A SENSITIVE METHOD FOR MEASUREMENT OF AMINO ACYL RIBONUCLEIC ACID SYNTHETASE ACTIVITIES

1962 ◽  
Vol 40 (5) ◽  
pp. 653-666 ◽  
Author(s):  
Murray J. Fraser
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Rat Liver ◽  
Ribonucleic Acid ◽  
Ehrlich Ascites Carcinoma ◽  
Carcinoma Cells ◽  
Ehrlich Ascites ◽  
Sensitive Method

A sensitive method for the measurement of amino acyl RNA synthetase activities (amino acid activating enzymes) is described. The method is based on measurements of the rates of labelling of soluble ribonucleic acid with14C-amino acids. Determinations of α-glutamyl-RNA, glutaminyl-RNA, and glycyl-RNA synthetase activities in the "pH 5 enzymes" fractions from rat liver and mouse Ehrlich ascites carcinoma cells have been made.

A SENSITIVE METHOD FOR MEASUREMENT OF AMINO ACYL RIBONUCLEIC ACID SYNTHETASE ACTIVITIES

1962 ◽  
Vol 40 (1) ◽  
pp. 653-666 ◽  
Author(s):  
Murray J. Fraser
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Rat Liver ◽  
Ribonucleic Acid ◽  
Ehrlich Ascites Carcinoma ◽  
Carcinoma Cells ◽  
Ehrlich Ascites ◽  
Sensitive Method

A sensitive method for the measurement of amino acyl RNA synthetase activities (amino acid activating enzymes) is described. The method is based on measurements of the rates of labelling of soluble ribonucleic acid with14C-amino acids. Determinations of α-glutamyl-RNA, glutaminyl-RNA, and glycyl-RNA synthetase activities in the "pH 5 enzymes" fractions from rat liver and mouse Ehrlich ascites carcinoma cells have been made.

COMPETITION BETWEEN AMINO ACIDS FOR TRANSPORT INTO EHRLICH ASCITES CARCINOMA CELLS

1961 ◽  
Vol 39 (11) ◽  
pp. 1717-1735 ◽  
Author(s):  
P. G. Scholefield
Keyword(s):  
Carbon Dioxide ◽  
Amino Acids ◽  
Amino Acid ◽  
Ehrlich Ascites Carcinoma ◽  
Carcinoma Cells ◽  
Transport Systems ◽  
Radioactive Carbon ◽  
Ehrlich Ascites ◽  
Competitive Inhibitors ◽  
Amino Acid Analogues

The cumulative entry of amino acids into Ehrlich ascites carcinoma cells is due to the presence of active transport systems, each with its own specific range of substrates. Several amino acids and amino acid analogues may have an affinity for the same transport system and thus may inhibit transport of other amino acids by acting as competitive inhibitors or competitive substrates. Loss of methionine from ascites cells takes place by a diffusion process which obeys Fick's law. Leucine accumulation by ascites cells is small and is increased on addition of certain other amino acids. The increase is not due to inhibition of leucine oxidation as increase in the rate of production of radioactive carbon dioxide from labeled leucine also occurs. Kinetic aspects of these results are discussed.

BIOCHEMICAL STUDIES ON 1-AMINOCYCLOPENTANE CARBOXYLIC ACID

1962 ◽  
Vol 40 (1) ◽  
pp. 1101-1110 ◽  
Author(s):  
K. Ahmed ◽  
P. G. Scholefield
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Carboxylic Acid ◽  
Brain Cortex ◽  
Ehrlich Ascites Carcinoma ◽  
Carcinoma Cells ◽  
Inhibitory Effects ◽  
Synthetic Amino Acid ◽  
Ehrlich Ascites ◽  
Biochemical Studies

The synthetic amino acid 1-aminocyclopentane carboxylic acid does not seem to be metabolized but is actively concentrated by slices of rat brain cortex and Ehrlich ascites carcinoma cells. Its transport into the ascites cells has much in common with that of methionine since they are both inhibited by similar groups of amino acids. Kinetic analysis of the inhibitory effects of glycine, D- and L-methionine, allyl glycine, and thienyl glycine on the transport of 1-aminocyclopentane carboxylic acid confirms the suggestion that this amino acid and methionine enter ascites cells as the result of the action of a common transport system.

