Multiple forms of β-galactosidase from the germinating seeds of Vigna radiata
TL;DR: Four forms of β-galactosidase were isolated and partially purified from the cotyledons of germinating seeds of Vigna radiata by ammonium sulphate fractionation and ion exchange chromatography through DEAE-cellulose and CM- cellulose columns, showing similar pH optima and temperature optima but differed from each other in ionic charge, kinetic parameters, activation energy and sensitivity towards heat.
Abstract: Four forms of β-galactosidase were isolated and partially purified from the cotyledons of germinating seeds of Vigna radiata by ammonium sulphate fractionation and ion exchange chromatography through DEAE-cellulose and CM-cellulose columns. These multiple forms showed similar pH optima and temperature optima but differed from each other in M r , ionic charge, kinetic parameters, activation energy and sensitivity towards heat, metal ions and certain chemical agents. From their K m values, p -nitrophenyl β- d -galactoside appeared to be a better substrate than o -nitrophenyl β- d -galactoside for two of these enzyme forms, whereas, the converse was found for the other two forms.
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TL;DR: Results indicate that the β-galactosidase of this study is solely responsible for the removal of galactose from the cell wall during ripening, and its in-vivo activity must be much greater than that observed in- vitro.
Abstract: A β-galactosidase (EC 3.2.1.23) capable of degrading a number of fruit cell-wall polysaccharides in vitro, was isolated from ripening kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson cv. Hayward). The enzyme has a molecular weight of approximately 60 kDa by gel permeation and consists of several basic isoforms. Several polypeptides were enriched during purification, with 33-, 46- and 67-kDa bands being predominant after sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The optimum activity of the enzyme against p-nitrophenyl-β-d-galactopyranoside was at pH 3.2, but against a galactan purified from kiwifruit cell walls, it was at pH 4.9. The enzyme was specific for galactosyl residues in the β-configuration, releasing galactose from a variety of kiwifruit cell-wall polysaccharide fractions including cell wall material, Na2CO3-soluble pectin, high-molecular-weight galactan, xyloglucan, and galactoglucomannan. A galactosylated glucuronomannan found throughout the kiwifruit plant was also a substrate for the enzyme. The results indicate that the enzyme attacks the non-reducing end of galactose side chains, cleaving single galactose residues which may be attached to the 2, 3, 4, or 6 position of the aglycone. Activity of the enzyme in-vitro was too low to account for the total loss of galactose from the cell walls during ripening. If the β-galactosidase of this study is solely responsible for the removal of galactose from the cell wall during ripening then its in-vivo activity must be much greater than that observed in-vitro.
128 citations
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...Others have also reported difficulty in maintaining active, purified 13-galactosidase in storage (Burns 1990; Dick et al. 1990; Kundu et al. 1990), and have observed the continued appearance of low-molecular-weight bands in SDS-PAGE of purified 13-galactosidase (Edwards et al....
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...Other authors have found differences in pH optima for various isoforms of 13-galactosidase against synthetic substrates (Pressey 1983; Burns 1990; Dick et al. 1990; Kundu et al. 1990)....
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TL;DR: The exo-galactanase is clearly a key enzyme in galactan mobilisation and may be the sole activity involved in depolymerising the dominant (1 → 4)-β-Galactan component of the cell wall.
Abstract: The main polysaccharide component of the thickened cell walls in the storage parenchyma of Lupinus angustifolius L. cotyledons is a linear (1 → 4)-β-linked d-galactan, which is mobilised after germination (L.A. Crawshaw and J.S.G Reid, 1984, Planta 160, 449–454). The isolation from the germinated cotyledons of a β-d-galactosidase or exo-(1 → 4)-β-d-galactanase with a high specificity for the lupin galactan is described. The enzyme, purified using diethylaminoethyl-cellulose, carboxymethyl-cellulose and affinity chromatography on lactose-agarose, gave two bands (major 60 kDa, minor 45 kDa) on sodium dodecyl sulphate-gel electrophoresis, and two similar bands on isoelectric focusing (major, pI 7.0, minor pI 6.7, both apparently possessing enzyme activity). The minor component cross-reacted with an antiserum raised against, and affinity-purified on, the major band. Both components had a common N-terminal sequence. The minor component was probably a degradation product of the major one. The enzyme had limited β-galactosidase action, catalysing the hydrolysis of p-nitrophenyl-β-d-galactopyranoside and (1→ 4)- and (1 → 6)-β-linked galactobioses. Lactose [β-d-galactopyranosyl-(1 → 4)-d-glucose] was hydrolysed only very slowly and methyl-β-d-galactopyranoside not at all. Lupin galactan was hydrolysed rapidly and extensively to galactose, whereas other cell-wall polysaccharides (xyloglucan and arabinogalactan) with terminal non-reducing β-d-galactopyranosyl residues were not substrates. A linear (1 → 4)-β-linked galactopentaose was hydrolysed efficiently to the tetraose plus galactose, but further sequential removals of galactose to give the tetraose and lower homologues occurred more slowly. Galactose, γ-galactonolactone and Cu+2 were inhibitory. No endo-β-d-galactanase activity was detected in lupin cotyledonary extracts, whereas exo-galactanase activity varied pari passu with galactan mobilisation. Exo-galactanase protein was detected, by Western immunoblotting of cotyledon extracts, just before the activity could be assayed and then increased and decreased in step with the enzyme activity. The exo-galactanase is clearly a key enzyme in galactan mobilisation and may be the sole activity involved in depolymerising the dominant (1 → 4)-β-galactan component of the cell wall.
83 citations
TL;DR: Double immunodiffusion analysis indicated thatbeta-galactosidases I, II, III and V are immunologically similar to each other, while beta-galactsosidase IV shares partially identical antigenic determinants with the other four isoforms.
