Amino Acid Pool Of Cells Research Articles

The Ubiquitin System, an Immense Realm

Among the functions of intracellular proteolysis are the elimination of misfolded or otherwise abnormal proteins; the maintenance of amino acid pools in cells affected by stresses, such as starvation; and the generation of protein fragments that act as hormones, antigens, or other effectors. Many intracellular proteins are either conditionally or constitutively short-lived, with in vivo half-lives that can be as brief as a few minutes. In some cases, a proteolytic pathway targets and destroys a protein cotranslationally, i.e., an emerging polypeptide chain can be degraded while it is still a ribosome-associated peptidyl-tRNA (1, 2). The regulated and processive degradation of intracellular proteins is carried out largely by the ubiquitin (Ub)- proteasome system (Ub system), in conjunction with molecular chaperones, autophagy, and lysosomal proteolysis. Other mediators of intracellular protein degradation include proteases such as caspases, calpains, and separases. These and other nonprocessive proteases can function as “upstream” components of the Ub system, generating protein fragments that are targeted and degraded to short peptides by Ub-mediated pathways. Proteins that are damaged, misfolded, or otherwise abnormal are often recognized as such and selectively destroyed by the Ub system. Physiologically important exceptions include conformationally perturbed proteins and/or their aggregates that are harmful but cannot be efficaciously repaired or removed. The resulting proteotoxicity underlies both aging and specific diseases, including neurodegeneration.

Chirality differences in amino acid retention and release from the acid-extractable pool of cultured mammalian cells

In previous work, no chiral differences were found between D and L enantiomers of Leu in their ability to displace one another from the acid-extractable pool in mammalian cells. Recent evidence suggested otherwise. Our aim is to examine whether, in physiological range, D-amino acids have an equivalent ability to displace L-amino acids from the acid-extractable pool of HeLa cells, and vice versa. In the millimolar range, D-Leu and L-Leu have similar uptake and displacement properties with regard to the acid-extractable pool in HeLa cells, despite only the latter isomer being incorporated into protein. Below millimolar concentrations however, a distinct difference was found in the displacement of tritium-labelled L-Leu from the pool by unlabelled D-Leu compared with unlabelled L-Leu. Thus, unlabelled L-Leu in the external medium at 10 −4 or 10 −5 M displaced an equivalent amount of label from the pool as D-Leu introduced at a concentration approx. one order of magnitude higher, respectively. Reciprocal experiments, in which the acid-extractable pool was preloaded with 3H-D-Leu, confirmed this finding. The chirality difference was noted whether pool prelabelling was carried out at 37 or 0°C; but in order to avoid the complications of active transport mechanisms, the competition work reported here was done at 0°C. Similar chirality differences were observed with other hydrophobic amino acids, including His, Ile and Phe, such as, preferential displacement by the L-Leu racemer compared with the D-Leu racemer below mM levels. This was also true for the D and L forms of the non-utilisable isomer of Leu, norleucine ( nLeu). We conclude that D-forms of hydrophobic amino acids have lower affinity for similar or the same intracellular binding sites involved in the acid-extractable pool than their L-forms. The significance of these chirality findings to amino acid pools in cells, and to the predominance of L-forms of amino acids in the biosphere is considered.

Ammonium-nitrogen partitioning under different balances of nitrogen and carbon in the cyanobacterium Anabaena cylindrica.

The composition of free amino acids in nitrogen-fixing cells of Anabaena cylindrica was changed by varying the ratio of N2 to CO2 in the gas phase of the medium. Glutamate, aspartate and alanine were the major components in the free amino acid pool of cells incubated in the light and with air supplemented with 0.5% CO2. When the cells were bubbled with pure N2, cellular glutamine, citrulline and arginine increased while glutamate decreased. Adding NH+4 in the dark stimulated the increase of glutamine but not citrulline. Light was required for the rapid synthesis and accumulation of citrulline. Carbonylcyanide metachlorophenylhydrazone (CCCP), which inhibits ATP synthesis, suppressed the NH+4- timulated increase in citrulline in the light. It is concluded that the balance in nitrogen and carbon supply and cellular ATP level determined the metabolism of glutamine, which changed the composition of the cellular free amino acid.

The sodium effect of Bacillus subtilis growth on aspartate.

aspH mutants of Bacillus subtilis have a constitutive aspartase activity and grow well on aspartate as sole carbon source. aspH aspT mutants, which are deficient in high affinity aspartate transport as a result of the aspT mutation, grow as well as aspH mutants in medium containing high concentrations of aspartate and Na+. This Na+ effect is not due to an enhancement of aspartate transport but is the result of increased cellular metabolism. The ability to grow rapidly in sodium aspartate is induced by prior growth in the presence of Na+. In potassium aspartate, the addition of arginine, citrulline, ornithine, delta 1-pyrroline-5-carboxylase or proline instead of Na+ also allows rapid growth; but in a mutant deficient in ornithine--oxo-acid aminotransferase, only pyrroline-carboxylate or proline can replace Na+. The amino acid pool of cells growing slowly in potassium aspartate contains proline at a low concentration which increases upon addition of proline (but not Na+) to the medium. Thus, Na+ addition does not increase the synthesis of proline, but proline or pyrroline-carboxylate acts similarly to Na+ either in preventing some inhibitory effect (by aspartate or the accumulating NH4+) or in overcoming some deficiency (e.g. in further proline metabolism.

Mode of Action of Pyocin R11

The effects of pyocin Rl on transport systems and syntheses of macromolecules in Pseudomonas aeruginosa strain P14 were studied under two conditions, 0.4 mM and 40 mM Magnesium ions. In the presence of 0.4 mM magnesium ions, the incorporation of [14C]leucine into protein and the uptake of [14C]leucine into the leucine pool were completely inhibited by pyocin. Furthermore, [14C]leucine which had been taken up into the leucine pool was depleted by pyocin. Comparing the concentration of amino acid within cells with that in the medium, it was concluded that the active transport in P14 cells was inhibited completely by pyocin. Such an effect of pyocin Rl on transport systems was not specific to leucine but common to many amino acids and uridine. On the other hand, under the condition of 40 mM magnesium ions, [14C]-leucine once accumulated was not depleted by pyocin, but uptake of [14C]leucine into the leucine pool stopped immediately while incorporation of [14C]leucine into protein continued at a considerably reduced rate; therefore the machinery for protein synthesis was not directly inhibited by pyocin. It is suggested that the loss from the amino acid pool in cells brings about the rapid cessation of protein synthesis under the condition of 0.4 mM magnesium.