On the Occurrence and Structure of Subunits of Endopolygalacturonase Isoforms in Mature-Green and Ripening Tomato Fruits

Endopolygalacturonase [poly(1,4-α-galacturonide) glycanohydrolase EC 3.2.1.151 occurs in tomato fruit in three molecular forms- PG1, PG2A, PG2B. Trace amounts of PG1, 1-10 pkat g-1 are shown to occur in mature-green fruit as compared to 17 nkat in ripe fruit. As polygalacturonase activity increases through ripening, the percentage of the activity due to PG1 decreases progressively from 100 to less than 20. On fully or partly demethylated substrates, PG1 is more active than PG2 when the ionic strength is that expected in the tissue apoplast. A method for purifying PGI from ripe fruit is described. PG1 preparations contain polypeptides of Mr 45, 43 and 38 thousand. The Mr 43 thousand and 45 thousand components correspond in size to PG2A and PG2B and are detected by antisera raised against PG2A. The M, 38 thousand polypeptide is immunologically distinct. From carbohydrate and amino acid analyses, this polypeptide appears to contain 2870 carbohydrate as glucosamine, mannose, xylose and fucose attached to a polypeptide of estimated Mr 28 342 that is rich in tyrosine and glycine. A method for purifying the subunits of PG1 by cation exchange chromatography in 6 M urea is described. PG2A and PG2B were separated by column chromatography and shown to have identical N-terminal sequences, and serine at the C-terminus. PG2A and PG2B are confirmed as two glycoforms of the one polypeptide. The possibility that PGl consists of populations of molecules containing either PG2A or PG2B coupled with the Mr 38 thousand polypeptide is discussed.

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Manipulating fruit chloroplasts as a strategy to improve fruit quality

10.32747/2013.7598148.bard ◽

2013 ◽

Author(s):

Alan B. Bennett ◽

Arthur A. Schaffer ◽

Ilan Levin ◽

Marina Petreikov ◽

Adi Doron-Faigenboim

Keyword(s):

Chloroplast Development ◽

Tomato Fruit ◽

Soluble Sugar ◽

Fruit Size ◽

Soluble Solids ◽

Nucleotide Polymorphisms ◽

Ripe Fruit ◽

Green Fruit ◽

Functional Alleles ◽

Sugar Levels

The Original Objectives were modified and two were eliminated to reflect the experimental results: Objective 1 - Identify additional genetic variability in SlGLK2 and IPin wild, traditional and heirloom tomato varieties Objective 2 - Determine carbon balance and horticultural characteristics of isogenic lines expressing functional and non-functional alleles of GLKsand IP Background: The goal of the research was to understand the unique aspects of chloroplasts and photosynthesis in green fruit and the consequences of increasing the chloroplast capacity of green fruit for ripe fruit sugars, yield, flavor and nutrient qualities. By focusing on the regulation of chloroplast formation and development solely in fruit, our integrated knowledge of photosynthetic structures/organs could be broadened and the results of the work could impact the design of manipulations to optimize quality outputs for the agricultural fruit with enhanced sugars, nutrients and flavors. The project was based on the hypothesis that photosynthetic and non-photosynthetic plastid metabolism in green tomato fruit is controlled at a basal level by light for minimal energy requirements but fruit-specific genes regulate further development of robust chloroplasts in this organ. Our BARD project goals were to characterize and quantitate the photosynthesis and chloroplast derived products impacted by expression of a tomato Golden 2- like 2 transcription factor (US activities) in a diverse set of 31 heirloom tomato lines and examine the role of another potential regulator, the product of the Intense Pigment gene (IP activities). Using tomato Golden 2-like 2 and Intense Pigment, which was an undefined locus that leads to enhanced chloroplast development in green fruit, we sought to determine the benefits and costs of extensive chloroplast development in fruit prior to ripening. Major conclusions, solutions, achievements: Single nucleotide polymorphisms in the promoter, coding and intronicSlGLK2 sequences of 20 heirloom tomato lines were identified and three SlGLK2 promoter lineages were identified; two lineages also had striped fruit variants. Lines with striped fruit but no shoulders were not identified. Green fruit chlorophyll and ripe fruit soluble sugar levels were measured in 31 heirloom varieties and fruit size correlates with ripe fruit sugars but dark shoulders does not. A combination of fine mapping, recombinant generation, RNAseq expression and SNP calling all indicated that the proposed localization of a single locus IP on chr 10 was incorrect. Rather, the IP line harbored 11 separate introgressions from the S. chmielewskiparent, scattered throughout the genome. These introgressions harbored ~3% of the wild species genome and no recombinant consistently recovered the IP parental phenotype. The 11 introgressions were dissected into small combinations in segregating recombinant populations. Based on these analyses two QTL for Brix content were identified, accounting for the effect of increased Brix in the IP line. Scientific and agricultural implications: SlGLK2 sequence variation in heirloom tomato varieties has been identified and can be used to breed for differences in SlGLK2 expression and possibly in the green striped fruit phenotype. Two QTL for Brix content have been identified in the S. chmielewskiparental line and these can be used for increasing soluble solids contents in breeding programs.

