Induction of (2'-5')oligoadenylate synthetase in serum-starved HeLa S3 cells by growth factors and its role in growth regulation

When serum-starved HeLa S3 cells were stimulated to proliferate by addition of fetal calf serum (FCS), (2'-5')oligoadenylate synthetase (2-5A synthetase) activity was induced. Although no interferon (IFN) activity was detectable in the HeLa S3 cell-conditioned culture medium after growth stimulation, addition of anti-IFN-beta monoclonal antibody inhibited both the expression of the 2-5A synthetase gene and the production of the enzyme, suggesting that endogenous IFN-beta was involved in 2-5A synthetase induction. Purified preparations of three growth factors, epidermal growth factor, platelet-derived growth factor, and insulin, also induced 2-5A synthetase through IFN-beta. When serum-starved HeLa S3 cells were treated with FCS, DNA synthesis was initiated synchronously, with peaks after 12 and 32 h, although the level of 2-5A synthetase reached a maximum after the first peak of DNA synthesis. Inhibition of 2-5A synthetase induction by anti-IFN-beta antibody enhanced the second, but not the first cycle of DNA synthesis. These results suggested that in HeLa S3 cells, after stimulation with growth factors the IFN/2-5A synthetase system played a role in cell growth negative regulatory mechanisms.     

Induction of hyaluronic acid synthetase activity in rat fibroblasts by medium change of confluent cultures

Hyaluronic acid synthesis in cultured cells usually occurs during the growth phase. The relation between hyaluronic acid synthetase activity and cell proliferation is studied. The synthetase activity in rat fibroblasts is high during the growth phase, but low in the stationary phase. When the old medium of stationary cultures is renewed with fresh medium containing 20% calf serum, DNA synthesis occurs synchronously between 12 and 20 hours, followed by cell division. Under these conditions, the hyaluronic acid synthetase activity is significantly induced within two hours, reaching a maximum level at 5–8 hours, and then decreases gradually. This induction of the synthetase, which shows a high turnover rate, requires continued synthesis of both RNA and protein. Furthermore, the induction of both DNA and hyaluronic acid synthesis is found to be caused by calf serum added in the medium. However, dialysis and ultrafiltration of the serum permit us to concentrate an active fraction with a high molecular weight, which induces the synthetase activity, but not DNA synthesis.     

The effect of cessation of growth on protein synthesis in a mutant of Chinese hamster ovary cells with a temperature-sensitive leucyl-tRNA synthetase

A temperature-sensitive mutant of Chinese hamster ovary cells with an altered leucyl-tRNA synthetase fails to grow and to incorporate amino acids into protein properly at or near the non-permissive temperature. This mutant was used to determine whether cessation of growth at the elevated temperature affected elongation factor EF-1, since the activity of EF-1 is markedly lower in non-growing cells in stationary phase than in rapidly-growing cells in exponential phase. Cell-free extracts prepared from cells maintained at 39°C for 24 h showed a marked decrease in the ability to translate natural mRNAs, compared to cells incubated at 34°C. However, the ability to translate poly(U), which requires elongation factor EF-1 (and EF-2), was not affected. Analyses of activities involved in the initiation of protein synthesis and in the activation of amino acids revealed that, with the exception of leucyl-tRNA synthetase, the rest of the components required for translation also appeared to be relatively stable even after 24 h at the elevated temperature. The effects of elevated temperature on cell-free extracts were also investigated. The results were similar to those obtained with intact cells; that is, except for leucyl-tRNA synthetase which was rapidly inactivated in vitro at 39°C, other aminoacyl-tRNA synthetases and translational components involved in chain initiation and elongation were relatively stable. Thus, no change in EF-1 activity was detected as a result of arrested cell growth, an inherent lability of the elongation factor, or metabolic degradation as a consequence of a rapid turnover rate in the absence of protein synthesis.     

