Purification of several pectin methyltransferases from cell suspension cultures of flax ( Linum usitatissimum L.)

• Published in: Comptes rendus de l'Académie des sciences, Série III, Sciences de la vie Vol. 324; no. 4; pp. 335 - 343
• Main Authors: Bourlard, Thierry, Bruyant-Vannier, Marie-Pierre, Schaumann, Annick, Bruyant, Philippe, Morvan, Claudine
• Format: Journal Article
• Language: English
• Published: Paris Elsevier B.V 01.04.2001; Elsevier
• Subjects: Agronomy. Soil science and plant productions; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Economic plant physiology; Electrophoresis, Polyacrylamide Gel; Enzymes; Enzymes and enzyme inhibitors; Flax - enzymology; Fundamental and applied biological sciences. Psychology; Isoelectric Focusing; Metabolism; Microsomes - enzymology; Molecular Weight; Nutrition. Photosynthesis. Respiration. Metabolism; pectin methyltransferase; pectine méthyltransférase; Photoaffinity Labels; Plant physiology and development; Plant Viral Movement Proteins; S-adenosyl-homocysteine; S-adenosylmethionine; Solubility; Substrate Specificity; Transferases; Viral Proteins - chemistry; Viral Proteins - isolation & purification; Viral Proteins - metabolism; αSAH = S-adenosyl-L-homocysteine-sepharose 4B affinity gel; S200-HR = Sephacryl S200-HR gel; pectine méthyltransférase; S-adenosyl-homocysteine; DME = degree of methylesterification; HMP = highly methylesterified pectins; S-adenosylmethionine; CLPA = cross linked pectate gel; LMP = low metylesterified pectins; pectin methyltransferase; SEC = size exclusion chromotography; PMT = pectin methyltransferase; SAH = S-adenosyl-homocysteine; SAM = S-adenosyl-methionine; Cell culture; Purification; Enzyme; Isozyme; Transferases; Fiber crop; Linum usitatissimum; Linaceae; Enzymatic activity; Isoelectrical focusing; Methyltransferases; Dicotyledones; Angiospermae; Spermatophyta; Optimum pH
• ISSN: 0764-4469
• DOI: 10.1016/S0764-4469(01)01309-9

Three pectin methyltransferases (PMT5, PMT7, PMT18; EC 2.1.1.6.x) were solubilized from the endo-membrane complex of flax cells, with 0.05 % Triton X-100. After a 3 step-chromatography procedure, PMT7 and PMT5 were purified to apparent homogeneity. PMT5 and PMT7 differed regarding their optimum pH (5 or 7), the methyl acceptor (low or highly methylesterified pectin), their focusing pH range (6–7 or 8–9) and relative molecular mass (40 ± 5 or 110 ± 10 kDa). SDS-PAGE of PMT5 and PMT7 did not reveal bands at 40 or 110 kDa but only a silver stained band of about 18 kDa. Two independent methods (photo labelling and enzymatic activity) showed that this silver-stained band corresponded to a methyltransferase with affinity for pectins. This polypeptide was of the same size as the enzyme designed PMT18 (18 ± 3 kDa; pI 4–4.5) recovered during size exclusion chromatography of either PMT7 or PMT5, suggesting that PMT18 bears the catalytic site of PMT5 and PMT7. Trois pectine méthyltransferases (PMT5, PMT7, PMT18; EC 2.1.1.6.x) se distinguant par leur pH optimal (5 ou 7) et par le degré de méthylestérification du substrat pectique, ont été solubilisées à partir du système endomembranaire de cellules de lin avec une solution de Triton X-100 (0,05 %). Après purification, PMT5 et PMT7 diffèrent par leur point isoélectrique (6–7 ou 8–9) et leur masse moléculaire relative (40 ± 5 ou 110 ± 10 kDa). Une électrophorèse SDS-PAGE de PMT5 et PMT7 ne révèle aucune protéine à 40 ou 110 kDa mais uniquement une bande colorée à lˈargent autour de 18 kDa. Lˈintensité de la coloration apparaît proportionnelle à celle du photomarquage et celle de lˈactivité enzymatique de la PMT soumise à lˈélectrophorèse. La masse relative de ce polypeptide, révélé après dénaturation de PMT5 ou PMT7, est similaire à celle de lˈenzyme PMT18 (18 ± 3 kDa; pI 4–4,5), collectée après chromatographie dˈexclusion par taille de PMT7 ou PMT5. Nos résultats suggèrent que PMT18 est la sous-unité catalytique de PMT7 et PMT5.