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Pectinesterases From Mature, Unripe Peach Fruit Bind Strongly to Pectic Polysaccharides

Functional Plant Biology ◽

10.1071/pp9950977 ◽

1995 ◽

Vol 22 (6) ◽

pp. 977 ◽

Cited By ~ 3

Author(s):

H Glover ◽

CJ Brady

Keyword(s):

Enzyme Activity ◽

Strong Association ◽

Exchange Chromatography ◽

Tissue Type ◽

Ripe Fruit ◽

Peach Fruit ◽

Pectic Polysaccharides ◽

Green Fruit ◽

Large Molecular Weight

Contrary to previous findings, the level of the pectin de-esterifying enzyme, pectinesterase (PE; EC 3.1 .1. 11), is shown to be much higher in mature, green peach fruit than in ripe fruit. Aqueous buffers readily extracted three pectinesterase isoforms from ripe fruit but only a portion of the activity from mature, green fruit. In mature, green fruit extracts the enzyme precipitated when the ionic strength was lowered; consequently isoforms could not be recovered by ion exchange chromatography. In extraction residues from mature, green fruit, residual PE could be measured as active enzyme and, when denatured, could be detected by immunological techniques. Extraction of the enzyme was enhanced after digestion of the tissue with pectin lyase. The extracted enzyme fractionated as a large molecular weight complex rich in uronic acid, rhamnose, galactose and arabinose. After further digestion with endo-β-1,4- galactanase, the enzyme was in two fractions of smaller size but with residual carbohydrate. When mature, green and ripe fruit tissue were co-extracted, the recovered activity was as predicted from independently extracted tissues demonstrating that enzyme activity was not influenced by inhibitors contributed by either tissue type. Isoforms known to be present in the ripe fruit were recovered from extracts of the mixed tissues. It is concluded that PE in extracts of mature, green fruit has a strong association with pectic polymers and this has lead to its underestimation in previous studies. It is suggested that such an association with pectin polymers in vivo may regulate enzyme activity and enzyme turnover.

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Easy and Rapid Purification of Highly Active Nisin

International Journal of Peptides ◽

10.1155/2011/175145 ◽

2011 ◽

Vol 2011 ◽

pp. 1-9 ◽

Cited By ~ 23

Author(s):

André Abts ◽

Antonino Mavaro ◽

Jan Stindt ◽

Patrick J. Bakkes ◽

Sabine Metzger ◽

...

Keyword(s):

Low Ph ◽

Exchange Chromatography ◽

Single Step ◽

Cation Exchange Chromatography ◽

Gram Positive Bacteria ◽

Highly Active ◽

One Step ◽

Rapid Purification ◽

The One ◽

Purification Protocol

Nisin is an antimicrobial peptide produced and secreted by several L. lactis strains and is specifically active against Gram-positive bacteria. In previous studies, nisin was purified via cation exchange chromatography at low pH employing a single-step elution using 1 M NaCl. Here, we describe an optimized purification protocol using a five-step NaCl elution to remove contaminants. The obtained nisin is devoid of impurities and shows high bactericidal activity against the nisin-sensitive L. lactis strain NZ9000. Purified nisin exhibits an IC50 of ~3 nM, which is a tenfold improvement as compared to nisin obtained via the one-step elution procedure.

