Polyribosome metabolism in Escherichia coli starved for an amino acid

Most polyribosomes are inferred to be inert in starving cells, but some are in a dynamic state, since (1) messenger RNA continues to enter polyribosomes; (2) about 20 to 40% of the polyribosomes are labile after rifampycin addition; (3) β-galactosidase can be induced with a lag period no more than three times as long as in growing cells; and (4) the apparent rate of synthesis of protein chains, judged by the distribution of pulse-labeled protein between ribosomes and soluble protein, is about half that in growing cells.     

Selective synthesis of mitochondrial proteins by Chinese hamster ovary cells severely starved for various amino acids

Chinese hamster ovary (CHO) cells were subjected to severe amino acid starvation for histidine, leucine, methionine, asparagine, tyrosine, glutamine, valine, and lysine, using amino acid analogs or mutations in specific aminoacyl-tRNA synthetases. At protein synthetic rates of less than 5%, in all cases, the newly synthesized proteins were found on two- dimensional electrophoretic gels to consist of a few intensely labeled spots, with the exception of lysine. This pattern could also be produced by strong inhibition of cytoplasmic protein synthesis with cycloheximide, and was abolished by preincubation with the mitochondrial protein synthesis inhibitor chloramphenicol. It appears therefore that the spots represent mitochondrial protein synthesis and that animal cells must have separate aminoacyl-tRNA synthetases for mitochondrial tRNAs corresponding to all these amino acids except, possibly, for lysine.     

RNA metabolism in nuclei isolated from HeLa cells.

Nuclei with low cytoplasmic contamination, capable of synthesizing RNA for an extended period of time, were prepared from HeLa cells. Besides elongating RNA chains already initiated in vivo, the nuclear preparation initiates the synthesis of new RNA chains. This was shown by labelling the newly synthesized RNA with [gamma-32P]GTP and by detecting the presence of labelled guanosine tetraphosphate among the alkaline hydrolysis products of synthesized RNA. By synthesizing RNA in the presence of each of the four gamma-32P-labelled nucleoside triphosphates, it was possible to conclude that RNA chain synthesis starts predominantly with a purine base. Both nucleolar and nucleoplasmic RNAs are made. The nuclear preparation methylates the nucleolar RNA by utilizing S-adenosyl-L-methionine as a methyl-group donor.     

Deoxyribonucleotide metabolism in Herpes simplex virus infected HeLa cells.

The effect of Rolly No. 11 strain herpes simplex virus infection of HeLa cells in culture on deoxynucleotide metabolism and the level of various enzymes concerned with the biosynthesis of DNA has been investigated. Of 18 enzyme activities studied, thymidine kinase, DNA polymerase and deoxyribonuclease were markedly augmented, a finding in agreement with previous reports. Deoxycytidine kinase, ribonucleotide reductase, thymidylate kinase and deoxycytidylate deaminase activities, in contrast with previous reports, did not increase; the activities of the other enzymes studied, also did not increase. Whereas most of the radioactivity derived from [14-C] thymidine in the acid-soluble fraction of the uninfected cells was present as deoxythymidine triphosphate, that present in the infected cells was primarily in the form of deoxythymidine monophosphate. Thus, in the infected cell deoxythymidylate kinase is a rate-limiting enzyme in the biosynthesis of deoxythymidine triphosphate. A marked increase in the pools of the four naturally occurring deoxynucleoside triphosphates (dTTP, dCTP, dATP, dGTP) was found. The rate of formation of the virus-induced enzymes was determined, as were the various nucleoside triphosphate pools and the other phosphorylated derivatives of thymidine; a maximum was reached for all these csmponents between 6 to 8 h post infection. Although an apparent greater synthesis of DNA occurred in the uninefected cells, when the specific activity of the radioactive deoxythymidine triphosphate was taken into account, there was actually a greater rate of DNA synthesis in the infected cells, with the peak at 8 h post infection.     

Metabolism of branched-chain amino acids in starved rats: the role of hepatic tissue

Parameters of branched-chain amino acids (BCAA; leucine, isoleucine and valine) and protein metabolism were evaluated using L-[1-(14)C]leucine and alpha-keto[1-(14)C]isocaproate (KIC) in the whole body and in isolated perfused liver (IPL) of rats fed ad libitum or starved for 3 days. Starvation caused a significant increase in plasma BCAA levels and a decrease in leucine appearance from proteolysis, leucine incorporation into body proteins, leucine oxidation, leucine-oxidized fraction, and leucine clearance. Protein synthesis decreased significantly in skeletal muscle and the liver. There were no significant differences in leucine and KIC oxidation by IPL. In starved animals, a significant increase in net release of BCAA and tyrosine by IPL was observed, while the effect on other amino acids was non-significant. We conclude that the protein-sparing phase of uncomplicated starvation is associated with decreased whole-body proteolysis, protein synthesis, branched-chain amino acid (BCAA) oxidation, and BCAA clearance. The increase in plasma BCAA levels in starved animals results in part from decreased BCAA catabolism, particularly in heart and skeletal muscles, and from a net release of BCAA by the hepatic tissue.     

