Purification and Partial Carbohydrate Analysis of Phytochrome from Avena sativa

Highly purified samples of phytochrome from etiolated oat (Arena satira L.) seedlings were analyzed for sugar content. Phytochrome was found to be a glycoprotein, with about 4% of its weight as carbohydrate. In addition, modifications in the procedure for purifying phytochrome are proposed here and certain details about the protocol are discussed that improve the yield and reproducibility of previously published methods.     

Isolation and Purification of an alpha-Mannosidase from Coleoptiles of Avena sativa

An alpha-mannosidase has been purified from the coleoptiles of Avena sativa L. var. Segrehavre. The enzyme, which is tightly associated with the cell wall, was solubilized with 3 m LiCl. The purification involves precipitation with (NH(4))(2)SO(4), gel filtration, ion exchange chromatography, and isoelectric focusing. The enzyme appears homogeneous when chromatographed on disc gels and on isoelectric focusing gels. The enzyme runs as a single protein of constant specific activity when chromatographed on Sephadex G-200. The estimated molecular weight of the enzyme is 630,000. The enzyme appears to have no metal ion cofactor requirement and is insensitive to p-chloromercuribenzoate. The pH optimum for the enzyme with p-nitrophenyl-alpha-d-mannoside as the substrate is 4.5 and the K(m) is 3.2 mm. The enzyme may have some carbohydrate associated with it as indicated by a positive periodate-Schiff reaction on disc gels.     

C-Glycosylflavones from Avena sativa

Avena sativa leaves, stems and inflorescences contain a range of new C-glycosylflavone 2″-O-glycosides, including vitexin and isoswertisin 2″-rhamnosides, isovitexin and isoorientin 2″-arabinosides. The structure of ‘vitexin 4′-rhamnoside’ from Crataegus oxyacantha is revised in vitexin 2″-rhamnoside.     

Purification and properties of apoplastic amylase from oat (Avena sativa) seedlings

The protein fraction extracted with a high ionic strength buffer from the cell wall preparation of oat ( Avena sativa L.) coleoptiles and first leaves contained starch-degrading (amylase) activity. The activity of apoplastic amylase in the coleoptiles and first leaves continued to increase in parallel with organ growth. One of the apoplastic amylases recovered from shoot cell wall preparations was purified by sequential ion exchange and gel filtration chromatography, and the catalytic properties of the enzyme were analysed. The purified enzyme gave a single 25 kDa protein band on SDS-PAGE. The enzyme exhibited maximum activity at pH 5.0 against maltooligosaccharides. The purified enzyme hydrolysed soluble starch and maltooligosaccharides larger than tetraose at maltose unit, but did not hydrolyse β -limit dextrin or p -nitrophenyl- α - d -glucopyranoside. These results as well as the findings that the molecular size and the catalytic properties of the purified enzyme are different from those of known amylases obtained from Gramineae caryopses suggest that this enzyme is a novel type of β -amylase present in cell walls of vigorously elongating Gramineae shoot organs.     

Purification and initial characterization of ubiquitin from the higher plant, Avena sativa

Ubiquitin is a highly conserved, 76-amino acid polypeptide recently demonstrated to be involved in ATP-dependent protein degradation in mammalian cells. From immunoblot analyses with anti-human-ubiquitin antibodies we have detected the presence of free ubiquitin in green leaves, etiolated shoots, and dry seeds of the higher plant, oats (Avena sativa L.). We also find that crude oat extracts contain protease(s) that rapidly degrade both oat and human ubiquitin (t1/2 approximately 10 min at 27 degrees C). This proteolysis apparently cleaves ubiquitin at the carboxyl-terminal glycine dipeptide and results in inactivation of the molecule with respect to ligation but does not affect its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using homogenization conditions that preclude this proteolysis (low pH and the addition of the protease inhibitor p-chloromercuribenzoate) and immunoblotting as an assay for the protein, a procedure for the purification of ubiquitin from etiolated oat shoots was developed. Characterization of purified oat ubiquitin by absorption spectra, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, radioimmunoassay with anti-human-ubiquitin antibodies, and kinetic analyses using the ubiquitin activating enzyme isolated from rabbit liver indicates that this protein is remarkably similar to the mammalian form. Small differences between the oat and human proteins have been observed by amino acid compositional analyses indicating that the two forms are not totally homologous. Immunoblotting of crude oat extracts has revealed the presence of high molecular weight proteins recognized by anti-ubiquitin antibodies that represent ubiquitin-protein conjugates formed in vivo. Taken together, these data provide evidence that higher plants contain a ubiquitin-dependent proteolytic pathway that is mechanistically identical to that present in animals.     

