ionic detergents. 2-Oxoglutarate and formaldehyde were also inhibitors. 7. Kinetic analysis confirmed previous workers' observations of a group transfer (Ping Pong) mechanism. 8. Spectral observations suggested that the partially purified preparation contained flavoprotein and a b-type cytochrome. 9. The role of the enzyme in the oxidation of methylamine is discussed.     

Solubilization and partial purification of dihydroxyacetone-phosphate acyltransferase from guinea pig liver

Dihydroxyacetone-phosphate:acyl coenzyme A acyltransferase (EC 2.3.1.42) was solubilized and partially purified from guinea pig liver crude peroxisomal fraction. The peroxisomal membrane was isolated after osmotic shock treatment and the bound dihydroxyacetone-phosphate acyltransferase was solubilized by treatment with a mixture of KCl-sodium cholate. The solubilized enzyme was partially purified by ammonium sulfate fractionation followed by Sepharose 6B gel filtration. The enzyme was purified 1200-fold relative to the guinea pig liver homogenate and 80- to 100-fold from the crude peroxisomal fraction, with an overall yield of 25–30% from peroxisomes. The partially purified enzyme was stimulated two- to fourfold by Asolectin (a soybean phospholipid preparation), and also by individual classes of phospholipid such as phosphatidylcholine and phosphatidylglycerol. The kinetic properties of the enzyme showed that in the absence of Asolectin there was a discontinuity in the reciprocal plot indicating two different apparent K_m values (0.1 and 0.5 mm) for dihydroxyacetone phosphate. The V_max was 333 nmol/min/mg protein. In the presence of Asolectin the reciprocal plot was linear, with a K_m = 0.1 mm and no change in V_max. The enzyme catalyzed both an exchange of acyl groups between dihydroxyacetone phosphate and palmitoyl dihydroxyacetone phosphate in the presence of CoA and the formation of palmitoyl [³H]coenzyme A from palmitoyl dihydroxyacetone phosphate and [³H]coenzyme A, indicating that the reaction is reversible. The partially purified enzyme preparation had negligible glycerol-3-phosphate acyltransferase (EC 2.3.1.15) activity.     

Solubilization and partial purification of squalene synthase from daffodil microsomal membranes

A squalene synthase was solubilized from daffodil (Narcissus pseudonarcissus L.) microsomes with CHAPS, a zwitterionic non-denaturating detergent. By successive chromatography on DEAE Sephacel and APP Sepharose a fraction enriched in this enzyme (21-fold) was prepared.     

Solubilization and partial purification of heme oxygenase from rat liver.

Hepatic microsomal heme oxygenase was solubilized, partially purified, and characterized from Co2+-treated rats. The enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis exhibited a minimum molecular weight of greater than or equal to 68,000. The solubilized enzyme was totally devoid of contamination with cytochrome P-450 or b5. The requirement for reduced pyridine nucleotides was absolute, and ascorbate could not support heme oxidative activity. However, both TPNH and DPNH could serve as electron donors, with TPNH being more effective. The presence of an appropriate flavoprotein reductase was essential for heme oxidation. The enzyme had an apparent Km of 40 micrometer, a pH optimum of 7.5, and lost substantial activity upon freezing and thawing. Methemoglobin was 30% as effective a substrate for the enzyme as was heme. Free porphyrins could not serve as substrates for the enzyme. The activity of the enzyme was inhibited by HgCl2, p-chloromercuribenzoate, iodoacetamide, mercaptoethanol, and dithiothrietol indicating that free -SH group(s) is necessary for enzyme activity.