Isolation of pl 4.6 extensin peroxidase from tomato cell suspension cultures and identification of Val—Tyr—Lys as putative intermolecular cross‐link site

Extensins and kindred hydroxyproline‐rich glycoproteins occur in dicot cell walls mainly as insoluble integral components that may form an intermolecularly cross‐linked network interpenetrated by other polymers. Extensins also occur in muro as a small pool of soluble monomeric precursors to network...

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Published in:The Plant journal : for cell and molecular biology 1996-04, Vol.9 (4), p.477-489
Main Authors: Schnabelrauch, Linda S., Kieliszewski, Marcia, Upham, Brad L., Alizedeh, Hasan, Lamport, Derek. T.A.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Analytical, structural and metabolic biochemistry
Biological and medical sciences
Cells, Cultured
Enzyme Stability
Enzymes
Enzymes and enzyme inhibitors
Fundamental and applied biological sciences. Psychology
Glycoproteins - chemistry
Glycoproteins - metabolism
Hydrogen-Ion Concentration
Isoelectric Point
Isoenzymes - isolation & purification
Isoenzymes - metabolism
Kinetics
Lycopersicon esculentum - enzymology
Metabolism
Molecular Sequence Data
Molecular Weight
Oxidoreductases
Peroxidases - isolation & purification
Peroxidases - metabolism
Plant physiology and development
Plant Proteins - chemistry
Plant Proteins - metabolism
Protein Conformation
Spectrophotometry
Substrate Specificity
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Summary:Extensins and kindred hydroxyproline‐rich glycoproteins occur in dicot cell walls mainly as insoluble integral components that may form an intermolecularly cross‐linked network interpenetrated by other polymers. Extensins also occur in muro as a small pool of soluble monomeric precursors to network extensin. These precursors were prepared in milligram quantities by salt elution from the surface of intact cells grown as tomato suspension cultures. Based on an FPLC (Superose‐6) gel filtration assay of cross‐linked extensin oligomers, a pl 4.6 extensin cross‐linking peroxidase isozyme was partially purified from the culture growth medium. Purification involved: volume reduction, ultracentrifugation to remove pectin and co‐adsorbed cationic peroxidase, followed by chromatography of anionic extensin peroxidase (pl 4.6) on DEAE—Trisacryl and TSK‐gel DEAE‐5PW columns. With tomato P1 extensin as substrate and 60 µM H2O2 as co‐substrate, at 23° pl 4.6 extensin peroxidase gave a K,m of 0.22 mM P1 and a Vmax of 70 µmol P1 cross‐linked min−1 mg−1 enzyme, at the optimum pH 5.5. Assayed with 12 different extensins from representative monocots, dicots, and gymnosperms, the pl 4.6 isozyme cross‐linked highly selectively, indicating two natural groups: cross‐linking or CL‐extensins and non‐cross‐linking or NCL‐extensins. CL‐extensins contained the X—Hyp—Val—Tyr—Lys motif and were also highly glycosylated. However, the simplest motif common to CL‐extensins but absent from NCL‐extensins was Val—Tyr—Lys. Thus, peroxidative coupling of extensin monomers and resistance of the resultant oligomers to depolymerization by anhydrous HF suggests that the intermolecular cross‐link involves tyrosine or lysine.
ISSN:0960-7412
1365-313X
DOI:10.1046/j.1365-313X.1996.09040477.x