Accumulation of glycinebetaine and its synthesis from radioactive precursors under salt-stress in the cyanobacterium Aphanothece halophytica

Growth in salt-stressed (2.0 M NaCl) Aphanothece halophytica was initially delayed during the first two days of cultivation and eventually attained the same growth rate as the control (0.5 M NaCl) cells. Glycinebetaine accumulation increased slightly in control cells but a dramatic increase of glycinebetaine occurred in salt-stressed cells during a growth period of six days. There was no apparent increase in the synthesis of [¹⁴C] glycinebetaine in the control cells, in contrast to the marked increase in its synthesis in the salt-stressed cells. Increasing NaCl concentration in the growth medium induced both the accumulation and the synthesis of glycinebetaine. Time course experiments provided evidence that [¹⁴C] choline was first oxidized to [¹⁴C] betaine aldehyde which was further oxidized to [¹⁴C] glycinebetaine in A. halophytica. The supporting data for such a pathway were obtained from the presence of choline and betaine aldehyde dehydrogenase activities found in the membrane and cytoplasmic fractions, respectively. The activities of these two enzymes were also enhanced upon increasing NaCl concentration in the growth medium from 0.5 M to 2.0 M. Under this condition an increaseof approximately 1.5-fold was observed for choline dehydrogenase activity as compared to 2.5-fold for betaine aldehyde dehydrogenase activity, suggesting a preferable induction of the latter enzyme by salt stress. A. halophytica was able to utilize [¹⁴C] ethanolamine and [¹⁴C] glycine for the synthesis of [¹⁴C] glycinebetaine. This revised version was published online in August 2006 with corrections to the Cover Date.     

Beta-adrenergic receptors and adenylate cyclase in hypertrophic and hyperplastic rat salivary glands

Isoproterenol induces both the secretion of protein and the stimulation of DNA synthesis and growth in rat salivary glands.The specific binding of the labelled beta-adrenergic antagonist [³H]dihydroalprenolol has been used to measure the number of beta-adrenergic receptors in rat parotid glands during isoproterenol-induced growth. Isoproterenol-enlarged glands display no change in the specific binding capacity per gland for [³H]-dihydroalprenolol compared with normal tissue.Catecholamine sensitive adenylate cyclase activity varies independently of the number of specific [³H]dihydroalprenolol binding sites during isoproterenol-induced growth.Previously-described differences in optimal isoproterenol doses which produce protein secretion and stimulation of DNA synthesis may reflect different responses to various rates of receptor occupancy, or may be due to the presence of more than one type of beta-adrenergic receptor.     

Rapid intracellular degradation of newly synthesized collagen by bone cells Effect of dichloromethylenebisphosphonate

Experiments were carried out to determine whether bone cells isolated from rat calvaria degrade newly synthesized collagen intracellularly prior to secretion and to assess the effect of dichloromethylenebisphosphonate, a compound shown to stimulate collagen synthesis during this event. The findings indicate that isolated bone cells grown in culture degraded a proportion (average 16%) of newly synthesizes collagen prior to secretion. This process was markedly reduced by exposure to dichloromethylenebisphosphonate in a dose-related manner. Concomitantly with the observed decrease of degradation, an increase of collagen synthesis was detected as determined by the incorporation of [³H]proline into collagenase-digestible proteins or by the conversion of [³H]proline into [³H]hydroxyproline. No similar enhancement on total non-collagenous protein synthesis was evident. Dichloromethylenebisphosphonate did not influence the extracellular degradation of collagen. Although the reduction in intracellular degradation accounted only for part of the bisphosphonate mediated increase in net collagen synthesis, it is conceivable that the rate of collagen synthesis is regulated, at least in part, by mechanisms that modulate the level of intracellular degradation.     

Cycles of protein synthesis during pupal diapause in the flesh fly,Sarcophaga crassipalpis

Protein synthesis is cyclic during pupal diapause in Sarcophaga crassipalpis. These cycles are in phase with infradian MO₂ cycles, which have a periodicity of about 4 days at 25°C. Mean incorporation of [³⁵S]methionine by diapausing pupae was 5.4% during the 2 days of highest MO₂ but dropped to 1.7% during the 2 days of low MO₂. Diapausing pupae treated with a juvenile hormone analog prior to pupariation had a constant high MO₂ similar to peak values observed in untreated pupae, and such pupae consistently incorporated [³⁵S]methionine at a high rate (7.7%). [³⁵S]Methionine incorporation by nondiapausing pupae and pharate adults was eightfold higher than the peak rates observed during diapause. Autoradiography of in vivo labeled proteins indicated quantitative and qualitative changes in the synthesis of proteins by diapausing pupae during different phases of the MO₂ cycle. Brains from diapausing pupae labeled in vitro showed higher incorporation at the peak of the MO₂ cycle than at the nadir of the cycle, but no such differences were detected for integument, fat body, or fat body supernatant. Theses differences in tissue response indicate that control of protein synthesis during diapause is not cell autonomous, but is a function of the metabolism of the intact organism.     

