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.
About: This article is published in Phytochemistry.The article was published on 1990-01-01. It has received 11 citations till now. The article focuses on the topics: Galactoside.
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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
Cites background from "Multiple forms of β-galactosidase f..."
...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.
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.
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.
37 citations
References
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TL;DR: In this paper, homogenates of guinea-pig polymorphonuclear leucocytes were separated by differential centrifugation into six particulate fractions and a soluble fraction.
Abstract: 1. Homogenates of guinea-pig polymorphonuclear leucocytes were separated by differential centrifugation into six particulate fractions and a soluble fraction. 2. The distributions in these fractions of protein, DNA, succinate dehydrogenase, beta-glucuronidase, peroxidase, alkaline phosphatase, acid phosphatase (against p-nitrophenyl phosphate and beta-glycerophosphate), cathepsin, and catalase were compared. 3. Almost all of the DNA sedimented in the first two pellets, indicating that the nuclei were relatively intact. 4. The four hydrolases and peroxidase showed different distribution patterns, although these activities were previously reported to be localized mainly in the single ;granule' fraction isolated from leucocytes. 5. The particles containing peroxidase, acid phosphatase and alkaline phosphatase all exhibited latency. Maximum activity for each enzyme was obtained at roughly similar concentrations of Triton X-100. 6. The acid phosphatase of these cells was distributed between two populations of particles that differed in both sedimentation characteristics and density. The acid phosphatase(s) of the two populations showed slightly different substrate specificities. This bimodal distribution was not an artifact of the procedure used to elicit the cells. 7. Catalase was recovered almost entirely in the soluble fraction and showed no latency in freshly prepared homogenates. No urate oxidase was detected. 8. We conclude that the ;granule' fraction of the polymorphonuclear leucocyte, as isolated by previous workers, contains at least three, probably more, populations of particles with different enzyme contents, and that these cells probably do not contain peroxisomes.
231 citations
TL;DR: The ability of β-galactosidase II to degrade the galactan and the increase in its activity during tomato ripening suggest a possible role for this enzyme in tomato softening.
Abstract: Tomatoes (Lycopersicon esculentum L.) contained a high level of beta-galactosidase activity which was due to three forms of the enzyme. During tomato ripening, the sum of their activities remained relatively constant, but the levels of the individual forms of beta-galactosidase changed markedly. The three enzymes were separated by a combination of chromatography of DEAE-Sephadex A-50 and Sephadex G-100. During ripening of tomatoes, beta-galactosidases I and III levels decreased but the beta-galactosidase II level increased more than 3-fold. The three enzymes were optimally active near pH 4, and all were inhibited by galactose and galactonolactone. However, the enzymes differed in molecular weight, K(m) value with p-nitrophenyl-beta-galactoside, and stability with respect to pH and temperature. beta-Galactosidase II was the only enzyme capable of hydrolyzing a polysaccharide that was isolated from tomatoes and that consisted primarily of beta-1, 4-linked galactose. The ability of beta-galactosidase II to degrade the galactan and the increase in its activity during tomato ripening suggest a possible role for this enzyme in tomato softening.
229 citations
01 Jan 1970
TL;DR: It is concluded that the ;granule' fraction of the polymorphonuclear leucocyte, as isolated by previous workers, contains at least three, probably more, populations of particles with different enzyme contents, and that these cells probably do not contain peroxisomes.
Abstract: cathepsin, andcatalase were compared. 3.AlmostalloftheDNA sedimented inthefirst twopellets, indicating thatthenuclei were relatively intact. 4.Thefourhydrolases andperoxidase showed different distribution patterns, although theseactivities were previously reported tobelocalized mainly inthesingle 'granule' fraction isolated fromleucocytes. 5.The particles containing peroxidase, acidphosphatase andalkaline phosphatase all exhibited latency. Maximumactivity foreachenzyme was obtained atroughly similar concentrations ofTriton X-100.6.Theacidphosphatase ofthese cellswas distributed between twopopulations ofparticles thatdiffered inbothsedimentation characteristics anddensity. Theacidphosphatase(s) ofthetwopopulations showed slightly different substrate specificities. Thisbimodaldistribution was notan artifact oftheprocedure usedtoelicit thecells.7.Catalase was recovered almost entirely inthesoluble fraction andshowed no latency infreshly prepared homogenates.No urateoxidase was detected. 8.We conclude thatthe'granule' fraction ofthepolymorphonuclear leucocyte, as isolated byprevious workers, contains atleast three, probably more, populations ofparticles withdifferent enzyme contents, andthatthese cells probably donotcontain peroxisomes.
212 citations
TL;DR: A β-galactosidase was purified 600-fold from bovine testes by ammonium sulfate precipitation, acetone fractionation, and affinity chromatography on agarose substituted with terminal thio-β-Galactopyranosyl residues, and exhibited a high affinity for nitrophenylGalactosides.
187 citations
TL;DR: Five enzymes have been purified from the germinating seeds of Phaseolus vulgaris and appear to be highly specific for the glycopyranosyl group and the anomeric configuration of the glycosidic linkage.
143 citations