Translational control of the embryonic cell cycle

The synthesis and destruction of cyclin B drives mitosis in eukaryotic cells. Cell cycle progression is also regulated at the level of cyclin B translation. In cycling extracts from Xenopus embryos, progression into M phase requires the polyadenylation-induced translation of cyclin B1 mRNA. Polyadenylation is mediated by the phosphorylation of CPEB by Aurora, a kinase whose activity oscillates with the cell cycle. Exit from M phase seems to require deadenylation and subsequent translational silencing of cyclin B1 mRNA by Maskin, a CPEB and eIF4E binding factor, whose expression is cell cycle regulated. These observations suggest that regulated cyclin B1 mRNA translation is essential for the embryonic cell cycle. Mammalian cells also display a cell cycle-dependent cytoplasmic polyadenylation, suggesting that translational control by polyadenylation might be a general feature of mitosis in animal cells.     

Evidence for urea cycle activity in Sporosarcina ureae

Sporosarcina ureae BS 860, a motile, sporeforming coccus, possesses the enzymes required for a functioning urea (ornithine) cycle. This is only the second known example of urea cycle activity in a prokaryote. Specific activities are reported for ornithine carbamoyltransferase, argininosuccinase, arginase, and urease. Although argininosuccinate synthetase activity could not be detected directly in crude cell extracts, indirect evidence from radiocarbon tracing data for arginine synthesis from the substrate, l-[1-¹⁴C]-ornithine, strongly suggest the presence of this or other similar enzyme activity. Furthermore, good growth in defined media containing either 1.0% glutamine, ornithine, or citrulline as sole carbon sources suggests argininosuccinate synthetase activity is necessary for arginine synthesis. The effect of varying pH on arginase and urease activities indicate that these two enzymes may function within the context of the urea cycle to generate ammonia for amino acid synthesis, as well as for raising the pH of the growth micro-environment.     

Immunochemical evidence for glutamine-mediated degradation of glutamine synthetase in cultured Chinese hamster cells.

The specific activity of glutamine synthetase in cultured Chinese hamster cells is inversely related to the concentration of glutamine in the surrounding solution. Enzyme specific activity increases 8- to 10-fold when glutamine is removed from serum-free F12 growth media. The induction of glutamine synthetase activity occurs only after glutamine removal and not after the removal of other amino acids (methionine, leucine, or isoleucine). The analysis of the glutamine-mediated decrease in glutamine synthetase activity has been simplified by the finding that depression proceeds in nutrient-free buffered saline solution (141 mM NaCl, 5.4 mM KCl and 30 mM Tricine (pH 7.4). Under these conditions, 0.1 mM cyanide blocks glutamine-mediated depression. The cyanide inhibition is reversed by the addition of 1.0 mM glucose which suggests that ATP is required for depression. Glutamine-mediated depression is temperature-dependent, occurring between 25 and 45 degrees with an optimum rate at 37 degrees. Studies of the time course of induction and depression as a function of glutamine concentration suggest that glutamine regulates the rate at which the enzyme is either modified or degraded. We have employed an antibody prepared against homogeneous Chinese hamster liver glutamine synthetase to measure the amount of glutamine synthetase protein in extracts of cells containing induced or depressed levels of enzyme activity. A highly sensitive immunoprecipitation procedure enables quantitation of nanogram amounts of glutamine synthetase protein. Glutamine synthetase in cell extracts containing induced levels of enzyme activity possesses the same molecular specific activity (ratio of activity to antigenicity) as homogeneous Chinese hamster liver glutamine synthetase. The molecular specific activity of glutamine synthetase is almost the same in extracts of cells with depressed levels of enzyme obtained by growth for short (2 hours) and long (24 hours) times in the presence of glutamine. These data suggest that glutamine-mediated depression of glutamine synthetase results from degradation of enzyme molecules.     