Prolyl hydroxylase activity in L-929 fibroblasts incubated with and without ascorbate

An improved procedure was used to assay prolyl hydroxylase activity in both early-log and late-log L-929 fibroblasts grown on plastic surfaces. When 40 μg/ml of ascorbate was added to early-log phase cultures, the rate of hydroxy-[¹⁴C] proline synthesis increased 2-fold within 4 h, but there was no change in prolyl hydroxylase activity per cell. The results indicated therefore that ascorbate did not “activate” prolyl hydroxylase in the sense of converting inactive enzyme protein to active enzyme protein. Instead ascorbate appeared to increase hydroxyproline synthesis in early-log L-929 fibroblasts because the prolyl hydroxylase reaction in such cells was limited by the availability of ascorbate or a similar cofactor. When 40 μg/ml of ascorbate was added to late-log phase cultures, there was essentially no effect on the rate of hydroxyl[¹⁴C]-proline synthesis or prolyl hydroxylase activity. The late-log phase cells, however, contained three times more enzyme activity and about two times more immuno-reactive enzyme protein than early-log phase cells. In addition, the rate of protein synthesis per cell in late-log phase cells was only one-tenth the rate in early-log phase cells. The results suggested that as the cells grew to confluency, collagen polypeptides were more completely hydroxylated in part because the rate of polypeptide synthesis decreased and at the same time prolyl hydroxylase activity per cell increased. The results appear to provide an alternate explanation for previous observations on the effects of ascorbate and “crowding” on hydroxy[roline synthesis in cultures of L-929 fibroblasts.     

Compartmentation of NADPH in rat liver.

Rat liver slices were incubated with specifically ³H-labeled glucoses and [2-³H]sorbitol, and the incorporations of ³H into fatty acids and cholesterol were determined. Incorporation of ³H from [1-³H]glucose relative to that from [3-³H]glucose via NADPH formed in the pentose cycle was similar into fatty acids and cholesterol. This indicates (1) the presence of a common pool of NADPH formed via the pentose cycle, from which is derived the reductive hydrogens for fatty acid and cholesterol synthesis; (2) the absence of a major separate pool of NADPH formed from glucose by microsomal glucose dehydrogenase (EC 1.1.1.47) catalysis for use in cholesterol synthesis. ³H from [4-³H]glucose and from [2-³H]sorbitol was incorporated into cholesterol more than into fatty acids relative to the incorporations of ³H from [3-³H]glucose. Assuming that the ³H from [4-³H]glucose and from [2-³H]sorbitol were incorporated via the conversion, catalyzed by malic enzyme, of NADH to NADPH, this indicates the Compartmentation of the NADPH formed via malic enzyme catalysis from that formed via the pentose cycle. Alternatively, NADH provides reductive hydrogens for cholesterol synthesis in greater measure than in fatty acid formation or the stereochemistry of the synthetic processes are such that [A-³H]NADPH has greater excess than [B-³H]NADPH to cholesterol synthesis relative to fatty acid synthesis.     

Nuclear protein synthesis and phosphorylation in Friend erythroleukemia cells stimulated with DMSO

Patterns of nuclear protein synthesis and phosphorylation have been investigated in Friend erythroleukemia cells. The rate of incorporation of [³H]leucine and [³²P]phosphate remains relatively constant during the first 48 h of dimethylsulfoxide (DMSO) stimulation, when more than 90% of the cells commit to erythroid differentiation, but falls to 20% by 120 h. Histone H2A phosphorylation is greatly increased during DMSO treatment, but no significant changes were found in the non-histone phosphoprotein patterns as determined by gel electrophoresis. There is also a small, but reproducible, change in the relative amounts of the two sub-fractions of histone H2A. There are no striking changes in the electrophoretic patterns of [¹⁴C]leucine-labelled nuclear proteins during the first 48 h, but the amount and the synthesis of two proteins of 46 000 and 280 000 D are increased somewhat during this period. Another protein, of molecular weight 65 000, appears to be induced in low amounts.     

