Phosphoenolpyruvate synthesis by fetal guinea-pig liver mitochondria

Liver mitochondria isolated from fetal and newborn guinea pigs synthesized phosphoenolpyruvate at 4–6 nmol/min per mg protein with 2 mM malate, succinate, and α-ketoglutarate as substrates. These rates were 90–110% of that by adult liver mitochondria and were not substantially altered in the second half of gestation or within 24 h after birth. Both palmitoyl- and octanoylcarnitine were inhibitory to phosphoenolpyruvate synthesis in adult and fetal preparations, but free octanoate was inhibitory only in adult liver mitochondria.     

Inhibition of RNA synthesis in animal cells by sodium selenite

The effect of selenium-containing compounds on RNA synthesis in rat liver cells was studied in vivo. All the selenium derivatives under study inhibited the rRNA synthesis in liver cells. The most potent inhibiting effect was exerted by sodium selenite. It was shown that in a cell-free system of RNA biosynthesis sodium selenite selectively inhibited the activity of RNA-polymerase I in isolated nuclei and purified enzyme preparations of normal and tumour cells. A feasible mechanism of inhibition of the RNA-polymerase I activity by selenium and a hypothesis on the anticarcinogenic effect of selenium are postulated.     

Characterization of phosphatidylserine synthesis and translocation in permeabilized animal cells

The synthesis of phosphatidylserine and its translocation to the mitochondria were examined in permeabilized Chinese hamster ovary (CHO)-K1 cells by following the metabolism of a [3H]serine precursor to [3H] phosphatidylserine (PtdSer) and [3H]phosphatidylethanolamine (PtdEtn). In physiological salt solutions approximating the intracellular ionic composition, both the synthesis of PtdSer and its translocation required ATP. The ATP requirement for PtdSer synthesis could be completely bypassed, and that for translocation could be partially bypassed at Ca2+ concentrations 10(3)-10(4) times the intracellular physiological level (i.e. 1 mM). The ATP-dependent synthesis of PtdSer could be inhibited by chelation of Ca2+ with EGTA, inhibition of Ca2+ sequestration with 2,5-di(tert-butyl)hydroquinone, mobilization of sequestered Ca2+ with ionomycin, and competition for [3H]serine with ethanolamine. The inhibition of the ATP-dependent synthesis of PtdSer by the aforementioned inhibitors provided an efficient method to rapidly arrest the incorporation of [3H]serine into [3H]PtdSer. By pulse-labeling the [3H]PtdSer pool and arresting further synthesis with inhibitors, the translocation of nascent PtdSer could be uncoupled from synthesis. The results of these pulse-labeling-arrest experiments provide unambiguous evidence that PtdSer translocation to the mitochondria is not driven by PtdSer synthesis. The addition of apyrase to ATP-supplemented, permeabilized cells abruptly terminates [3H]serine incorporation into [3H]PtdSer and the decarboxylation of [3H]PtdSer to [3H]PtdEtn, thereby demonstrating that a specific ATP requirement exists for the translocation of nascent PtdSer to the mitochondria in permeabilized cells. The translocation of nascent PtdSer to the mitochondria was unaffected by 45-fold dilution of the standard reaction thus indicating that the translocation intermediate was unlikely to be a freely diffusible complex. The requirements for translocation of nascent phosphatidylserine are different from those for the vesicular movement of proteins insofar as the lipid movement does not require cytosol and is unaffected by the addition of Ca2+, GTP, or GTP gamma S. From these studies, we conclude that: 1) the synthesis and translocation of PtdSer can be readily studied in permeabilized cells, 2) the ATP-dependent synthesis of PtdSer is functionally coupled to the ATP-dependent sequestration of Ca2+ by the endoplasmic reticulum or closely related membranes, 3) PtdSer translocation is independent of its synthesis, and 4) there is a specific requirement for ATP in the translocation of PtdSer to the mitochondria.     

