Peptide Mapping Reveals Considerable Sequence Homology among the Three Polypeptide Subunits of G1 Storage Protein from French Bean Seed

Evidence is presented that French-bean (Phaseolus vulgaris) seed ferritin is composed of one type of subunit with an apparent Mr of 26500. In normal and iron-loaded leaf tissues it is detected immunologically with an antiserum raised against purified bean seed ferritin and migrates in SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis with the same mobility as the bean seed ferritin subunit. The biosynthetic pathway of ferritin in normal and iron-loaded leaves was investigated. RNA was extracted, fractionated into polyadenylated RNA and translated in a cell-free rabbit reticulocyte lysate and a wheat-germ-extract system. The products were identified by SDS/polyacrylamide-gel electrophoresis after indirect immunoprecipitation. In all cases the ferritin product had an Mr 5000 higher than that of the native subunit. Uptake and processing of the precursor form of ferritin from iron-loaded leaves by intact chloroplasts was demonstrated. This indicates that, in iron-loaded leaves, ferritin acts as a chloroplast protein. We propose that the ferritin precursor in normal leaves follows the same biosynthetic pathway. This suggests that the iron-buffering function of ferritin in plants takes place in the chloroplast and that non-functional cellular iron will accumulate in this cell organelle.

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Inducible UDP-glucose dehydrogenase from French bean (Phaseolus vulgaris L.) locates to vascular tissue and has alcohol dehydrogenase activity

Biochemical Journal ◽

10.1042/bj3130311 ◽

1996 ◽

Vol 313 (1) ◽

pp. 311-317 ◽

Cited By ~ 25

Author(s):

Duncan ROBERTSON ◽

Colin SMITH ◽

G. Paul BOLWELL

Keyword(s):

Alcohol Dehydrogenase ◽

Dehydrogenase Activity ◽

Vascular Tissue ◽

Cultured Cells ◽

Peptide Mapping ◽

Glucose Dehydrogenase ◽

Gel Filtration ◽

French Bean ◽

Suspension Cultured Cells ◽

Alcohol Dehydrogenase Activity

UDP-glucose dehydrogenase is responsible for channelling UDP-glucose into the pool of UDP-sugars utilized in the synthesis of wall matrix polysaccharides and glycoproteins. It has been purified to homogeneity from suspension-cultured cells of French bean by a combination of hydrophobic-interaction chromatography, gel filtration and dye-ligand chromatography. The enzyme had a subunit of Mr 40000. Km values were measured for UDP-glucose as 5.5±1.4 mM and for NAD+ as 20±3 μM. It was subject to inhibition by UDP-xylose. UDP-glucose dehydrogenase activity co-purified with alcohol dehydrogenase activity from suspension-cultured cells, elicitor-treated cells and elongating hypocotyls, even when many additional chromatographic steps were employed subsequently. The protein from each source was resolved into virtually identical patterns of isoforms on two-dimensional isoelectric focusing/PAGE. However, a combination of peptide mapping and sequence analysis, gel analysis using activity staining and kinetic analysis suggests that both activities are a function of the same protein. An antibody was raised and used to immunolocalize UDP-glucose dehydrogenase to developing xylem and phloem of French bean hypocotyl. Together with data published previously, these results are consistent with an important role in the regulation of carbon flux into wall matrix polysaccharides.

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