|
rdf:type |
|
|
lifeskim:mentions |
|
|
pubmed:issue |
15
|
|
pubmed:dateCreated |
1995-10-2
|
|
pubmed:abstractText |
We describe a convenient, simple and novel continuous spectrophotometric method for the determination of aminoacyl-tRNA synthetase activity. The assay relies upon the measurement of inorganic pyrophosphate generated in the first step of the aminoacylation of a tRNA. Pyrophosphate release is coupled to inorganic pyrophosphatase, to generate phosphate, which in turn is used as the substrate of purine nucleoside phosphorylase to catalyze the N-glycosidic cleavage of 2-amino 6-mercapto 7-methylpurine ribonucleoside. Of the reaction products, ribose 1-phosphate and 2-amino 6-mercapto 7-methylpurine, the latter has a high absorbance at 360 nm relative to the nucleoside and hence provides a spectrophotometric signal that can be continuously followed. The non-destructive nature of the spectrophotometric assay allowed the re-use of the tRNAs in question in successive experiments. The usefulness of this method was demonstrated for glutaminyl-tRNA synthetase (GlnRS) and tryptophanyl-tRNA synthetase. Initial velocities measured using this assay correlate closely with those assayed by quantitation of [3H]Gln-tRNA or [14C]Trp-tRNA formation respectively. In both cases amino acid transfer from the aminoacyl adenylate to the tRNA represents the rate determining step. In addition, aminoacyl adenylate formation by aspartyl-tRNA synthetase was followed and provided a more sensitive means of active site titration than existing techniques. Finally, this novel method was used to provide direct evidence for the cooperativity of tRNA and ATP binding to GlnRS.
|
|
pubmed:commentsCorrections |
http://linkedlifedata.com/resource/pubmed/commentcorrection/7659511-1109585,
http://linkedlifedata.com/resource/pubmed/commentcorrection/7659511-1534409,
http://linkedlifedata.com/resource/pubmed/commentcorrection/7659511-1565639,
http://linkedlifedata.com/resource/pubmed/commentcorrection/7659511-2459391,
http://linkedlifedata.com/resource/pubmed/commentcorrection/7659511-2478363,
http://linkedlifedata.com/resource/pubmed/commentcorrection/7659511-3003503,
http://linkedlifedata.com/resource/pubmed/commentcorrection/7659511-3098294,
http://linkedlifedata.com/resource/pubmed/commentcorrection/7659511-359044,
http://linkedlifedata.com/resource/pubmed/commentcorrection/7659511-382994,
http://linkedlifedata.com/resource/pubmed/commentcorrection/7659511-3911010,
http://linkedlifedata.com/resource/pubmed/commentcorrection/7659511-4312459,
http://linkedlifedata.com/resource/pubmed/commentcorrection/7659511-4339100,
http://linkedlifedata.com/resource/pubmed/commentcorrection/7659511-4910305,
http://linkedlifedata.com/resource/pubmed/commentcorrection/7659511-5327073,
http://linkedlifedata.com/resource/pubmed/commentcorrection/7659511-5327074,
http://linkedlifedata.com/resource/pubmed/commentcorrection/7659511-6356984,
http://linkedlifedata.com/resource/pubmed/commentcorrection/7659511-6989402,
http://linkedlifedata.com/resource/pubmed/commentcorrection/7659511-8317708,
http://linkedlifedata.com/resource/pubmed/commentcorrection/7659511-8352600,
http://linkedlifedata.com/resource/pubmed/commentcorrection/7659511-8369295,
http://linkedlifedata.com/resource/pubmed/commentcorrection/7659511-8385989
|
|
pubmed:language |
eng
|
|
pubmed:journal |
|
|
pubmed:citationSubset |
IM
|
|
pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/Adenosine Triphosphate,
http://linkedlifedata.com/resource/pubmed/chemical/Amino Acyl-tRNA Synthetases,
http://linkedlifedata.com/resource/pubmed/chemical/Aspartate-tRNA Ligase,
http://linkedlifedata.com/resource/pubmed/chemical/Aspartic Acid,
http://linkedlifedata.com/resource/pubmed/chemical/Diphosphates,
http://linkedlifedata.com/resource/pubmed/chemical/Dithionitrobenzoic Acid,
http://linkedlifedata.com/resource/pubmed/chemical/Inorganic Pyrophosphatase,
http://linkedlifedata.com/resource/pubmed/chemical/Purines,
http://linkedlifedata.com/resource/pubmed/chemical/Pyrophosphatases,
http://linkedlifedata.com/resource/pubmed/chemical/RNA, Transfer, Glu,
http://linkedlifedata.com/resource/pubmed/chemical/RNA, Transfer, Trp,
http://linkedlifedata.com/resource/pubmed/chemical/Tryptophan-tRNA Ligase,
http://linkedlifedata.com/resource/pubmed/chemical/glutaminyl-tRNA synthetase
|
|
pubmed:status |
MEDLINE
|
|
pubmed:month |
Aug
|
|
pubmed:issn |
0305-1048
|
|
pubmed:author |
|
|
pubmed:issnType |
Print
|
|
pubmed:day |
11
|
|
pubmed:volume |
23
|
|
pubmed:owner |
NLM
|
|
pubmed:authorsComplete |
Y
|
|
pubmed:pagination |
2886-92
|
|
pubmed:dateRevised |
2009-11-18
|
|
pubmed:meshHeading |
pubmed-meshheading:7659511-Acylation,
pubmed-meshheading:7659511-Adenosine Triphosphate,
pubmed-meshheading:7659511-Amino Acyl-tRNA Synthetases,
pubmed-meshheading:7659511-Aspartate-tRNA Ligase,
pubmed-meshheading:7659511-Aspartic Acid,
pubmed-meshheading:7659511-Binding Sites,
pubmed-meshheading:7659511-Diphosphates,
pubmed-meshheading:7659511-Dithionitrobenzoic Acid,
pubmed-meshheading:7659511-Escherichia coli,
pubmed-meshheading:7659511-Inorganic Pyrophosphatase,
pubmed-meshheading:7659511-Purines,
pubmed-meshheading:7659511-Pyrophosphatases,
pubmed-meshheading:7659511-RNA, Transfer, Glu,
pubmed-meshheading:7659511-RNA, Transfer, Trp,
pubmed-meshheading:7659511-Spectrophotometry, Ultraviolet,
pubmed-meshheading:7659511-Tryptophan-tRNA Ligase
|
|
pubmed:year |
1995
|
|
pubmed:articleTitle |
A broadly applicable continuous spectrophotometric assay for measuring aminoacyl-tRNA synthetase activity.
|
|
pubmed:affiliation |
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
|
|
pubmed:publicationType |
Journal Article,
Research Support, U.S. Gov't, P.H.S.,
Research Support, Non-U.S. Gov't
|