Phosphoenolpyruvate carboxykinase is induced in growth-arrested hepatoma cells

Phosphoenolpyruvate carboxykinase (PEPCK) mRNA is elevated in H4IIEC3 rat hepatoma cells cultured at high density, suggesting that PEPCK expression and growth arrest may be coordinately regulated. Induction of growth arrest either by contact inhibition (high culture density) or by serum deprivation correlated with significant increases in PEPCK protein and its mRNA. The observation that PEPCK mRNA was induced by contact inhibition in the presence of serum indicates that the effect of high density is independent of insulin or any other serum component. The magnitudes of the changes in PEPCK expression during growth arrest were greatly enhanced in KRC-7 cells, an H4IIEC3 subclone that is much more sensitive to growth arrest than its parental cell line. Restimulation of proliferation in growth-arrested KRC-7 cells, either by addition of serum or insulin to serum-deprived cells or by replating contact-inhibited cells at low density, caused a rapid decrease in PEPCK expression. However, PEPCK mRNA is not always reduced in proliferating cells since treatment of serum-starved cells with epidermal growth factor stimulated entry into the cell cycle but did not affect PEPCK mRNA levels. Finally, dexamethasone induction of PEPCK mRNA was blunted in cells cultured at high density but was unaffected by the presence or absence of serum. Collectively, these data suggest the possibility of cross-talk between the control of PEPCK expression and growth arrest in KRC-7 cells.     

Uridine transport and RNA synthesis in growing and in density-inhibited animal cells

Both chick embryo fibroblasts and mouse 3T3 cells reduce the rate at which they incorporate H³ uridine into RNA as their growth becomes inhibited at high cell density. This reduction occurs as a function of the cell population density, and with chick embryo cells (in contrast to 3T3 cells) it is not accompanied by significant medium alterations. This indicates the importance of the cell population density in the control of cellular metabolism. The decline in H³ uridine incorporation is paralleled by a decline in the rate of uptake of the isotope into the acid-soluble pool, suggesting that decreased entry of H³ uridine into the cell, rather than a decreased rate of RNA synthesis, is responsible for the reduced rate of incorporation into RNA of density-inhibited cells. This suggestion was confirmed by finding that when the restriction on uridine uptake was overcome by increasing the concentration of uridine in the medium, the density-dependent inhibition of uridine incorporation was largely reversed. We conclude that, even though the rate of H³ uridine incorporation into RNA is reduced three- to five-fold in density-inhibited cells, the rate of synthesis of pulse-labeled RNA continues at 70 to 85% of the rapidly-growing rate.     

Nitric oxide: comparative synthesis and signaling in animal and plant cells

Since its identification as an endothelium-derived relaxing factor in the 1980s, nitric oxide has become the source of intensive and exciting research in animals. Nitric oxide is now considered to be a widespread signaling molecule involved in the regulation of an impressive spectrum of mammalian cellular functions. Its diverse effects have been attributed to an ability to chemically react with dioxygen and its redox forms and with specific iron- and thiol-containing proteins. Moreover, the effects of nitric oxide are dependent on the dynamic regulation of its biosynthetic enzyme nitric oxide synthase. Recently, the role of nitric oxide in plants has received much attention. Plants not only respond to atmospheric nitric oxide, but also possess the capacity to produce nitric oxide enzymatically. Initial investigations into nitric oxide functions suggested that plants use nitric oxide as a signaling molecule via pathways remarkably similar to those found in mammals. These findings complement an emerging body of evidence indicating that many signal transduction pathways are shared between plants and animals.     

The regulation of protein synthesis in animal cells by serum factors.

We have investigated the regulation of protein synthesis in animal cells by serum factors. Withdrawal of serum from the medium of actively dividing Vero cells resulted in an immediate decline in the rate of peptide chain elongation (Hassell and Engelhardt, 1973). Assay of elongation factor I (EFI) activity in the post-ribosomal supernatant as well as that associated with the ribosomes revealed that serum deprivation resulted also in reduction in the activity of this factor. The decline in the activity of EFI after serum deprivation occurred to the same extent and at the same time as the decline in the in vivo rate of protein synthesis and the in vitro peptide synthetic capacity of cell-free extracts. A temporal correlation therefore exists among the in vivo rate of protein synthesis, the peptide synthetic activity of cell-free extracts, and the activity of EFI. The activity of peptidyl transferase was not altered by serum deprivation. The loss of extract peptide synthetic activity resulting from serum deprivation was reversible since serum addition to previously serum-starved cultures resulted in full restoration of activity for polyphenylalanine (polyPhe) synthesis within 3 h. Moreover, RNA synthesis was not required for this turn-on of polyPhe synthesis. Vased on these data we conclude that a translational control mechanism is operative in Vero cells deprived of serum.