Apple beta-galactosidase. Activity against cell wall polysaccharides and characterization of a related cDNA clone
| Autor: | G S Ross, Teresa F. Wegrzyn, Elspeth A. MacRae, Robert J. Redgwell |
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| Rok vydání: | 1994 |
| Předmět: |
Malus
DNA Complementary Physiology Molecular Sequence Data Gene Expression Plant Science Chromatography Affinity Substrate Specificity Affinity chromatography Cell Wall Polysaccharides Complementary DNA Vegetables Genetics Amino Acid Sequence Cloning Molecular Peptide sequence chemistry.chemical_classification Gel electrophoresis biology Base Sequence Sequence Homology Amino Acid food and beverages Plants biology.organism_classification beta-Galactosidase Molecular biology Enzyme assay Amino acid Molecular Weight Kinetics Biochemistry chemistry Fruit biology.protein Electrophoresis Polyacrylamide Gel Beta-galactosidase activity Research Article |
| Zdroj: | Plant physiology. 106(2) |
| ISSN: | 0032-0889 |
| Popis: | A [beta]-galactosidase was purified from cortical tissue of ripe apples (Malus domestica Borkh. cv Granny Smith) using a procedure involving affinity chromatography on lactosyl-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that two polypeptides of 44 and 32 kD were present in the fraction that showed activity against the synthetic substrate p-nitrophenol-[beta]-D-galactopyranoside. The enzyme preparation was incubated with polysaccharide extracts from apple cell walls containing [beta]-(1–>4)-linked galactans, and products of digestion were analyzed by gas chromatography. Small amounts of monomeric galactose were released during incubation, showing that the enzyme was active against native substrates. Amino acid sequence information was obtained from the purified protein, and this showed high homology with the anticipated polypeptide coded by the ethylene-regulated SR12 gene in carnation (K.G. Raghothama, K.A. Lawton, P.B. Goldborough, W.R. Woodson [1991] Plant Mol Biol 17: 61–71) and a harvest-related pTIP31 cDNA from asparagus (G. King, personal communication). Using the asparagus cDNA clone as a probe, an apple homolog (pABG1) was isolated. This clone contains a 2637-bp insert, including an open reading frame that codes for a polypeptide of 731 amino acids. Cleavage of an N-terminal signal sequence would leave a predicted polypeptide of 78.5 kD. Genomic DNA analysis and the isolation of other homologous apple clones suggest that pABG1 represents one member of an apple [beta]-galactosidase gene family. Northern analysis during fruit development and ripening showed accumulation of pABG1-homologous RNA during fruit ripening. Enzyme activity as measured in crude extracts increased during fruit development to a level that was maintained during ripening. |
| Databáze: | OpenAIRE |
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