Characterization of an ammonium transport protein from the peribacteroid membrane of soybean nodules

Science

Volumn 281, Issue 5380, 1998, Pages 1202-1206

  Kaiser, Brent N ^a   Finnegan, Patrick M ^a   Tyerman, Stephen D ^a   Whitehead, Lynne F ^a   Bergersen, Fraser J ^a   Day, David A ^a   Udvardi, Michael K ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

AMMONIA; CARBON 14; CARRIER PROTEIN; COMPLEMENTARY DNA; METHYLAMMONIUM; NITROGEN;

ARTICLE; BACTERIAL MEMBRANE; CONTROLLED STUDY; MEMBRANE CHANNEL; MEMBRANE TRANSPORT; NITROGEN FIXATION; NONHUMAN; NUCLEOTIDE SEQUENCE; PATCH CLAMP; PRIORITY JOURNAL; RHIZOBIUM; SOYBEAN; SYMBIOSIS; YEAST CELL;

BACTERIA (MICROORGANISMS); GLYCINE MAX; NEGIBACTERIA; RHIZOBIUM;

EID: 0032555513     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.281.5380.1202     Document Type: Article

References (35)

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16. + nylon membrane (Amersham), and hybridized with full-length digoxigenin-labeled (Boehringer Mannheim) GmSAT1 antisense RNA. Membranes were washed at high stringency [68°C for 30 min in 0.1 X SSC (70) containing 1% (w/v) SDS] before autoradiography.
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18. 6 (Qiagen) demonstrated that soluble E. coli DH10B cell fractions from pBK1HIS1 or pBK1HIS2 transformed cells contained IPTG-inducible immunoreactive proteins of 42 and 26 kD, respectively (results not shown). Approximately 3 kD of each of these fusion proteins is encoded by the pTrcHisB vector.
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20. 2, 1 mM PMSF] and the soluble (supernatant) fraction from nodules was concentrated by ammonium acetate-methanol precipitation. PBM was obtained from symbiosomes isolated from the nodules of 3.5-week-old soybean plants [D. A. Day and M. K. Udvardi. Aust. J. Plant. Physiol. 16, 69 (1989)]. PBM purity was verified routinely by microscopy and enzymatic assays (NADH-oxidase as a mitochondrial marker, NADPH-cytochrome c reductase as a plasma membrane marker, carotene as a plastid membrane marker) as described [L-J. OuYang, M. K. Udvardi, D. A. Day, Planta 182, 437 (1990)]. PBM proteins were extracted from the membrane with phenol [W. J. Hurkman and C. K. Tanaka, Plant Physiol. 81, 802 (1986)]. Proteins were separated by SDS-polyacrylamide gel electrophoresis [U. K. Laemmli, Nature 227, 680 (1970)] and transferred to Hybond C-extra nitrocellulose membranes (Amersham) with a semidry blotter (Millipore). Immunoblots were probed with antiserum to GmSAT1 at a dilution of 1:5000. Immunoreactive proteins were visualized by chemiluminescence with a commercially available kit (Boehringer Mannheim).
21. 2, 1 mM PMSF] and the soluble (supernatant) fraction from nodules was concentrated by ammonium acetate-methanol precipitation. PBM was obtained from symbiosomes isolated from the nodules of 3.5-week-old soybean plants [D. A. Day and M. K. Udvardi. Aust. J. Plant. Physiol. 16, 69 (1989)]. PBM purity was verified routinely by microscopy and enzymatic assays (NADH-oxidase as a mitochondrial marker, NADPH-cytochrome c reductase as a plasma membrane marker, carotene as a plastid membrane marker) as described [L-J. OuYang, M. K. Udvardi, D. A. Day, Planta 182, 437 (1990)]. PBM proteins were extracted from the membrane with phenol [W. J. Hurkman and C. K. Tanaka, Plant Physiol. 81, 802 (1986)]. Proteins were separated by SDS-polyacrylamide gel electrophoresis [U. K. Laemmli, Nature 227, 680 (1970)] and transferred to Hybond C-extra nitrocellulose membranes (Amersham) with a semidry blotter (Millipore). Immunoblots were probed with antiserum to GmSAT1 at a dilution of 1:5000. Immunoreactive proteins were visualized by chemiluminescence with a commercially available kit (Boehringer Mannheim).
22. 2, 1 mM PMSF] and the soluble (supernatant) fraction from nodules was concentrated by ammonium acetate-methanol precipitation. PBM was obtained from symbiosomes isolated from the nodules of 3.5-week-old soybean plants [D. A. Day and M. K. Udvardi. Aust. J. Plant. Physiol. 16, 69 (1989)]. PBM purity was verified routinely by microscopy and enzymatic assays (NADH-oxidase as a mitochondrial marker, NADPH-cytochrome c reductase as a plasma membrane marker, carotene as a plastid membrane marker) as described [L-J. OuYang, M. K. Udvardi, D. A. Day, Planta 182, 437 (1990)]. PBM proteins were extracted from the membrane with phenol [W. J. Hurkman and C. K. Tanaka, Plant Physiol. 81, 802 (1986)]. Proteins were separated by SDS-polyacrylamide gel electrophoresis [U. K. Laemmli, Nature 227, 680 (1970)] and transferred to Hybond C-extra nitrocellulose membranes (Amersham) with a semidry blotter (Millipore). Immunoblots were probed with antiserum to GmSAT1 at a dilution of 1:5000. Immunoreactive proteins were visualized by chemiluminescence with a commercially available kit (Boehringer Mannheim).
