title: A biotin-coupled bifunctional enzyme exhibiting both glutamine synthetase activity and glutamate decarboxylase activity.
abstract: OBJECTIVE
  To purify and study native form and enzymatic activity of the 42 kDa biotin-coupled protein (p42), which is related to glutamate action in chick retina.
  
  
  METHODS
  p42 was purified using molecular filtration in the presence of 0.7 M sodium chloride. Purity and identification of p42 were studied by SDS-PAGE, 2D-PAGE, LC-MS/MS, and MALDI-TOF MS. The native form of p42 was investigated using native-PAGE and Ferguson plot. Biotin-coupled property was examined by Western blot analysis. Enzymatic actions of p42 were studied using glutamate as substrate in the presence or absence of glutamine.
  
  
  RESULTS
  p42 was successfully purified from chick retinal protein solution using the molecular filtration. Western blot analysis with avidin showed that p42 was a biotin-coupled protein. Using SDS-PAGE, 2D-PAGE, LC-MS/MS, and MALDI-TOF MS, purified p42 was identified as a glutamine synthetase with four isoforms. Native-PAGE, followed by Ferguson plot analysis, showed two molecular forms of p42 corresponding to homotetramers and homooctamers. Enzymatic reaction followed by paper chromatography showed that p42 catalyzed the synthesis of glutamine from glutamate in the presence of ammonium ion, ATP, and magnesium ion. At prolonged reaction time, gamma-aminobutyric acid (GABA) was also formed. With glutamate and glutamine present at equal concentrations in the reaction mixture, GABA could be rapidly detected, but GABA could not be detected when glutamate concentration was more than four-fold that of glutamine. The results indicated that p42 also had glutamate decarboxylase activity. Both enzymatic activities were inhibited by avidin. High concentrations of Mn(2+) inhibited synthetase activity of p42 but not decarboxylase activity.
  
  
  CONCLUSIONS
  p42 was purified from chick retinal protein solution using molecular filtration in the presence of sodium chloride. The protein was a biotin-coupled bifunctional enzyme that contained glutamine synthetase activity and glutamate decarboxylase activity. Biotin was possibly involved in these activities. Mn(2+) showed different effects on the two activities.
authors.affiliation: Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Khomkrit
authors.initials: K
authors.lastname: Arunchaipong
authors.forename: Nison
authors.initials: N
authors.lastname: Sattayasai
authors.forename: Jintana
authors.initials: J
authors.lastname: Sattayasai
authors.forename: Jisnuson
authors.initials: J
authors.lastname: Svasti
authors.forename: Thipwarin
authors.initials: T
authors.lastname: Rimlumduan
mesh_terms: D000818:Animals; D001360:Avidin / Q000378:metabolism; D001710:Biotin / Q000378:metabolism*; D002645:Chickens; D005968:Glutamate Decarboxylase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D005973:Glutamine / Q000096:biosynthesis; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D055503:Protein Multimerization; D012160:Retina / Q000201:enzymology*; D013379:Substrate Specificity; D014462:Ultrafiltration / Q000379:methods; D005680:gamma-Aminobutyric Acid / Q000096:biosynthesis
chemical_list: D005973:Glutamine; D001360:Avidin; D018698:Glutamic Acid; D005680:gamma-Aminobutyric Acid; D001710:Biotin; D005968:Glutamate Decarboxylase; D005974:Glutamate-Ammonia Ligase; D008274:Magnesium