Preparation of crude transfer RNA and chromatographic purification of five transfer RNAs from calf liver

Pearson, R.L.; Hancher, C.W.; Weiss, Joseph F.; Holladay, D.W.; Kelmers, A.D.

doi:10.1016/0005-2787(73)90296-7

Cited by 28 publications

(7 citation statements)

References 37 publications

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“…Combination of BD-cellulose and RPC-5 chromatography proved, in agreement with the suggestion of other authors (15,16), to be effective in purification of specific tRNAs also from plant material. BD-cellulose was found to be very useful in bulk separation of tRNAPhe from most of the other tRNAs.…”

Section: Dlscussidnsupporting

confidence: 90%

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Isolation and chromatographic behaviour of phenylalanine tRNA from barley embryos

Labuda¹,

Janowicz²,

Haertlé³

et al. 1974

Nucl Acids Res

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Two fractions of phenylalanine tRNA (tRNA(Phe) (1) and tRNA(Phe) (2)) were purified by BD-cellulose and RPC-5 chromatography of crude tRNA isolated from barley embryos. Successive RPC-5 rechromatography runs of tRNA(Phe) (2) showed its conversion into more stable tRNA(Phe) (1), suggesting that the two fractions have essentially the same primary structure. Both tRNA(Phe) (1) and tRNA(Phe) (2) had about the same acceptor activity, but tRNA(Phe) (2) was aminoacylated much faster than tRNA(Phe) (1). RPC-5 chromatography of crude aminoacylated tRNA showed higher contents of phe-tRNA(Phe) (2) than of phe-tRNA(Phe) (1) but the ratio of these two fractions estimated by relative fluorescence intensity was about 1. Fluorescence spectra of tRNA(Phe) from barley embryos suggest that it contains Y base similar to Y(w) from wheat tRNA(Phe).

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Section: Dlscussidnsupporting

confidence: 90%

“…HD-cellulose chromatography was carried out according to (13), and RPC-5 chromatography according to (14,15), Details are given in legends to the figures. The pumps and hydraulic system of an aminoacid analyser were used for running RPC-5 columns.…”

mentioning

confidence: 99%

Isolation and chromatographic behaviour of phenylalanine tRNA from barley embryos

Labuda¹,

Janowicz²,

Haertlé³

et al. 1974

Nucl Acids Res

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“…With highly purified B. mori alanine tRNA synthetase, we observed aminoacylation of the wild-type B. mori tRNAAI,/CUA but no aminoacylation of the G3-C70 variant (Figure 2a). Because the purified human enzyme is not available, we prepared a crude HeLa cell extract and enriched for aminoacyl tRNA synthetases by chromatography (Pearson et al, 1973). With this extract, we detected alanine incorporation into the human wild-type tRNAAla/CUA but not into the G3-C70 variant (Figure 2b).…”

Section: Resultsmentioning

confidence: 99%

Evidence that a major determinant for the identity of a transfer RNA is conserved in evolution

Hou

Schimmel²

1989

Biochemistry

118

106

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We observed recently that a single G3.U70 base pair in the amino acid acceptor stem of an Escherichia coli alanine tRNA is a major determinant for its identity. Inspection of tRNA sequences shows that G3.U70 is unique to alanine in E. coli and is present in eucaryotic cytoplasmic alanine tRNAs. We show here that single nucleotide changes of G3.U70 to A3.U70 or to G3.C70 eliminate in vitro aminoacylation of an insect and of a human alanine tRNA by the respective homologous synthetase. Compared to the influence of G3.U70, other sequence variations in tRNAAla have a relatively small effect on aminoacylation by the insect and human enzymes. In addition, while these eucaryotic tRNAs have nucleotide differences from E. coli alanine tRNA, they are heterologously charged only with alanine when expressed in E. coli. The results indicate a functional role for G3.U70 that is conserved in evolution. They also suggest that the sequence differences between E. coli and the eucaryotic alanine tRNAs at sites other than the conserved G3.U70 do not create major determinants for recognition by any other bacterial enzyme.

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“…This value can be compared with the maximum acceptor site (pool-size) for highly purified leucyl-tRNA, which is in the range of 100-120 nmol/100 g tissue weight, whereas the maximum capacity for aminoacyl-tRNA synthetase activity (the enzyme) is -100-fold higher (20)(21)(22). The above values shall be compared with leucine uptake across the leg, which was 216±25 nmol/min per 100 g at the highest infusion rate (0.8 g N/kg per d, Table II).…”

Section: Resultsmentioning

confidence: 99%

Transport kinetics of amino acids across the resting human leg.

Lundholm¹,

Bennegård²,

Zachrisson³

et al. 1987

J. Clin. Invest.

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Flux rates of amino acids were measured across the leg after an overnight fast in resting human volunteers. A balanced amino acid solution was, after a primed infusion, continuously infused for 2 h at each of three step-wise and increasing rates corresponding to 83, 16.7, 33.2 Arterial steady state levels were obtained for most amino acids within 30 to 45 min after the primed constant infusion. Leg flux of amino acids switched from a net efflux after an overnight fast to a balanced flux between infusion rates corresponding to 0.2-0.4 g N/kg per d. At 0.8 g N/kg per d essentially all amino acids showed uptake. The infusion of amino acids stimulated leg uptake of glucose and lactate production and decreased FFA release. Oxygen uptake and leg blood flow increased significantly with increased infusion of amino acids. There was significant variability in transport rate among individual amino acids. Branched chain amino acids showed rapid transport and methionine slow transport rate. Only small changes in the muscle tissue concentration of certain amino acids were registered after 6 h of amino acid infusion despite uptake for several hours. When amino acids were infused at a rate corresponding to 0.8 g N/kg per d, the leg uptake of amino acids was 6% and the simultaneous whole body oxidation of infused amino acids was -10%. Net uptake of leucine across the leg per hour was 62% of the muscle pool of free leucine when amino acids were infused at a rate corresponding to 0.4 g N/kg per d. Multiple regression analysis showed that the arterial concentration of an amino acid was the most important factor for uptake, more so than insulin concentration and blood flow.It is concluded that leg exchange of amino acids is large enough to rapidly change the pool size of the amino acids in skeletal muscle, if not counter-regulated by changes in rates of protein synthesis and degradation. Estimates of the capacity for protein synthesis and transfer RNA acceptor sites in muscles agree in order of magnitude with the net uptake of amino

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Preparation of crude transfer RNA and chromatographic purification of five transfer RNAs from calf liver

Cited by 28 publications

References 37 publications

Isolation and chromatographic behaviour of phenylalanine tRNA from barley embryos

Isolation and chromatographic behaviour of phenylalanine tRNA from barley embryos

Evidence that a major determinant for the identity of a transfer RNA is conserved in evolution

Transport kinetics of amino acids across the resting human leg.

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