23. 2, 1 mM PMSF] and the soluble (supernatant) fraction from nodules was concentrated by ammonium acetate-methanol precipitation. PBM was obtained from symbiosomes isolated from the nodules of 3.5-week-old soybean plants [D. A. Day and M. K. Udvardi. Aust. J. Plant. Physiol. 16, 69 (1989)]. PBM purity was verified routinely by microscopy and enzymatic assays (NADH-oxidase as a mitochondrial marker, NADPH-cytochrome c reductase as a plasma membrane marker, carotene as a plastid membrane marker) as described [L-J. OuYang, M. K. Udvardi, D. A. Day, Planta 182, 437 (1990)]. PBM proteins were extracted from the membrane with phenol [W. J. Hurkman and C. K. Tanaka, Plant Physiol. 81, 802 (1986)]. Proteins were separated by SDS-polyacrylamide gel electrophoresis [U. K. Laemmli, Nature 227, 680 (1970)] and transferred to Hybond C-extra nitrocellulose membranes (Amersham) with a semidry blotter (Millipore). Immunoblots were probed with antiserum to GmSAT1 at a dilution of 1:5000. Immunoreactive proteins were visualized by chemiluminescence with a commercially available kit (Boehringer Mannheim).
24. 2, 1 mM PMSF] and the soluble (supernatant) fraction from nodules was concentrated by ammonium acetate-methanol precipitation. PBM was obtained from symbiosomes isolated from the nodules of 3.5-week-old soybean plants [D. A. Day and M. K. Udvardi. Aust. J. Plant. Physiol. 16, 69 (1989)]. PBM purity was verified routinely by microscopy and enzymatic assays (NADH-oxidase as a mitochondrial marker, NADPH-cytochrome c reductase as a plasma membrane marker, carotene as a plastid membrane marker) as described [L-J. OuYang, M. K. Udvardi, D. A. Day, Planta 182, 437 (1990)]. PBM proteins were extracted from the membrane with phenol [W. J. Hurkman and C. K. Tanaka, Plant Physiol. 81, 802 (1986)]. Proteins were separated by SDS-polyacrylamide gel electrophoresis [U. K. Laemmli, Nature 227, 680 (1970)] and transferred to Hybond C-extra nitrocellulose membranes (Amersham) with a semidry blotter (Millipore). Immunoblots were probed with antiserum to GmSAT1 at a dilution of 1:5000. Immunoreactive proteins were visualized by chemiluminescence with a commercially available kit (Boehringer Mannheim).
25. 2, 1 mM PMSF] and the soluble (supernatant) fraction from nodules was concentrated by ammonium acetate-methanol precipitation. PBM was obtained from symbiosomes isolated from the nodules of 3.5-week-old soybean plants [D. A. Day and M. K. Udvardi. Aust. J. Plant. Physiol. 16, 69 (1989)]. PBM purity was verified routinely by microscopy and enzymatic assays (NADH-oxidase as a mitochondrial marker, NADPH-cytochrome c reductase as a plasma membrane marker, carotene as a plastid membrane marker) as described [L-J. OuYang, M. K. Udvardi, D. A. Day, Planta 182, 437 (1990)]. PBM proteins were extracted from the membrane with phenol [W. J. Hurkman and C. K. Tanaka, Plant Physiol. 81, 802 (1986)]. Proteins were separated by SDS-polyacrylamide gel electrophoresis [U. K. Laemmli, Nature 227, 680 (1970)] and transferred to Hybond C-extra nitrocellulose membranes (Amersham) with a semidry blotter (Millipore). Immunoblots were probed with antiserum to GmSAT1 at a dilution of 1:5000. Immunoreactive proteins were visualized by chemiluminescence with a commercially available kit (Boehringer Mannheim).
26. 2, 1 mM PMSF] and the soluble (supernatant) fraction from nodules was concentrated by ammonium acetate-methanol precipitation. PBM was obtained from symbiosomes isolated from the nodules of 3.5-week-old soybean plants [D. A. Day and M. K. Udvardi. Aust. J. Plant. Physiol. 16, 69 (1989)]. PBM purity was verified routinely by microscopy and enzymatic assays (NADH-oxidase as a mitochondrial marker, NADPH-cytochrome c reductase as a plasma membrane marker, carotene as a plastid membrane marker) as described [L-J. OuYang, M. K. Udvardi, D. A. Day, Planta 182, 437 (1990)]. PBM proteins were extracted from the membrane with phenol [W. J. Hurkman and C. K. Tanaka, Plant Physiol. 81, 802 (1986)]. Proteins were separated by SDS-polyacrylamide gel electrophoresis [U. K. Laemmli, Nature 227, 680 (1970)] and transferred to Hybond C-extra nitrocellulose membranes (Amersham) with a semidry blotter (Millipore). Immunoblots were probed with antiserum to GmSAT1 at a dilution of 1:5000. Immunoreactive proteins were visualized by chemiluminescence with a commercially available kit (Boehringer Mannheim).
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28. -1 (Sigma) instead of a mixture of zymolase and glucoronidase.
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35. 15N analysis; S. Howitt for her assistance with sequence analysis; M. Ludwig for helpful discussions; and R. Thomson for assistance with the membrane preparations. Supported by Australian Research Council grants to D.A.D., S.D.T., and M.K.U. and an Australian National University postgraduate scholarship from the Cooperative Research Centre for Plant Science to B.N.K.