Factors affecting protein synthesis in vitro in rabbit reticulocytes

Rabbit reticulocytes in vitro rapidly incorporate labeled amino acids into their proteins. The process is accelerated by the plasma of every mammal investigated and also by extracts of normal erythrocytes, rabbit reticulocytes, liver, spleen, and yeast (1). We have described two sets of stimulating factors: one of these sets consists of certain amino acids (1), the other of fructose-amino acids in liver (2-4). The latter set is ineffective without the addition of iron to the reaction medium. The effect of iron has been referred to in preceding publications (2-5), but without detail. After the necessity of adding iron was recognized, in order to obtain a maximal rate of protein synthesis the reaction mixture was improved further by adding to it certain substances which depend upon added iron for their effect. These increased the effect of plasma. Eventually the total (potential as well as actual) accelerating effects of plasma and liver extract were accounted for by known substances. This led to the devising of a reaction mixture formula in which the amino acid incorporation is about five times as fast as that observed when the cells are incubated in saline

The incorporation of labeled lysine into the proteins of guinea pig liver homogenate

When C14-labeled lysine is incubated with guinea pig liver homogenate, α-aminoadipic, α-ketoadipic, and glutaric acids are formed from the lysine (1). These transformations were established by finding the radioactivity of the C14 tracer in the metabolic products. The homogenate proteins coagulated by boiling at pH 5 also contained radioactivity. The counts given by the proteins corresponded to about 0.02 to 0.03 per cent of that added as lysine; the extent of lysine incorporation into the proteins was of the same order of magnitude as Melchior and Tarver (2) had found after incubating S35-labeled methionine and Winnick et al. (3, 4) C14-labeled glycine with rat tissue homogenates. Yet we could not satisfy ourselves that the radioactivity remaining in the proteins in our experiments, although it persisted through exhaustive extraction, did not come from traces of adsorbed radioactive lysine. Some counts were found in the protein when the homogenate was boiled prior to incubation with isotopic lysine

Isolation of a peptide in guinea pig liver homogenate and its turnover of leucine

Leucine was synthesized with C14 in the carboxyl group. 10 mg. of the radioactive amino acid (DL) and 0.66 gm. (wet weight) of guinea pig liver homogenate were added to a reaction mixture containing 1.3 per cent of an amino acid mixture corresponding to the composition of casein and 0.005 M fumarate, all in a final volume of 4 ml. of isotonic saline solution(1) at pH 7.4. The reaction was carried out under oxygen for 6 hours at 38°

Incorporation in vitro of labeled amino acids into bone marrow cell proteins

Nearly all experiments on the incorporation of labeled amino acids into tissue proteins in vitro have been done on tissues whose cell structure has been partially or completely disintegrated, e.g. tissue slices, segments, or homogenates. Since cell destruction reduces or abolishes the uptake of labeled amino acids (1), it seemed worth while to carry out studies on intact cells in vitro. Bone marrow cells were found to be suitable for this purpose. The labeled amino acids used were glycine-1-C14, L-leucine-1-C14, L-lysine-1-C14, and L-lysine-6-C14

The degradation of L-lysine in guinea pig liver homogenate: formation of alpha-aminoadipic acid

A summary of the little that is known of the metabolism of lysine in animals is as follows: it is indispensable in the diet, its α-amino group does not participate in reversible transamination reaction in vivo (2), neither the L nor D form is attacked by the appropriate amino acid oxidase, certain ε-nitrogen-substituted derivatives can replace lysine in the diet and their α-amino groups are oxidized by amino acid oxidases (3, 4), no α-nitrogen-substituted derivatives yet prepared can substitute for lysine in the diet (4-6)

Incorporation in vitro of labeled amino acids into proteins of rabbit reticuloytes

Continuing our work on the incorporation of labeled amino acids into proteins (1), we have begun a study of the incorporation in vitro of C14-labeled glycine, L-histidine, L-leucine, and L-lysine into the proteins of rabbit reticulocytes. In preliminary experiments the incorporation into the hemoglobin isolated from the reticulocytes was determined. But, after it was found that plasma contains factors accelerating amino acid incorporation, it was decided to proceed as rapidly as possible toward the identification of these factors; we have, therefore, measured incorporation into the total proteins of the reticulocytes, since isolation of the hemoglobin was time-consuming. The results obtained with hemoglobin and with the total proteins are essentially the same, indicating that the other proteins of the reticulocytes incorporate amino acids at approximately the same rate as hemoglobin

Alpha-aminoadipic acid: A product of lysine metabolism

As part of a study of protein and peptide metabolism lysine was synthesized with C14 in the ε position and resolved into the L and D isomers. 10 mg. of labeled lysine dihydrochloride (either L- or D-) and 0.66 gm. (wet weight) of guinea pig liver homogenate were added to a reaction mixture containing 1.3 per cent of an amino acid mixture corresponding to the composition of casein except for lysine and 0.01 M α-ketoglutarate, all in a final volume of 4 ml. of isotonic saline solution.(1) The reaction was carried out under oxygen for 6 hours at 38°

A peptide fraction in liver

We reported in a preliminary communication (1) the isolation of a peptide fraction from guinea pig liver. The following points of interest appeared at once: many different amino acids were obtained on hydrolysis; the peptide fraction contained most of the indispensable amino acids, which indicated that it probably is important in protein metabolism; when guinea pig liver homogenate was incubated with C14-labeled glycine, leucine, or lysine, these were rapidly incorporated into this peptide fraction, which is further evidence that it is metabolically active; the peptide fraction had not been described hitherto; a fraction containing one or more large peptides can be separated from so complex a mixture as liver homogenate by starch chromatography

The uptake in vitro of C14-labeled glycine, L-leucine, and L-lysine by different components of guinea pig liver homogenate

We have reported (1) that L-lysine labeled with C14 can be incorporated into the proteins of guinea pig liver homogenate under two different conditions. In the one case the enzyme used was the whole homogenate, the optimum pH was near 6.2, there was an obligatory requirement of calcium, and the incorporation was independent of oxygen. This set of conditions is designated below as the “acid calcium” condition. In the other case the enzyme system was the precipitate obtained by centrifuging the homogenate diluted 15-fold with Ringer’s solution at 2500 X g, the optimum pH was near to 7.3, the reaction was accelerated a little by calcium but the presence of calcium was not obligatory, and the incorporation was a little less under nitrogen than under oxygen. This set of conditions is designated below as the “alkaline” condition

A Key Role of the Basal Ganglia in Pain and Analgesia - Insights Gained Through Human Functional Imaging

The basal ganglia (BG) are composed of several nuclei involved in neural processing related to the execution of motor, cognitive and emotional activities. Preclinical and clinical data have implicated a role for these structures in pain processing. Recently neuroimaging has added important information on BG activation in conditions of acute pain, chronic pain and as a result of drug effects. Our current understanding of alterations in cortical and sub-cortical regions in pain suggests that the BG are uniquely involved in thalamo-cortico-BG loops to integrate many aspects of pain. These include the integration of motor, emotional, autonomic and cognitive responses to pain