Aminolaevulinate synthetase of Micrococcus denitrificans. Purification and properties of the enzyme, and the effects of growth conditions on the enzyme activity in cells

1. 5-Aminolaevulinate synthetase was detected in extracts of the non-photosynthetic bacterium Micrococcus denitrificans. 2. Activity is high in cells grown anaerobically in a defined nitrate medium, but is low in cells grown in an iron-deficient medium, and in cells grown aerobically. 3. Aminolaevulinate synthetase was purified extensively; it has a molecular weight of about 68000; apparent K(m) values for glycine, succinyl-CoA and pyridoxal phosphate are 12mm, 10mum and 11mum respectively; 2mum-haemin and 14mum-protoporphyrin inhibit by 50%. 4. The low activity of aminolaevulinate synthetase in iron-deficient cells increases on adding iron salts to cells only under conditions where protein synthesis can occur. 5. In defined nitrate medium with a high Ca(2+) concentration anaerobic growth yield is higher, but aminolaevulinate synthetase activity is lower than in cells grown in the medium with a low Ca(2+) concentration. In medium made from AnalaR constituents, growth yield is low and aminolaevulinate synthetase activity is high even in the presence of high concentrations of Ca(2+); on adding Cu(2+) (0.1mum) to the medium growth yield and aminolaevulinate synthetase activity become the same as in non-AnalaR medium. 6. Cells incubated under conditions where protein synthesis does not occur but where electron transport does, lose their aminolaevulinate synthetase activity rapidly. 7. The activities of aminolaevulinate dehydratase and succinic thiokinase do not change under any of the conditions of growth examined. 8. The possible mechanisms controlling aminolaevulinate synthetase activity and the role of this enzyme in controlling the synthesis of haem in this organism are discussed.     

Unusual Valyl-Transfer Ribonucleic Acid Synthetase Mutant of Escherichia coli

Escherichia coli strain NP2907 was isolated as a spontaneous mutant of strain NP29, which possesses a thermolabile valyl-transfer ribonucleic acid (tRNA) synthetase. The valyl-tRNA synthetase of the new mutant, unlike that of its immediate parent, retains enzymatic activity in vitro but differs from the wild-type enzyme in stability and apparent K(m) for adenosine triphosphate. The new mutant locus, valS-102, cotransduces with pyrB at the same frequency as does the parental locus, valS-1. Cultures of strain NP29 cease growth immediately in any medium when shifted from 30 to 40 C. The new mutant grows normally at 30 C, and upon a shift to 40 C growth quickly accelerates exactly as for normal cells. Exponential growth, however, cannot be sustained at 40 C. At a point characteristic for each medium, growth becomes linear with time. This transition occurs almost immediately in rich media and after 1.5 generations in glucose minimal medium. Net synthesis of valyl-tRNA synthetase ceases in the new mutant as soon as the temperature is raised to 40 C, irrespective of the growth medium. We conclude that it is the amount of valyl-tRNA synthetase activity that limits the rate of growth in the linear phase at 40 C. This property of the mutant makes it possible to evaluate the in vivo efficiency of this enzyme at different growth rates and thereby to determine the concentration that is necessary for a given rate of protein synthesis. The results of our measurements indicate that cells of E. coli growing in minimal medium normally possess a functional excess of valyl-tRNA synthetase with respect to protein synthesis and to repression of threonine deaminase.     

Variations in activity of aminoacyl-tRNA synthetases as a function of development in Bacillus subtilis

Extracts from Bacillus sublilis cells at various stages of growth and spores were assayed for aminoacyl-tRNA synthetase and methionyl-tRNA transformylase activity. There was no major change in any synthetase activity or in methionyl-tRNA transformylase activity during the sporulation cycle, which implies that these are not sporulation induced enzymes. However, extracts from B. subtilis cultures showed a burst of activity of aminoacyl-tRNA synthetases during exponential growth.Preparations from dormant spores possessed the same kinds of aminoacyl-tRNA synthetase activities as vegetative cells for all the amino acids which were studied. Spores also contained methionyl-tRNA transformylases. These findings suggest that spores ought to be able to aminoacylate tRNA and formylate the initiator. N-formylmethionyl-tRNA, immediately upon germination.     

