Immunochemical analysis of multiple subunit polypeptides of glutamine synthetase in methionine sulfoximine-sensitive and tolerant tobacco cell cultures

Tomoyuki Yamaya, Hirohiko Ishida, Kazunari Kamachi, Kunihiko Ojima

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

A methionine sulfoximine (MSX) tolerant cell line of tobacco (Nicotiana tabacum L. cv. Xanthi) cells was selected by culturing the wild-type cells in suspension media in the presence of MSX at a step-wise increase in its concentration (0.3 to 5 μM). Fifty per cent inhibition of growth occurred at 0.18 μM MSX for the wild-type cells whereas 4.65 μM was required for the tolerant cells. The tolerant cells possessed about 1.5-fold increase in glutamine synthetase (GS) activity. Kinetic experiments showed that an inhibitor constant for MSX was identical between GS isolated from these two cell types. Subunit polypeptides of GS in both cell types were analyzed with an immunoblotting method by using polyclonal antibody raised against a chloroplastic GS in spinach. A single polypeptide (41 kDa) was recognized by the antibody in wild-type cells, whereas two predominant polypeptides of 41 and 40 kDa were seen in the MSX tolerant cells. When the GS subunit polypeptides in the wild-type cells were examined with two-dimensional gel electrophoresis, two major and four minor polypeptides associating distinct charge at 41 kDa were detected. The extract from the MSX-tolerant cells had the same set of polypeptides at 41 kDa and in addition two major and some minor spots at 40 kDa. These results indicate that 1) tobacco GS is consisted of heterogeneous subunit polypeptides in surface charge and 2) MSX causes formation of additional multiple 40 kDa polypeptides which may be related to the tolerant nature of the selected cell line.

Original languageEnglish
Pages (from-to)325-331
Number of pages7
JournalPlant and Cell Physiology
Volume31
Issue number3
Publication statusPublished - 1990 Dec 1

Keywords

  • Glutamine synthetase
  • Immunoblotting
  • Methionine sulfoximine
  • Stress selection
  • Tobacco cell

ASJC Scopus subject areas

  • Physiology
  • Plant Science
  • Cell Biology

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