Purification and properties of human liver monoamine oxidase

Human liver monoamine oxidase [monoamine: O₂ oxidoreductase (deaminating), E. C. 1.4.3.4] was purified by a method which does not depend on the isolation of mitochondria, and in which vacuum dialysis, during which the enzyme separates out as a yellow precipitate, is an important step in purification. By this method a final specific activity of 550 and fold purification of 40 was attained. A single peak was obtained with the analytical ultracentrifuge, and a sedimentation constant of 6.78S noted. A single active band was observed by polyacrylamide gel electrophoresis. The enzyme exhibits optimum activity at pH 8.7, with no activity below pH 5.5 or above pH 11.8. Using benzylamine hydrobromide as the substrate, in 0.05 m phosphate buffer (pH 7.4) at 27 °C, the Michaelis constant was found to be 1.7 × 10^?3m. The enzyme, which is quite stable, is a flavo-protein, as shown by absorption and fluorescence spectra. The C-terminal group is glycine. The molecular weight, as determined by SDS polyacrylamide-gel electrophoresis, is 64,000. Repeated attempts to determine the N-terminal group were unsuccessful.     

Purification of human blood platelet monoamine oxidase

Monoamine oxidase B has been purified from human blood platelets 185-fold to a specific activity of 113 nmole/min/mg protein by a combination of Triton X-100 solubilization and ion exchange chromatography. A protein fraction corresponding to 58,000 Da on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was identified as monoamine oxidase by its ability to bind [3H]Pargyline.     

Purification and properties of mitochondrial monoamine oxidase type A from human placenta

A high yield purification scheme for monoamine oxidase A from human placental mitochondria is described. The enzyme is solubilized by a combination of treatment with phospholipase A and C and extraction with Triton X-100 and further purified by partitioning between dextran and polyethylene glycol polymers. The enzyme was obtained in 35% yield and high purity on DEAE-Sepharose CL-6B chromatography. This product, 90% catalytically active, showed a single major and several minor bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Further purification could be achieved by additional chromatography using Bio-Gel HTP, but concomitant loss of catalytic activity occurred (enzyme remained about 60% active). The difference extinction coefficient for flavinox--flavinred at 456 nm was 10,800 +/- 350 m-1 cm-1. A sulfhydryl to flavin ratio of 7.5 was obtained when enzyme was denatured with sodium dodecyl sulfate, reduced with 2-mercaptoethanol, and titrated with 2,2'-dipyridyl disulfide. Anaerobic titration with 0.5 eq of sodium dithionite gave rise to the red anionic flavin radical, and full reduction was observed on further addition of reagent. The Km value for kynuramine was essentially the same for mitochondria (0.12 mM) and enzyme after DEAE-Sepharose CL-6B chromatography (0.17 mM). The concentration of clorgyline and deprenyl required for 50% inactivation also remained essentially unchanged. Incubation of the enzyme with 2,2'-dipyridyl disulfide caused inactivation in a biphasic manner with apparent second-order rate constants of 1230 M-1 min-1 and 235 M-1 min-1 for the rapid and slow phase, respectively. This inactivation was largely abolished by the inclusion of the competitive inhibitor amphetamine (Ki = 20 microM) in the incubation mixture. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a subunit molecular mass of 60-64 kDa, about 1.5-2.5 kDa higher than human liver monoamine oxidase B.     

Purification and immunochemical characterization of monoamine oxidase from rat and human liver.