Evidence that the lipid carrier for N-acetylglucosamine is different from that for mannose in mung beans and cotton fibers

Cell-free enzyme particles from mung beans (Phaseolus aureus) or cotton (Gossypium hirsutum L.) fibers catalyze the incorporation of mannose from GDP-[¹⁴C]mannose and N-acetylglucosamine from UDP-[³H]-N-acetylglucosamine into polyprenyl-type lipids. These lipids have been synthesized and purified and the lipid moieties compared to each other as well as to dolichyl phosphate and to lipids isolated from similar mannoseand N-acetylglucosamine-containing lipids from liver and aorta.

The following lines of evidence indicate that in plants, the lipid carrier for N-acetylglucosamine is different from the lipid carrier for mannose: [List: see text]

We propose that the apparent difference in the lipid carrier for these two sugars may be a point of control of glycoprotein synthesis.

     

Emetine Binding to Ribosomes of Entamoeba histolytica—Inhibition of Protein Synthesis and Amebicidal Action*

After demonstration that emetine is amebicidal by inhibiting protein synthesis, the question arose whether active protein synthesis is required for emetine's amebicidal effect. The answer appears to be “no,” as derived from experiments on intact amebae. Responses were compared for log- and stationary-growth phase amebae. In the latter, protein synthesis is significantly slower, and sensitivity to emetine, i.e. degree of inhibition of protein synthesis, was maintained independently of rate of protein synthesis. Both stages equally bound tritiated emetine to their ribcsomes. Binding of [³H]emetine was not affected by certain drugs that interfere with energy metabolism, protein synthesis, and/or ribosomal function, e.g. dinitrophenol, puromycin, chloroquine, and acriflavin. High concentrations of EDTA combined with puromycin (which disaggregates ribosomes into their subunits) lowered binding by 50%. In chase experiments the ribosomes of intact amebae were prelabeled with [³H]emetine or [³H]isoemetine, then exposed to relatively high concentrations of unlabeled emetine. Labeled isoemetine was displaced almost completely, whereas no displacement of [³H]emetine occurred; evidently, the high stability of the emetine-ribosome binding is due in part to a hydrogen-bonding reaction of the C-1' atom of the emetine molecule with the chain-elongation site. Finally, evidence was obtained that capacity to bind emetine is an index of drug resistance.     

Inhibition of isocitrate lyase from Pseudomonas indigofera by itaconate

The effect of the inhibitor itaconate on the activity of purified isocitrate lyase from Pseudomonas indigofera was examined for the reaction in both directions. Itaconate was found to equilibrate very slowly with its enzyme-bound form, so that a rapid change in itaconate concentration produced a gradual change in reaction velocity which eventually reached a new steady state. Kinetic studies of this relaxation phenomenon indicated that itaconate inhibited by binding the enzyme only after prior binding of glyoxylate, thus mimicking the kinetic behavior of succinate. On the basis of these studies, the dissociation constants for itaconate and glyoxylate from their respective enzyme-bound forms were calculated. More than half of the isocitrate lyase was complexed by glyoxylate during cleavage of saturating isocitrate. The rate constant for release of itaconate from the enzyme was calculated to be about 0.2 min^?1. Direct binding of [¹⁴C]itaconate and [¹⁴C]succinate to isocitrate lyase at pH 6.8 was measured. Some binding of both ligands was found in the absence of glyoxylate, which was stimulated by the presence of 1 mm glyoxylate. These results suggest that there are up to three or more binding sites per active subunit, but that only one of these is catalytic.     

Co-ordinated synthesis of phytoalexin biosynthetic enzymes in biologically-stressed cells of bean (Phaseolus vulgaris L.)