Effects of external osmolality on polyamine metabolism in HeLa cells.

The polyamine content of Escherichia coli is inversely related to the osmolality of the growth medium. The experiments described here demonstrate that a similar phenomenon occurs in mammalian cells. When grown in media of low NaCl concentration, HeLa cells and human fibroblasts were found to contain high levels of putrescine, spermidine, and spermine. The putrescine content of HeLa cells was a function of the osmolality of the medium, as shown by growing cells in media containing mannitol or additional glucose. External osmolality per se had no effect on the contents of spermidine and spermine. For all media, the total cellular polyamine content could be correlated with the activity of ornithine decarboxylase, the first enzyme in polyamine biosynthesis. Different levels of enzyme activity appear to result solely from variations in the rate of enzyme degradation. A sudden increase in a NaCl concentration produced rapid loss of ornithine decarboxylase activity and a gradual loss of putrescine and spermidine. A sudden decrease in NaCl concentration led to rapid and substantial increases in ornithine decarboxylase activity and putrescine.     

Hepatocyte heterogeneity in the metabolism of amino acids and ammonia.

With respect to hepatocyte heterogeneity in ammonia and amino acid metabolism, two different patterns of sublobular gene expression are distinguished: 'gradient-type' and 'strict- or compartment-type' zonation. An example for strict-type zonation is the reciprocal distribution of carbamoylphosphate synthase and glutamine synthase in the liver lobule. The mechanisms underlying the different sublobular gene expressions are not yet settled but may involve the development of hepatic architecture, innervation, blood-borne hormonal and metabolic factors. The periportal zone is characterized by a high capacity for uptake and catabolism of amino acids (except glutamate and aspartate) as well as for urea synthesis and gluconeogenesis. On the other hand, glutamine synthesis, ornithine transamination and the uptake of vascular glutamate, aspartate, malate and alpha-ketoglutarate are restricted to a small perivenous hepatocyte population. Accordingly, in the intact liver lobule the major pathways for ammonia detoxication, urea and glutamine synthesis, are anatomically switched behind each other and represent in functional terms the sequence of the periportal low affinity system (urea synthesis) and a previous high affinity system (glutamine synthesis) for ammonia detoxication. Perivenous glutamine synthase-containing hepatocytes ('scavenger cells') act as a high affinity scavenger for the ammonia, which escapes the more upstream urea-synthesizing compartment. Periportal glutaminase acts as a pH- and hormone-modulated ammonia-amplifying system in the mitochondria of periportal hepatocytes. The activity of this amplifying system is one crucial determinant for flux through the urea cycle in view of the high Km (ammonia) of carbamoylphosphate synthase, the rate-controlling enzyme of the urea cycle. The structural and functional organization of glutamine and ammonia-metabolizing pathways in the liver lobule provides one basis for the understanding of a hepatic role in systemic acid base homeostasis. Urea synthesis is a major pathway for irreversible removal of metabolically generated bicarbonate. The lobular organization enables the adjustment of the urea cycle flux and accordingly the rate of irreversible hepatic bicarbonate elimination to the needs of the systemic acid base situation, without the threat of hyperammonemia.     

Reversed transport of amino acids in Ehrlich cells.

Gramicidin induces a marked Na+-dependent efflux of amino acids from Ehrlich cells. In absence of Na+, gramicidin does not alter the efflux. In presence gramicidin, glycine efflux is inhibited by methionine and less so by leucine. Glycine efflux caused by HgCl2 is neither Na+ dependent nor inhibitable by amino acids. Neither efflux of inositol which is transported by an Na+-dependent route, nor efflux of several other solutes which are transported by Na+-independent routes, is affected by gramicidin. The antibiotic appears to permit a reversal in the direction of of the operation of the Na+-dependent amino acid transport system. The increased efflux is partly, but not entirely, due to an increase in the cellular Na+ concentration and a reduction of the electrochemical potential difference for Na+.