Purification from barley

T. B. Frandsen, F. Lok, E. Mirgorodskaya, P. Roepstorff, and B. Svensson, Purification, enzymatic characterisation and nucleotide sequence of a high-isoelectric-point a-glucosidase from barley malt, Plant Physiol., 123 (2000) 275-286. 

McMorrow, E.M. Bradbeer, J.W. Separation, purification, and comparative properties of chloroplast and cytoplasmic phosphoglycerate kinase from barley leaves. Plant Physiol., 93, 374-383 (1990)... 

Kotake, T., Nakagawa, N., Takeda, K., and Sakurai, N., Purification and characterization of wall-bound exo-1,3-beta-D-glucanase from barley (Hordeum vulgare L.) seedlings. Plant Cell Physiol 1997, 38 (2), 194-200. 

SA in Table II - nmoles/hr/mg protein and N.D - not detectable. Tobacco, soy cultures and barley seedlings were the best source of ALS, both in terms of specific activity and total units. The enzyme preparations from all sources were unstable in buffer solutions in spite of protective thiol agents. The inactivation of ALS in the crude extract of tobacco showed a distinct biphasic kinetics, implying the presence of at least two isozymes (unpublished observations). The presence of two ALS genes in tobacco (29) and at least three in microorganisms (18) has also been noted by other workers. ALS from barley was most amenable to purification. Table III gives a profile for the rapid purification of this enzyme with high recovery. 

Limit Dextrins from Barley Starch. In one experiment, barley starch paste was treated with malt extract, in a second experiment with purified malt amylase (Table XVII). The purified enzyme has yielded limit dextrins with chain lengths greater than those given by the malt extract, possibly because certain carbohydrases capable of attacking the limit dextrins have been removed in the purification or because of a lower stability of the purified enzyme. 

Boisen, S., and Djurtoft, R. (1982). Protease inhibitor from barley embryo inhibiting trypsin and trypsin-like microbial proteases. Purification and characterisation of two isoforms. J. Sci. 

Partial separation of 6-ketoacyl-ACP synthetase, 6-ketoacyl-ACP reductase, acetyl CoA ACP transacylase and malonyl-CoAiACP transacylase was achieved from barley chloroplasts . From avocado fruit, Caughey and Kekwlck purified the B-ketoacyl-ACP reductase and malonyl-CoA. ACP acyltransferase to homogeneity and also purified the enoyl-ACP reductase. However, the most thorough study was that by Shlmakata and Stumpf mainly with spinach leaves. Purifications of acetyl CoArACP transacylase, B-ketoacyl-ACP synthetase B-ketoacyl-ACP synthetase II, B-ketoacyl-ACP reductase... 

In 1833 an amylase from germinating barley was recovered and called diastase (1). Like malt itself, this product converted gelatinized starch into sugars, primarily maltose. Shordy thereafter, BerzeHus proclaimed the existence of non-living catalysts, and Schwaim (2) reported on his observation and purification of pepsin. 

K. Higuchi, K. Kanazawa, N. Nishizawa, M. Chino, and S. Mori, Purification and characterization of nicotianamine synthase from Fe-deficient barley roots. Plant Soil 765 173 (1994). 

Examination of the ratios of the dextrinogenic to the saccharogenic activities of malted barley extracts before and after treatment shows that the results of the Ohlsson procedures23 are not always predictable.8 The concentration of the amylases in the extracts, and the kinds and concentrations of substances which accompany them, influence the results. The presence or the absence of calcium ions is an important factor. Calcium ions increase the inactivation of beta amylase of malted barley and protect the alpha amylase from inactivation at unfavorable temperatures and also at unfavorable hydrogen ion activities.28 With purification, both amylases become increasingly thermolabile and increasingly sensitive to unfavorable hydrogen ion activities.78... 

Lee, R. C., Burton, R. A., Hrmova, M., and Fincher, G. B., Barley arabinoxylan arabinofuranohydrolases purification, characterization and determination of primary structures from cDNA clones. Biochem 72001, 356 (Pt 1), 181-9. 

Additions of valine, leucine, and isoleucine were found to relieve the growth inhibitory effects of TP on Bacillus cell cultures, soybean cell cultures, and Arabidopsis seedlings. ALS isolated from a number of sources was found to be sensitive to TP at nM levels. The barley enzyme has been amenable to purification. A purification procedure that gives >60 % recovery and 235-fold purification is described. 

There have been only a few kinetic studies on plant /3-D-manno-sidases, because of the apparently limited distribution of this enzyme in various species, and also because of the paucity of attempts at purification. The enzyme from malted barley has been purified 41-fold by fractionation with ammonium sulfate, and chromatography302 on Biogel P-100, DEAE-cellulose, and CM-cellulose. Some properties of /3-D-mannosidases from various tissues are summarized in Table II. 

Figure 2. Radiochromatograms for -metabolites of WPh-6E0 eluted from RP-HPLC columns with linear gradients (25 min) left, water-soluble NH -treated fraction from excised barley leaves ]50-100% CH3OH at 2.0 mL/min ( )] right, purification of peak C (8 mm cartridge, 25-100% CH CN at 2.5 mL/min).

Hrmova M, Harvey AJ, Wang J, Shirley NJ, Jones GP, Stone BA, Hoj PB, Fincher GB (1996) Barley /3-D-Glucan Exohydrolase with /J-D-Glucosidase Activity. Purification, Characterization, and Determination of Primary Structure from a cDNA Clone. J Biol Chem 271 5277... 

The cultures of P. convolvulus were grown on moist barley grains at room temperature for a period of 28-30 days. Methanol extraction of the infected grains, followed by solvent partitioning of the metabolite mixture, led to the separation of the steroid compounds from metabolites 3, 4, 5 and 6 which dissolve readily in basic aqueous soivent. While the final purification of metabolites 1 and 2 was easily achieved by silica gel chromatography, the purification of the polar metabolites 3-6... 

Kolossov, V., and Rebeiz, C. (2001). Chloroplast Biogenesis 84 Solubilization and Partial Purification of Membrane-bound (4-vinyl) Chlotophyllide, a Reductase from Etiolated Barley Leaves, Anal. Biochem. 295 214-219. 

Acetamido-4-amino-2,4-dideoxy-D- glucose (40) Affinity chromatographic purification of a 8-D-2-acetamido-2-deoxy-glucosidase from malted barley 332 s... 

The final reaction in the biosynthesis of threonine involves a /8-y rearrangement and the loss of phosphate from O-phosphohomoserine (Fig. 2). Threonine synthases have been isolated from Lemna (Schnyder et al., 1975) radish, sugarbeet (Madison and Thompson, 1975), peas (Schnyder et al., 1975 Thoen et al., 1978b), and barley (Aames, 1978). None of these enzymes has been extensively characterized but a requirement for pyridoxyl-5 -phosphate was demonstrated after partial purification of the barley and pea enzymes. Unlike several other enzymes associated with threonine synthesis, the activity of threonine synthase was not stimulated by monovalent cations. However, all of the plant enzymes are strongly activated by 5-adeno-sylmethionine (Section III,B,5). 

Barley leaf PGK isoenzymes were separated by DEAE Sephacel chromatography followed by purification on ATP Sepharose (5). The yeast enzyme was obtained from baker s yeast and the main electrophoretic component was used (10). 

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