BIOCHEMICAL STUDIES ON 1-AMINOCYCLOPENTANE CARBOXYLIC ACID

1962 ◽  
Vol 40 (8) ◽  
pp. 1101-1110 ◽  
Author(s):  
K. Ahmed ◽  
P. G. Scholefield
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Carboxylic Acid ◽  
Brain Cortex ◽  
Ehrlich Ascites Carcinoma ◽  
Carcinoma Cells ◽  
Inhibitory Effects ◽  
Synthetic Amino Acid ◽  
Ehrlich Ascites ◽  
Biochemical Studies

The synthetic amino acid 1-aminocyclopentane carboxylic acid does not seem to be metabolized but is actively concentrated by slices of rat brain cortex and Ehrlich ascites carcinoma cells. Its transport into the ascites cells has much in common with that of methionine since they are both inhibited by similar groups of amino acids. Kinetic analysis of the inhibitory effects of glycine, D- and L-methionine, allyl glycine, and thienyl glycine on the transport of 1-aminocyclopentane carboxylic acid confirms the suggestion that this amino acid and methionine enter ascites cells as the result of the action of a common transport system.

THE ACTIVATION OF GLUTAMIC ACID AND GLUTAMINE IN MAMMALIAN TISSUES

1963 ◽  
Vol 41 (5) ◽  
pp. 1135-1145 ◽  
Author(s):  
M. A. Alford ◽  
M. Brotman ◽  
M. A. Chudy ◽  
M. J. Fraser
Keyword(s):  
Amino Acid ◽  
Glutamine Synthetase ◽  
Glutamic Acid ◽  
Rat Liver ◽  
Ehrlich Ascites Carcinoma ◽  
Carcinoma Cells ◽  
Specific Enzyme ◽  
Ehrlich Ascites ◽  
Mammalian Tissues ◽  
Made In

Measurements of α-glutamyl-RNA synthetase and glutaminyl-RNA synthetase activities have been made in fractions derived from 105,000 × g supernatants of homogenates of rat liver and of mouse Ehrlich ascites carcinoma cells. Evidence is presented which indicates that each amino acid is activated by a specific enzyme. Both enzymes catalyze amino acid dependent ATP-32PP exchange. The α-glutamyl-RNA synthetase purified 42-fold from rat liver 105,000 × g supernatant is free of glutamine synthetase.

THE ACTIVATION OF GLUTAMIC ACID AND GLUTAMINE IN MAMMALIAN TISSUES

1963 ◽  
Vol 41 (1) ◽  
pp. 1135-1145 ◽  
Author(s):  
M. A. Alford ◽  
M. Brotman ◽  
M. A. Chudy ◽  
M. J. Fraser
Keyword(s):  
Amino Acid ◽  
Glutamine Synthetase ◽  
Glutamic Acid ◽  
Rat Liver ◽  
Ehrlich Ascites Carcinoma ◽  
Carcinoma Cells ◽  
Specific Enzyme ◽  
Ehrlich Ascites ◽  
Mammalian Tissues ◽  
Made In

Measurements of α-glutamyl-RNA synthetase and glutaminyl-RNA synthetase activities have been made in fractions derived from 105,000 × g supernatants of homogenates of rat liver and of mouse Ehrlich ascites carcinoma cells. Evidence is presented which indicates that each amino acid is activated by a specific enzyme. Both enzymes catalyze amino acid dependent ATP-32PP exchange. The α-glutamyl-RNA synthetase purified 42-fold from rat liver 105,000 × g supernatant is free of glutamine synthetase.