Thymidine 5′-Monophosphate-Requiring Mutants of Saccharomyces cerevisiae Are Deficient in Thymidylate Synthetase

Thymidylate synthetase activity was measured in crude extracts of the yeast Saccharomyces cerevisiae by a sensitive radiochemical assay. Spontaneous non-conditional mutants auxotrophic for thymidine 5'-monophosphate (tmp1) lacked detectable thymidylate synthetase activity in cell-free extracts. In contrast, the parent strains (tup1, -2, or -4), which were permeable to thymidine 5'-monophosphate, contained levels of activity similar to those found in wild-type cells. Specific activity of thymidylate synthetase in crude extracts of normal cells or of cells carrying tup mutations was essentially unaffected by the ploidy or mating type of the cells, by the medium used for growth, by the respiratory capacity of the cells, by concentrations of exogenous thymidine 5'-monophosphate as high as 50 mug/ml, or by subsequent removal of thymidine 5'-monophosphate from the medium. Extracts of a strain bearing the temperature-sensitive cell division cycle mutation cdc21 lacked detectable thymidylate synthetase activity under all conditions tested. Its parent and another mutant (cdc8), which arrests with the same terminal phenotype under restrictive conditions, had normal levels of the enzyme. Cells of a temperature-sensitive thymidine 5'-monophosphate auxotroph arrested with a morphology identical to the cdc21 strain at the nonpermissive temperature and contained demonstrably thermolabile thymidylate synthetase activity. Tetrad analysis and the properties of revertants showed that the thymidylate synthetase defects were a consequence of the same mutation causing, in the auxotrophs, a requirement for thymidine 5'-monophosphate and, in the conditional mutants, temperature sensitivity. Complementation tests indicated that tmp1 and cdc21 are the same locus. These results identify tmp1 as the structural gene for yeast thymidylate synthetase.     

Membrane association of the hyaluronate stimulatory factor from LX-1 human lung carcinoma cells

LX-1 human lung carcinoma cells interact with human fibroblasts in culture to cause an increase in hyaluronate production (Knudson et al: Proceedings of the National Academy of Sciences of the United States of America 81:6767, 1984). It is shown here that a similar increase in hyaluronate production also occurs when membranes derived from LX-1 cells, or detergent extracts thereof, are added to cultures of the human fibroblasts. However, no stimulation occurs when membranes or extracts from fibroblasts are added to cultures of the LX-1 cells. The hyaluronate stimulatory factor present in the detergent extracts is a heat- and trypsin-sensitive protein, requires more than 12 h for its action on fibroblasts, causes an elevation in hyaluronate synthetase activity in membranes derived from the fibroblasts, and can be reconstituted into artificial lipid vesicles. Thus, it is concluded that the stimulatory factor is a membrane-bound protein present on the surface of the LX-1 cells and that it interacts with fibroblasts to induce increased hyaluronate synthesis.     

Intracellular localization of mannan synthetase activity in budding baker's yeast

In extracts from budding yeast cells mannan synthetase is present at a much higher activity than in extracts from stationary cells. This activity is largely sedimentable. It is associated with fragments of the plasmalemma, with vesicles known to be involved in the local secretion of glucanases at the site of budding, and with ‘light membranes’ representing a mixed fraction which probably contains fragments of the endoplasmic reticulum. The possible involvement of these structures in the synthesis and secretion of mannanprotein is discussed.     

Glyoxylate metabolism in growth and sporulation of Bacillus cereus

Megraw, Robert E. (Iowa State University, Ames), and Russell J. Beers. Glyoxylate metabolism in growth and sporulation of Bacillus cereus. J. Bacteriol. 87:1087-1093. 1964.-Isocitrate lyase and malate synthetase were found in cell-free extracts of Bacillus cereus T. The patterns of synthesis of enzymes of the glyoxylic acid cycle were dependent upon the medium in which the organism was grown. Cells grown in acetate or in an acetate precursor, such as glucose, produced enzymes of the glyoxylic acid cycle in greatly diminished quantities, as compared with cells grown in media containing glutamate or yeast extract as principal carbon sources. Glutamate-grown cells had high isocitrate lyase activity but very low malate synthetase activity. Glyoxylate produced in this situation is metabolized by alternate pathways: conversion to tartronic semialdehyde and the latter to glyceric acid, thus providing evidence for a glycerate pathway; and reduction to glycolate (the reverse of this reaction was present at a low rate). Enzymatic activity of the glyoxylic acid cycle declines at the point where sporogenesis begins, indicating a metabolic shift for the synthesis of spore material.     

Carbon and ammonia metabolism of Spirillum lipoferum.