Abstract: Five isoforms of beta-galactosidase (EC 3.2.1.23), designated as beta-galactosidases I-V, were isolated from five-day-old mung bean (Vigna radiata) seedlings. Beta-galactosidases II and III were purified to electrophoretic homogeneity by a procedure involving acid precipitation, ammonium sulfate fractionation, chromatography on diethylaminoethyl-cellulose (DEAE-Cellulose) and con A-Sepharose. and chromatofocusing. Beta-galactosidases I, II and III have the same molecular mass of 87 kDa. comprising two nonidentical subunits with molecular masses of 38 and 48 kDa, while beta-galactosidases IV and V have molecular masses of 45 and 73 kDa, respectively. All the enzymes were active against p-nitrophenyl-beta-D-galactoside, and to a lesser extent, p-nitrophenyl-alpha-L-arabinoside and p-nitrophenyl-beta-D-fucoside. The enzymes were inhibited by D-galactono-1,4-lactone, D-galactose, Hg2+, Ag+ and sodium dodecyl sulfate (SDS). Beta-galactosidases I, II and III were shown to be competitively inhibited by either D-galactono-1, 4-lactone or D-galactose. Isoforms I, II and III have a common optimal pH of 3.6, while isoforms IV and V have pH optima at 3.8 and 4.0, respectively. Isoelectric points of isoforms I, II and III were 7.7, 7.5 and 7.3, respectively. Double immunodiffusion analysis indicated that beta-galactosidases I, II, III and V are immunologically similar to each other, while beta-galactosidase IV shares partially identical antigenic determinants with the other four isoforms. The purified beta-galactosidases II and III were capable of releasing D-galactose residue from the hemicellulose fraction isolated from mung bean seeds.
70 citations
TL;DR: The β-galactosidase isolated from cotyledons of germinating seeds of Copaifera langsdorffii seems to perform a key role in xyloglucan degradation since it is responsible for the retrieval of a major sterical hindrance for further hydrolysis of the oligosaccharides and therefore essential for completion of xylglucan mobilisation.
Abstract: The storage xyloglucan of germinating seeds of Copaifera langsdorffii is degraded by the action of β-galactosidase, endo-β-glucanase, α-xylosidase and β-glucosidase, producing free galactose, glucose and xylose. One of the β-galactosidases from cotyledons of germinating seeds of C. langsdorffii was purified by ion exchange and gel chromatography (Biogel P-60), leading to a single polypeptide (molecular mass 40 kDa). The enzyme has optimum activity at pH 3.2 (stable from pH 2.3 to 6.0) and is active on p -NP-β-gal ( K m 3.5 mM) and lactose but not on o -NP-β-gal or p -NP-β-gal. Small amounts of galactose were released from xyloglucan of seeds of C. langsdorffii , Tamarindus indica and less from Hymenaea courbaril . No galactose was released after incubation with β-1,4-linked galactan from Lupinus angustifolius cotyledons. Much higher activity was observed on oligosaccharides obtained by hydrolysis of C. langsdorffii xyloglucan with Trichoderma viride cellulase. The purified β-galactosidase attacked XLLG and XLXG specifically, producing a mixture of XXXG and XXLG (unsubstituted glucose is assigned G ; glucose branched with xylose is assigned X and if galactose is branching xylose, the trisaccharide is assigned L ). Considering the recent discovery by Crombie and co-workers that (L) at the non-reducing end of the oligosaccharides prevents β-glucosidase from acting on G L XG or G L LG but not on GXLG or GXXG, the β-galactosidase isolated in this work seems to perform a key role in xyloglucan degradation since it is responsible for the retrieval of a major sterical hindrance (L) for further hydrolysis of the oligosaccharides and therefore essential for completion of xyloglucan mobilisation.
43 citations
TL;DR: β -Galactosidase activity in coffee berries showed a progressive increase of more than four-fold as the fruit developed from the immature to ripe stage, with a slight decrease in fully ripe fruit, suggesting that β-galactsidase plays a role in cell wall degradation such as occurs during fruit ripening.
Abstract: β-Galactosidase (EC 3.2.1.23) from ripe coffee berries was purified and characterized. The enzyme displayed activity against p -nitrophenyl-β- d -galactopyranoside (PNPG) ( K m 0.33 mM), lactose ( K m 40 mM), arabinogalactan and galactan. Purification was carried out using (NH 4 ) 2 SO 4 precipitation, gel-filtration using Bio-Gel P-2 and P-200, ion-exchange chromatography on Cellex-CM and affinity chromatography on p -aminophenyl-β- d -thiogalactopyranoside-agarose. Activity was highest within a pH range of 2.5–6, with an optimum at pH 4.4. The M r was estimated to be 2.9 × 10 4 by SDS-PAGE. The enzyme appeared to be a dimer of two identical subunits. It was inhibited by galactose (competitive, K i 0.26 mM); p -chloromercuribenzoate (PCMB) at 0.29 mM completely abolished activity. The enzyme catalysed release of galactose from galactan and arabinogalactan; pectin yielded galactose only when the action of β-galactosidase was combined with that of an endopolygalacturonase from Aspergillus niger. β -Galactosidase activity in coffee berries showed a progressive increase of more than four-fold as the fruit developed from the immature to ripe stage, with a slight decrease in fully ripe fruit. The same trend was observed with three other glycosidases, but not for N -acetyl glucosaminidase. It is suggested that β-galactosidase plays a role in cell wall degradation such as occurs during fruit ripening.
37 citations
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TL;DR: Procedures are described for measuring protein in solution or after precipitation with acids or other agents, and for the determination of as little as 0.2 gamma of protein.
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TL;DR: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products.
Abstract: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
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TL;DR: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products as mentioned in this paper.
Abstract: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
203,017 citations
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