AMINO ACID ACCUMULATION IN EHRLICH ASCITES CARCINOMA CELLS

1960 ◽  
Vol 38 (1) ◽  
pp. 1311-1326 ◽  
Author(s):  
A. Tenenhouse ◽  
J. H. Quastel
Keyword(s):  
Amino Acids ◽  
Steady State ◽  
Amino Acid ◽  
Active Transport ◽  
Ehrlich Ascites Carcinoma ◽  
Carcinoma Cells ◽  
Ion Concentration ◽  
Metabolic Inhibitors ◽  
Ehrlich Ascites ◽  
Glycine Transport

Measurements of the transport of amino acids into Ehrlich ascites carcinoma cells have shown that the following relationship exists between the intracellular steady-state concentration of the amino acid (Cx) and the extracellular concentration (C0):[Formula: see text]where Cm is the maximum intracellular concentration (formed when C0 is large) and Em is a constant. It is shown that Em is identical with Km, the Michaelis constant, if a carrier enzyme is involved in the process of active transport and is, therefore, a measure of the affinity of the amino acid for the effective agent involved in the transport phenomenon.The ratio of the steady-state intracellular and extracellular concentrations of amino acids exceeds unity with all amino acids examined. The responses of L-S-ethylcysteine transport to changes of potassium ion concentration and to changes of temperature differ from those of glycine transport and indicate that different carriers are involved in the active transport of these amino acids into Ehrlich ascites cells. This conclusion is supported by the fact that, whereas glycine and L-serine compete with each other for concentrative uptake, such mutual competition does not occur between S-ethylcysteine and glycine or L-serine or L-leucine.Effects of the metabolic inhibitors, 2,4-dinitrophenol, iodoacetate, and stilbestrol show that these substances exercise inhibitory effects on active transport of amino acids by suppression of respiratory or glycolytic energy. Stilbestrol, which is a particularly potent inhibitor, is more effective under aerobic conditions (in the absence of glucose) than under anaerobic conditions (in the presence of glucose). It is reasonable to account for these results on the hypothesis that the carrier responsible for amino acid transport is ATP dependent and that the carrier breaks down, and is no longer available for amino acid transfer, if the ATP content of the cell is depleted.

AMINO ACID ACCUMULATION IN EHRLICH ASCITES CARCINOMA CELLS

1960 ◽  
Vol 38 (11) ◽  
pp. 1311-1326 ◽  
Author(s):  
A. Tenenhouse ◽  
J. H. Quastel
Keyword(s):  
Amino Acids ◽  
Steady State ◽  
Amino Acid ◽  
Active Transport ◽  
Ehrlich Ascites Carcinoma ◽  
Carcinoma Cells ◽  
Ion Concentration ◽  
Metabolic Inhibitors ◽  
Ehrlich Ascites ◽  
Glycine Transport

Measurements of the transport of amino acids into Ehrlich ascites carcinoma cells have shown that the following relationship exists between the intracellular steady-state concentration of the amino acid (Cx) and the extracellular concentration (C0):[Formula: see text]where Cm is the maximum intracellular concentration (formed when C0 is large) and Em is a constant. It is shown that Em is identical with Km, the Michaelis constant, if a carrier enzyme is involved in the process of active transport and is, therefore, a measure of the affinity of the amino acid for the effective agent involved in the transport phenomenon.The ratio of the steady-state intracellular and extracellular concentrations of amino acids exceeds unity with all amino acids examined. The responses of L-S-ethylcysteine transport to changes of potassium ion concentration and to changes of temperature differ from those of glycine transport and indicate that different carriers are involved in the active transport of these amino acids into Ehrlich ascites cells. This conclusion is supported by the fact that, whereas glycine and L-serine compete with each other for concentrative uptake, such mutual competition does not occur between S-ethylcysteine and glycine or L-serine or L-leucine.Effects of the metabolic inhibitors, 2,4-dinitrophenol, iodoacetate, and stilbestrol show that these substances exercise inhibitory effects on active transport of amino acids by suppression of respiratory or glycolytic energy. Stilbestrol, which is a particularly potent inhibitor, is more effective under aerobic conditions (in the absence of glucose) than under anaerobic conditions (in the presence of glucose). It is reasonable to account for these results on the hypothesis that the carrier responsible for amino acid transport is ATP dependent and that the carrier breaks down, and is no longer available for amino acid transfer, if the ATP content of the cell is depleted.

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