Intact cells and extracts from Spirillum lipoferum rapidly oxidized malate, succinate, lactate, and pyruvate. Glucose, galactose, fructose, acetate, and citrate did not increase the rate of O2 uptake by cells above the endogenous rate. Cells grown on NH+/4 oxidized the various substrates at about the same rate as did cells grown on N2. Added oxidized nicotinamide adenine dinucleotide generally enhanced O2 uptake by extracts supplied organic acids, whereas oxidized nicotinamide adenine dinucleotide phosphate had little effect. Nitrogenase synthesis repressed by growth of cells in the presence of NH+/4 was derepressed by methionine sulfoximine or methionine sulfone. The total glutamine synthetase activity from N2-grown cells was about eight times that from NH+/4-grown S. lipoferum; the response of glutamate dehydrogenase was the opposite. The total glutamate synthetase activity from N2-grown S. lipoferum was 1.4 to 2.6 times that from NH+/4-grown cells. The levels of poly-beta-hydroxybutyrate and beta-hydroxybutyrate dehydrogenase were elevated in cells grown on N2 as compared with those grown on NH+/4. Cell-free extracts capable of reducing C2H2 have been prepared; both Mg2+ and Mn2+ are required for good activity.     

Metabolic stability of the two forms of initiation factor IF-3 in Escherichia coli during the growth cycle.

Possible alteration in the ratio of the long and short forms of initiation factor IF-3 (FEBS Lett. 79, 264-275, 1977) during the growth cycle of Escherichia coli was examined. The ratio was found to remain unchanged between the exponential and stationary growth phases. Contrary to an earlier report (Eur. J. Biochem. 29, 319-325, 1972), the total amount of IF-3 relative to the ribosome content in stationary phase cells was essentially the same as in midlogarithmic phase cells. The activity of IF-3, assayed after its separation from other initiation factors by chromatography, was also the same in extracts from midlogarithmic and stationary phase cells. The data show that in Escherichia coli the ratio of IF-3/ribosome is maintained constant. The ribosomes themselves have been shown to retain virtually full activity in vitro during this transition indicating that growth-cycle-dependent biochemical modifications of the ribosome do not affect its protein synthetic capacity per se.     

Control of δ-aminolaevulate synthetase activity in Rhodopseudomonas spheroides

1. delta-Aminolaevulate synthetase from Rhodopseudomonas spheroides grown semi-anaerobically undergoes a spontaneous activation during the first hour after the disruption of cells when homogenates are stored at 4 degrees . 2. After cultures of R. spheroides growing semi-anaerobically are oxygenated no activation of delta-aminolaevulate synthetase occurs in cell extracts. Cessation of activation in extracts is almost complete 10min. after oxygenation of cells has begun. 3. A heat-stable fraction of low molecular weight from semi-anaerobic cells reactivates delta-aminolaevulate synthetase in extracts of oxygenated cells and appears to contain a compound responsible for the spontaneous activation. 4. A heat-stable fraction of low molecular weight from oxygenated cells inhibits the spontaneous activation in extracts of semi-anaerobic cells. 5. The effect of oxygen on the rate of bacteriochlorophyll synthesis in R. spheroides may be mediated through alterations in the concentrations of a low-molecular-weight activator and inhibitor of delta-aminolaevulate synthetase.     

CHOLINE METABOLISM AND MEMBRANE FORMATION IN RAT HEPATOMA CELLS GROWN IN SUSPENSION CULTURE : II. Phosphatidylcholine Synthesis during Growth Cycle and Fluctuation of Mitochondrial Density

