Use of a single tissue extract to determine cellular protein and nucleic acid concentrations and rate of amino acid incorporation

Abstract The leukocytes of chronic granulocytic leukemia incorporate labeled valine and leucine at a higher rate than normal leukocytes. 6-mercaptopurine causes significant decrease in the rate of amino acid incorporation into cellular protein of leukemic cells. The onset of a sharp decrease in the amino acid incorporation rate by granulocytic leukemic cells occurs only after several days of therapeutic oral doses of 6-mercaptopurine. The decrease in incorporation rate precedes the decrease in circulating leukocytes by several days, indicating that damage to a vital function of these cells occurs before their disappearance from the blood stream. The decrease in the amino acid incorporation rate persists as long as the leukemia is in remission and even after therapy has been stopped; it exists until exacerbation occurs. Increase in incorporation accompanies exacerbation of the leukemic cell count. A possible action of 6-mercaptopurine is its role in interference with amino acid incorporation into cellular protein of chronic granulocytic leukemia cells.

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A STABILIZED ENZYME SYSTEM FOR AMINO ACID INCORPORATION

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)70686-8 ◽

1957 ◽

Vol 228 (1) ◽

pp. 23-39

Author(s):

Howard Sachs

Keyword(s):

Amino Acid ◽

Enzyme System ◽

Amino Acid Incorporation ◽

Acid Incorporation

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AMINO ACID INCORPORATION BY INTACT AND DISRUPTED RIBONUCLEOPROTEIN PARTICLES

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)70638-8 ◽

1957 ◽

Vol 229 (1) ◽

pp. 535-546

Author(s):

George C. Webster

Keyword(s):

Amino Acid ◽

Amino Acid Incorporation ◽

Acid Incorporation ◽

Ribonucleoprotein Particles

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Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency

Amino Acids ◽

10.1007/s00726-020-02927-z ◽

2020 ◽

Author(s):

Thomas L. Williams ◽

Debra J. Iskandar ◽

Alexander R. Nödling ◽

Yurong Tan ◽

Louis Y. P. Luk ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Genetic Code ◽

Unnatural Amino Acids ◽

Trna Synthetase ◽

Unnatural Amino Acid ◽

Amino Acid Incorporation ◽

Acid Incorporation ◽

Genetic Code Expansion ◽

Incorporation Efficiency

AbstractGenetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

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