Changes in the rates of synthesis of three enzymes of phenyl-propanoid biosynthesis in Phaseolus vulgaris L. (dwarf French bean) have been investigated by immunoprecipitation of [³⁵S]methionine-labeled enzyme subunits with mono-specific antisera. Elicitor causes marked, rapid but transient co-ordinated increases in the rate of synthesis of phenyl-alanine ammonia-lyase, chalcone synthase and chalcone isomerase concomitant with the phase of rapid increase in enzyme activity at the onset of accumulation of phenyl-propanoid-derived phytoalexin antibiotics in suspension cultures of P. vulgaris. Co-ordinate induction of enzyme synthesis is also observed in hypocotyl tissue during race:cultivar-specific interactions with Colletotrichum lindemuthianum, causal agent of anthracnose. In an incompatible interaction (host resistant) there are early increases apparently localized to the initial site of infection prior to the onset of phytoalexin accumulation and expression of hypersensitive resistance. In contrast, in a compatible interaction (host susceptible) there is no induction of synthesis in the early stages of infection, but a delayed widespread response at the onset of lesion formation associated with attempted lesion limitation. It is concluded that expression of the phytoalexin defense response in biologically stressed cells of P. vulgaris characteristically involves co-ordinate induction of synthesis of phytoalexin biosynthetic enzymes.     

Protein metabolism in senescing wheat leaves : determination of synthesis and degradation rates and their effects on protein loss

Wheat leaves (Triticum aestivum L.) at the moment of their maximum expansion were detached and put in darkness. Their protein, RNA and DNA contents, as well as their rates of protein synthesis and degradation, were measured at different times from 0 to 5 days after detachment. Rates of protein synthesis were measured by incorporation into proteins of large amounts of [³H]leucine. Fractional rates of protein degradation were estimated either from the difference between the rates of synthesis and the net protein change or by the disappearance of radioactivity from proteins previously labeled with [³H]leucine or [¹⁴C]proline.

Protein loss reached a value of 20% during the first 48 hours of the process. RNA loss paralleled that of protein, whereas DNA content proved to be almost constant during the first 3 days and decreased dramatically thereafter.

Measurements of protein synthesis and degradation indicate that, in spite of a slowdown in rate of protein synthesis, an increased rate of protein breakdown is mainly responsible for the observed rapid protein loss.

     

The turnover of deoxyuridine triphosphate during the HeLa cell cycle

The synthesis and breakdown of deoxyuridine triphosphate (dUTP) was studied to determine whether a dUTP pool is present at any stage of the HeLa cell cycle. Although cell extracts were found to be capable of phosphorylating dUMP to dUTP, only minimal quantities of intracellular dUMP, dUDP or dUTP could be detected. When thymidylate synthetase was blocked with FUdR the dUMP pool increased but no substantial increase in dUDP or dUTP was seen. A powerful and specific dUTP nucleotidohydrolase (dUTPase, EC3.6.1.23) which hydrolyses dUTP to dUMP and PPi was detected. The activity of this enzyme as well as that of the dUTP synthesizing enzymes was low in G1, rose through S and G2 and reached a maximum just prior to cell division. Pulsing experiments with [5-³H]UdR and [¹⁴C]TdR suggest that the size of the dUTP pool is 1% of the dTTP pool.     

Protein and glycerolipid biosynthesis in isolaged intestinal epithelial cells of normal and bile-fistula rats.

Protein and glycerolipid biosynthesis was studied in isolated epithelial cells of the intestinal mucosa of normal and 1-day bile-fistula rats. In cells from fistula rats, protein synthesis from [1-¹⁴C]leucine was decreased 40–45% and glycoprotein synthesis from [1-¹⁴C]glucosamine 25–30%. Under comparable conditions, the synthesis of phosphatidylcholine from a variety of precursors was decreased 40–45% while no change was observed in the formation of either phosphatidylethanolamine or triacylglycerols. Chylomicron release was inhibited 70–80% in the cells from the bile-fistula rats. In vitro addition of either lysophosphatidylcholine or choline to the cells from the bile-fistula animals resulted in greatly increased phosphatidylcholine and protein biosynthesis and an effective release of chylomicrons. It is suggested that this stimulation of incorporation of label is due to a net synthesis of phosphatidylcholine required for membrane and lipoprotein repairs in the fistula cells. These results provide further evidence, that, in the rat, biliary phosphatidylcholine may play an essential role in the formation and clearance of chylomicrons from the intestinal mucosa.     

Hormonal effects on RNA synthesis by stage 6 oocytes of Xenopus laevis.