1. Monoamine oxidase from rat and human liver was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. 2. The enzyme activity was extracted from mitochondrial preparations by Triton X-100. The enzyme was purified by (NH4)2SO4 fractionation followed by chromatography on DEAE-cellulose, Sepharose 6B, spheroidal hydroxyapatite, and finally chromatography on diazo-coupled tyramine-Sepharose. 3. Distinct differences occur in the chromatographic behaviour of the two enzymes on both DEAE-cellulose and spheroidal hydroxyapatite. 4. It is unlikely that the purification of the enzymes on tyramine-Sepharose is due to affinity chromatography and reasons for this are discussed. 5. The purified enzymes did not oxidize-5-hydroxytryptamine and the relative activities of the enzymes with benzylamine were increased approx. 1.25-fold compared with the enzyme activities of mitochondrial preparations. 6. Immunotitration of enzyme activity in extracts of mitochondrial preparations from rat liver was carried out with 5-hydroxytryptamine, tyramine and benzylamine. The enzyme activities were completely immunoprecipitated by the same volume of antiserum. Similar results were obtained with the antiserum to the enzyme from human liver.     

Pigment-free NADPH:protochlorophyllide oxidoreductase from Avena sativa L. Purification and substrate specificity.

The enzyme NADPH:protochlorophyllide oxidoreductase (POR) is the key enzyme for light-dependent chlorophyll biosynthesis. It accumulates in dark-grown plants as the ternary enzyme-substrate complex POR-protochlorophyllide a-NADPH. Here, we describe a simple procedure for purification of pigment-free POR from etioplasts of Avena sativa seedlings. The procedure implies differential solubilization with n-octyl-beta-D-glucoside and one chromatographic step with DEAE-cellulose. We show, using pigment and protein analysis, that etioplasts contain a one-to-one complex of POR and protochlorophyllide a. The preparation of 13 analogues of protochlorophyllide a is described. The analogues differ in the side chains of the macrocycle and in part contain zinc instead of the central magnesium. Six analogues with different side chains at rings A or B are active substrates, seven analogues with different side chains at rings D or E are not accepted as substrates by POR. The kinetics of the light-dependent reaction reveals three groups of substrate analogues with a fast, medium and slow reaction. To evaluate the kinetic data, the molar extinction coefficients in the reaction buffer had to be determined. At concentrations above 2 mole substrate/mole enzyme, inhibition was found for protochlorophyllide a and for the analogues.     

NADH oxidase from the extreme thermophile Thermus aquaticus YT-1. Purification and characterisation

A protein with NADH oxidase activity from the extreme thermophile Thermus aquaticus YT-1 was purified and characterised. The enzyme was found to have a relative molecular mass of 110,000 and be composed of two subunits of identical size. FAD was found to be present at a concentration of 0.7 mol/mol dimer and was required for activity. During the oxidation of NADH, oxygen uptake takes place with the production of hydrogen peroxide. The enzyme had, with the exception of a higher glutamic acid and tryptophan content, a similar amino acid composition as the NADH oxidase isolated from the mesophile Bacillus megaterium. Purified NADH oxidase was found to have a Km of 39 microM for beta-NADH and a Vmax of 4.68 mumol NADH mg-1 min-1 and was still active at 95 degrees C. Enzymatic activity was found to be independent of pH between 5.0 and 10.5.     

Purification of rat liver monoamine oxidase by octyl glucoside extraction and reconstitution

Catalytically active isoenzymes of rat liver monoamine oxidase have been copurified from the outer mitochondrial membrane by a novel method involving repetitive solubilization with octyl-β-d-glucopyranoside followed by reconstitution into lipid vesicles. As analyzed using sodium dodecyl sulfate-gel electrophoresis, the purified enzyme migrates as a single band of protein of molecular weight 60,000. The preparation is capable of metabolizing 576 nmol serotonin and 777 nmol β-phenylethylamine/min/mg protein. Apparent K_m values and sensitivity to the inhibitor clorgyline are very similar for the purified and outer mitochondrial membrane-bound enzyme when determined with the substrates β-phenylethylamine, serotonin, and tyramine.     

