A BROADLY APPLICABLE CONTINUOUS SPECTROPHOTOMETRIC ASSAY FOR MEASURING AMINOACYL-TRANSFER-RNA SYNTHETASE-ACTIVITY

被引:50
作者
LLOYD, AJ [1 ]
THOMANN, HU [1 ]
IBBA, M [1 ]
SOLL, D [1 ]
机构
[1] YALE UNIV,DEPT MOLEC BIOPHYS & BIOCHEM,NEW HAVEN,CT 06520
基金
美国国家卫生研究院;
关键词
D O I
10.1093/nar/23.15.2886
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 nondestructive 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 [H-3]Gln-tRNA or [C-14]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.
引用
收藏
页码:2886 / 2892
页数:7
相关论文
共 24 条
[1]  
BATTACHARYYA T, 1993, BIOCHEMISTRY-US, V32, P9268
[2]   MOLECULAR AND CELLULAR STUDIES OF TRYPTOPHANYL-TRANSFER RNA-SYNTHETASE USING MONOCLONAL-ANTIBODIES - EVALUATION OF A COMMON ANTIGENIC DETERMINANT IN EUKARYOTIC, PROKARYOTIC AND ARCHAEBACTERIAL ENZYMES WHICH MAPS OUTSIDE THE CATALYTIC DOMAIN [J].
BERESTEN, SF ;
ZARGAROVA, TA ;
FAVOROVA, OO ;
RUBIKAITE, BI ;
RYAZANOV, AG ;
KISSELEV, LL .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1989, 184 (03) :575-581
[3]   SYNTHESIS AND PMR STUDIES OF SOME METHYLATED 6-THIOPURINE NUCLEOSIDES [J].
BROOM, AD ;
MILNE, GH .
JOURNAL OF HETEROCYCLIC CHEMISTRY, 1975, 12 (01) :171-174
[4]   COGNITION, MECHANISM, AND EVOLUTIONARY RELATIONSHIPS IN AMINOACYL-TRANSFER RNA-SYNTHETASES [J].
CARTER, CW .
ANNUAL REVIEW OF BIOCHEMISTRY, 1993, 62 :715-748
[5]   ACTIVE-SITE TITRATION AND AMINOACYL ADENYLATE BINDING STOICHIOMETRY OF AMINOACYL-TRANSFER-RNA SYNTHETASES [J].
FERSHT, AR ;
ASHFORD, JS ;
BRUTON, CJ ;
JAKES, R ;
KOCH, GLE ;
HARTLEY, BS .
BIOCHEMISTRY, 1975, 14 (01) :1-4
[6]  
FERSHT AR, 1978, BIOCHEMISTRY-US, V17, P3748
[7]   CHEMICALLY MODIFIED ATP DERIVATIVES FOR THE STUDY OF AMINOACYL-TRANSFER RNA-SYNTHETASES FROM BAKERS-YEAST - ATP ANALOGS WITH FIXED CONFORMATIONS OR MODIFIED TRIPHOSPHATE CHAINS IN THE AMINOACYLATION REACTION [J].
FREIST, W ;
WIEDNER, H ;
CRAMER, F .
BIOORGANIC CHEMISTRY, 1980, 9 (04) :491-504
[8]   2 INTERCONVERTIBLE FORMS OF TRYPTOPHANYL SRNA IN E COLI [J].
GARTLAND, WJ ;
SUEOKA, N .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1966, 55 (04) :948-&
[9]  
HOBEN P, 1985, METHOD ENZYMOL, V113, P55
[10]   THE GLUTAMINYL-TRANSFER RNA-SYNTHETASE OF ESCHERICHIA-COLI - PURIFICATION, STRUCTURE AND FUNCTION RELATIONSHIP [J].
KERN, D ;
POTIER, S ;
LAPOINTE, J ;
BOULANGER, Y .
BIOCHIMICA ET BIOPHYSICA ACTA, 1980, 607 (01) :65-80