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Assessment of locally produced waxing materials on the shelf life and fruit quality of two tomato varieties (Solanum lycopersicum)

Journal of Energy and Natural Resource Management ◽

10.26796/jenrm.v4i1.73 ◽

2017 ◽

Vol 4 (1) ◽

pp. 36-47

Author(s):

R. Osae G. Essilfie J. O. Anim

Keyword(s):

Shelf Life ◽

Tomato Fruit ◽

Chemical Properties ◽

Storage Period ◽

Cassava Starch ◽

Soluble Solids ◽

Shea Butter ◽

Tomato Fruits ◽

Randomized Design ◽

Colour Development

The study was conducted to assess the effect of different waxing materials on the quality attributes of tomato fruits. A 2 x8 factorial experiment layout in complete randomized design with 16 treatment combinations and 3 replication was adopted.The materials that were used for the experiment are two (2) varieties of tomatoes (Pectomech and Power Rano) and seven(7) waxing material (shea butter, cassava starch, beeswax, and a combination of shea butter + cassava starch, shea butter + beeswax, cassava starch + beeswax, shea butter + cassava starch + beeswax) and a control. Results from the experiment indicated that all waxing treatments delayed the development of weight loss, firmness, pH, total soluble solids, and total titrable acidity. The results also suggested that edible wax coatings delayed the ripening process and colour development of tomato fruits during the storage period and extended the shelf life. However Beewax treatment and its combinations performed better than the other treatments. It was therefore recommended that locally produced wax such as Beewax, Shea butter, Cassava Starch treatments and their combinations could be a good technology for preserving the quality and extending the shelf life of fresh tomato fruit as well as maintaining the physical and chemical properties.

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Preparation and properties of three analogues of [5-leucine]enkephalin, substrates for enzyme conversion studies

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19851329 ◽

1985 ◽

Vol 50 (6) ◽

pp. 1329-1334

Author(s):

Jaroslav Vičar ◽

Linda Servítová ◽

Martin Flegel ◽

Karel Hauzer ◽

Tomislav Barth

Keyword(s):

High Pressure ◽

Liquid Chromatography ◽

Guinea Pig ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Guinea Pig Ileum ◽

Opioid Activity ◽

C Terminus ◽

N Terminus ◽

Fragment Condensation

Analogues of [5-Leu]enkephalin, prolonged by methionine on the N-terminus or, by lysine or methionine on the C-terminus were prepared by fragment condensation, purified by ion exchange chromatography or high-pressure liquid chromatography. The substances were characterised by their opioid activity in a test on guinea-pig ileum in comparison with the activity of [5-Leu]enkephalin.

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Evaluation of transgenic tomato fruit with reduced polygalacturonase activity in combination with the rin mutation

Postharvest Biology and Technology ◽

10.1016/0925-5214(94)00049-x ◽

1995 ◽

Vol 6 (1-2) ◽

pp. 91-101 ◽

Cited By ~ 5

Author(s):

A.J. Murray ◽

C.R. Bird ◽

W.W. Schuch ◽

G.E. Hobson

Keyword(s):

Tomato Fruit ◽

Transgenic Tomato ◽

Polygalacturonase Activity

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Chemical identity of tryptensin with angiotensin

Biochemical Journal ◽

10.1042/bj1870647 ◽

1980 ◽

Vol 187 (3) ◽

pp. 647-653 ◽

Cited By ~ 16

Author(s):

K Arakawa ◽

M Yuki ◽

M Ikeda

Keyword(s):

Amino Acid ◽

Thin Layer ◽

Gel Filtration ◽

Deae Cellulose ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Cation Exchange Chromatography ◽

Human Plasma Protein ◽

Plasma Protein Fraction ◽

Layer Chromatography

Tryptensin, a vasopressor substance generated from human plasma protein fraction IV-4 by trypsin, has been isolated and the amino acid composition analysed. The procedures used for the isolation were: (a) adsorption of the formed tryptensin on Dowex 50W (X2; NH4+ form); (b) gel filtration through Sephadex G-25; (c) cation-exchange chromatography on CM-cellulose; (d) anion-exchange chromatography on DEAE-cellulose; (e) re-chromatography on CM-cellulose; (f) gel filtration on Bio-Gel P-2; (g) partition chromatography on high-pressure liquid chromatography. The homogeneity of the isolated tryptensin was confirmed by thin-layer chromatography and thin-layer electrophoresis. The amino acid analysis of the hydrolysate suggested the following proportional composition: Asp, 1; Val, 1; Ile, 1; Tyr, 1; Phe, 1; His, 1; Arg, 1; Pro, 1. This composition is identical with that of human angiotensin.

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