The development and ageing of membranes from etioplasts of Avena sativa

A growth period from day 4 to day 11 after sowing of dark-grown oat seedlings ( Avena sativa L. cv. Flämingskrone) was studied. Etioplasts were prepared from various stages. By means of one and two dimensional gel eletrophoresis developmental changes in the polypeptide pattern of the prolamellar body-prothylakoid complex of etioplasts were studied. Polypeptides were described by their molecular weights and pI values after isoelectric focusing. The polypeptide map changes continuously with age, with the onset of effects of senescence around day 7. Concomitantly, proto-chlorophyllide and proteins are degraded, with a higher degradation rate for proto-chlorophyllide. An analysis of protochlorophyllide fluorescence displayed a constant ratio (fluorescence emission at 650 nm/fluorescence emission at 630 nm) from day 6 to day 11. It is concluded, that the best stages for greening studies of dark-grown oat seedlings occur between day 4 and day 6 after sowing.     

A cell-free xylan synthesizing enzyme system from Avena sativa

A cell-free paniculate preparation from oat seedlings showedxylan synthetase activity, producing xylan from uridine diphosphatexylose. With high substrate concentration or prolonged incubation,three polysaccharides other than neutral xylan were synthesized.It is suggested that one of these might be a glycolipid andthe other two might be glucuronoxylans. ¹Present address: Department of Fruit and Vegetable Storage,The Agricultural Research Organization, The Volcani Center,P.O.B. 6, Bet Dagan, Israel. (Received December 5, 1972; )     

Respiration of Mitochondria Isolated from Leaves and Protoplasts of Avena sativa

Mitochondria isolated from mesophyll protoplasts differed from mitochondria isolated directly from leaves of Avena sativa in that protoplast mitochondria (a) had a lower overall respiratory capacity, (b) were less able to use low concentrations of exogenous NADH, (c) did not respond rapidly or strongly to added NAD, (d) appeared to accumulate more oxaloacetate, and (e) oxidized both succinate and tetramethyl-p-phenylene-diamine (an electron donor for cytochrome oxidase) more slowly than did leaf mitochondria. It is concluded that cytochrome oxidase activity was inhibited, the external NADH dehydrogenase had a reduced affinity for NADH, succinate oxidation was inhibited, NAD and oxaloacetate porters were probably inhibited, and accessibility to respiratory paths may have been reduced in protoplast mitochondria. The results also suggest that there was a reduced affinity of a succinate porter for this substrate in oat mitochondria. In addition, all oat mitochondria required salicylhydroxamic acid (SHAM) as well as cyanide to block malate and succinate oxidation. Malate oxidation that did not appear to saturate the cytochrome pathway was sensitive to SHAM in the absence of cyanide, suggesting that the oat mitochondria studied had concomitant alternative and subsaturating cytochrome oxidase pathway activity.     

Metabolism of diclofop-methyl in cell cultures of Avena sativa and Avena fatua

The metabolism of the herbicide, diclofop-methyl (methyl-2-[4-(2', 4'-dichlorophenoxy) phenoxy]propanoate), in cell suspension cultures of Avena sativa L. (cv. Garry) and in callus of Avena fatua L. (transferred to liquid) was determined as a function of time (8 h to about 3 weeks) and was compared to previous metabolism data from intact plants. A. fatua metabolized ¹⁴C-labeled diclofop-methyl more rapidly than A. sativa, but the metabolites formed were similar if not identical. Within 2 days, approximately 50% of the total ¹⁴C recovered was in A. fatua cells whereas less than 15% was in A. sativa cells. In older cultures of A. fatua, the amounts of ¹⁴C in the cells and in the medium were about 45% each; 10 to 12% was in the non-extractable cell residue. The ¹⁴C recovered from A. sativa cells increased to a maximum of about 35% at 7 days and then slowly decreased to about 18% by 21 days, whereas the ¹⁴C in the medium of A. sativa decreased to about 60% at 7 days and then increased to over 75% by 21 days. The nonextractable ¹⁴C residue was 5% or less even after 21 days. Major metabolites in methanolic extracts of cells of both A. sativa and A. fatua were diclofop (2-[4-(2', 4'-dichlorophenoxy)phenoxy] propanoate), diclofop hydroxylated at an undetermined position on the 2,4-dichlorophenyl ring (ring OH-diclofop), and conjugates of diclofop and ring-OH diclofop.