The incorporation of methyl-labeled choline into phosphorylcholine and phosphatidylcholine of cellular membranes by Novikoff rat hepatoma cells (line N1S1-67) during growth in suspension culture was investigated. Upon initiation of a fresh culture at 10⁵ cells/ml, the rate of synthesis of phosphorylcholine by the cells was four to five times greater than that of the synthesis of phosphatidylcholine. While the rate of synthesis of the latter remained relatively constant, the rate of phosphorylation of choline decreased progressively during the course of the growth cycle of the culture to 10–20% of the initial rate when the culture reached stationary phase at 3 x 10⁶ cells/ml. The decrease in phosphorylcholine synthesis during the growth cycle was not due to depletion of choline in the medium or a decrease in its concentration, but was correlated with a decrease in choline kinase activity of the cells as measured in cell-free extracts. Newly synthesized phosphatidylcholine was detectable in cells only as an integral part of cellular membranes. Its distribution among various cytoplasmic membrane structures separated by isopycnic centrifugation in sucrose density gradients remained relatively constant during the growth cycle. About 50% was associated with the mitochondria, and the remainder with plasma membrane fragments and other membranous structures with mean densities of about 1.15 and 1.13 g/cm³, respectively. However, the density of the mitochondria increased from about 1.167 g/cm³ in early exponential phase cells to about 1.190 g/cm³ in stationary phase cells. The finding that the density of the entire propulation of mitochondria changed simultaneously and progressively is in agreement with the view that mitochondria grow by addition of phospholipids and structural proteins and increase in number by division.     

Differential effects of Mg2+ on various hydrolytic and synthetic activities of multifunctional glucose-6-phosphatase

The adaptive synthesis of fatty acid synthetase in the livers of rats fed a fat-free diet following 48 hr of fasting has been studied using immunochemical methods. The development of fatty acid synthetase activity during adaptive synthesis occurs about 3 hr following feeding, whereas the synthesis of material precipitable by anti-fatty acid synthetase serum, as judged by the incorporation of ³H-labeled amino acids into the immunoprecipitate, commenced within 1 hr. Extracts of liver of rats fed a fat-free diet for 1–3 hr following fasting contain increasing amounts of material which competes with purified fatty acid synthetase for antibody binding sites, even though they have no fatty acid synthetase activity. This suggests the presence of enzymatically inactive precursors of fatty acid synthetase in the liver extracts. The incorporation of [¹⁴C]pantothenate into fatty acid synthetase during adaptive synthesis follows the same pattern as the development of enzyme activity, indicating that these enzymatically inactive precursors of fatty acid synthetase may represent an apoenzyme which is converted to the enzymatically active holoenzyme by the incorporation of the 4′-phosphopantetheine prosthetic group. The subcellular site of synthesis of fatty acid synthetase was shown to be in the pool of polysomes that are not membrane bound, rather than in the rough endoplasmic reticulum.     

Phospholipid Alterations During Growth of Escherichia coli

As cultures of Escherichia coli progressed from the exponential growth phase to the stationary growth phase, the phospholipid composition of the cell was altered. Unsaturated fatty acids were converted to cyclopropane fatty acids, and phosphatidyl glycerol appears to have been converted to cardiolipin. With dual isotope label experiments, the kinetics of synthesis of cyclopropane fatty acid for each of the phospholipids was examined in vivo. The amount of cyclopropane fatty acid per phospholipid molecule began to increase in phosphatidyl ethanolamine at a cell density below the density at which this increase was observed in phosphatidyl glycerol or cardiolipin. The rate of this increase in phosphatidyl glycerol or in cardiolipin was faster than the rate of increase in phosphatidyl ethanolamine. After a few hours of stationary-phase growth, all the phospholipids were equally rich in cyclopropane fatty acids. It is suggested that the phospholipid alterations observed are a mechanism to protect against phospholipid degradation during stationary phase growth. Cyclopropane fatty acid synthetase activity was assayed in cultures at various stages of growth. Cultures from all growth stages examined had the same specific activity in crude extracts.     

Effect of alpha factor pheromone on the activity of enzymes which catalyze the synthesis and hydrolysis of cell wall structural polymers from Saccharomyces cerevisiae

Cells of Saccharomyces cerevisiae of the a mating type treated with alpha factor contain an increased amount of structural polymers (beta-glucans and chitin) in their cell walls and, consequently, exhibit higher glucan synthetase and chitin synthetase activities than untreated cells. However, alpha factor has no detectable effect on the activities of these enzymes when they are assayed, "in vitro", in the presence of the pheromone. On the other hand, the activity of beta-glucanases remains constant during the time that growth of a cells is kept arrested by alpha factor at the G1 phase of the cell division cycle and starts to increase when budding of the cells is reinitiated.