RNA synthesis has been studied in “large” oocytes of Xenopus laevis, both as a function of time after injection of females with human chorionic gonadotropin (HCG) and in relation to the induction of maturation with progesterone in vitro. Rates of RNA synthesis were measured by analyzing the kinetics of incorporation of exogenous [³H]guanosine, and microinjected [³H]- or [¹⁴C]GTP, into acid-precipitable material, coupled with measurements of precursor pool specific activity. The kinetics of incorporation into RNA of injected precursor are biphasic, indicating the synthesis of both stable and unstable RNA species. Estimates of the total rate of synthesis (stable and unstable) were derived from fitting a linear function to data over the first 60–90 min, while a linear function fit to the data beyond 90 min represented largely the synthesis of stable RNA species.Exposure of oocytes to progesterone had no effect on initial synthetic rates, but maturing oocytes synthesized stable RNA at 1.4–1.6 times the rate in control oocytes. A comparison of data obtained with oocytes from unstimulated (no prior HCG treatment) and HCG-stimulated females indicated that HCG has no substantial effect on rates of RNA synthesis. The significance of continued RNA synthesis in large full grown oocytes is discussed.     

The inhibition of oxalate biosynthesis in isolated perfused rat liver by DL-phenyllactate and n-heptanoate

Glycolate oxidase was isolated and partially purified from human and rat liver. The enzyme preparation readily catalyzed the oxidation of glycolate, glyoxylate, lactate, hydroxyisocaproate and α-hydroxybutyrate. The oxidation of glycolate and glyoxylate by glycolate oxidase was completely inhibited by 0.02 m dl-phenyllactate or n-heptanoate. The oxidation of glyoxylate by lactic dehydrogenase or xanthine oxidase was not inhibited by 0.067 m dl-phenyllactate or n-heptanoate. The conversion of [U-¹⁴C] glyoxylate to [¹⁴C] oxalate by isolated perfused rat liver was completely inhibited by dl-phenyllactate and n-heptanoate confirming the major contribution of glycolate oxidase in oxalate synthesis. Since the inhibition of oxalate was 100%, lactic dehydrogenase and xanthine oxidase do not contribute to oxalate biosynthesis in isolated perfused rat liver. dl-Phenyllactate also inhibited [¹⁴C] oxalate synthesis from [1-¹⁴C] glycolate, [U-¹⁴C] ethylene glycol, [U-¹⁴C] glycine, [3-¹⁴C] serine, and [U-¹⁴C] ethanolamine in isolated perfused rat liver. Oxalate synthesis from ethylene glycol was inhibited by dl-phenyllactate in the intact male rat confirming the role of glycolate oxidase in oxalate synthesis in vivo and indicating the feasibility of regulating oxalate metabolism in primary hyperoxaluria, ethylene glycol poisoning, and kidney stone formation by enzyme inhibitors.     

De novo synthesis and hormonal regulation of a dipeptidase in Cucurbita maxima

The following evidence was obtained for the de novo synthesis of dipeptidase in squash (Cucurbita maxima Duch. var. Hubbard) cotyledons during germination: (i) the amount of [¹⁴C]leucine incorporated into the dipeptidase was greater than that found in other proteins; (ii) the enzyme coincided with a peak of radioactivity in DEAE column chromatography; and (iii) the specific radioactivity of the enzyme increased with purification. There was also a positive correlation between the rate of [¹⁴C]leucine incorporation into dipeptidase and the rate of dipeptidase development. Four plant growth regulators, gibberellic acid (GA) benzyladenine (BA), indol-3-acetic acid (IAA), and abscisic acid (ABA) were examined for their effect on the development of dipeptidase activity at 5 × 10^?6 and 5 × 10^?5 M. None of these regulators affected the activity of the isolated dipeptidase per se. In intact see ds, BA and IAA inhibited the development of dipeptidase activity at the higher concentration, ABA reduced the activity at both concentrations; however, GA enhanced its development at the higher concentration. In distal-half cotyledons, BA and GA stimulated enzyme development but they showed no synergistic effect. IAA suppressed the development of enzyme activity at the higher concentration and ABA inhibited development at both levels.     

Direct evidence for de novo synthesis of rat liver phenylalanine: pyruvate transaminase after glucagon treatment.

A specific antibody to phenylalanine:pyruvate transaminase has been used to show that the number of enzyme molecules and the rate of enzyme synthesis are increased by glucagon and N⁶,O^2′-dibutyryl cyclic AMP. Cycloheximide given simultaneously with glucagon or dibutyryl cyclic AMP blocked the increase in [³H]leucine incorporation when it was injected along with glucagon, but had no effect when given 4 h after the glucagon. This finding suggests that the mRNA synthesis for phenylalanine:pyruvate transaminase may be completed in 4 h.