Effects of salicylic acid on protein content and <Superscript>14</Superscript>C-amino acid incorporation into proteins of pea roots

Pea (Pisum sativum L.) root treatment with salicylic acid (SA) changed the content of some proteins and incorporation of ¹⁴C-amino acids into proteins. The analysis of changes in these indices allowed us to subdivide all proteins into the four groups: (1) most abundant SA-independent proteins; (2) SA-dependent proteins, which content and ¹⁴C-amino acids incorporation both increased; (3) SA-dependent proteins, which content and ¹⁴C-amino acids incorporation both decreased; and (4) SA-dependent proteins, which content was not essentially changed (referred earlier to SA-independent proteins) but ¹⁴C-amino acids incorporation into these proteins was strongly suppressed. It is very likely that proteolysis of the proteins referred to the fourth group is very low and even a strong inhibition of their synthesis (incorporation of ¹⁴C-amino acids) does not result in the substantial decrease in their contents. Some SA-dependent proteins were identified by means of modern methods of proteomics: phosphoglyceromutase, S-adenosylmethionine synthase 3, enolase, chalcone isomerase, nucleoside diphosphate kinase 1, and tioredoxin h.     

Tunicamycin--an inhibitor of yeast glycoprotein synthesis

Tunicamycin, a glucosamine-containing antibiotic, halted synthesis of the external glycoproteins invertase, acid phosphatase and mannan by yeast protoplasts within 30 min; formation of two intracellular proteins, alpha-glucosidase and alkaline phosphatase, and of glucan continued at the control rate for at least 60–80 min. No accumulation of mannan-free acid phosphatase or invertase was evident in treated cells. Utilization of hexoses and incorporation of ¹⁴C-amino acids into protein were not affected. Incorporation of ³H-glucosamine into trichloroacetic acid-insoluble products was only partially reduced. In yeast tunicamycin acts primarily as an inhibitor of glycoprotein synthesis and not of general glucosamine metabolism.     

Changes in Amino Acid Metabolism in vivo with Time after Feeding

Changes in the metabolism in vivo of amino acids with the lapse of time after feeding a diet were investigated by measuring the incorporation of ¹⁴C into some body components one hour after injection with ¹⁴C-amino acid mixture.

The incorporation of ¹⁴C into protein in the liver and carcass was rather constant, but that into blood sugar, liver glycogen, and lipids in the liver and carcass showed a change with the lapse of time after feeding a 25% casein diet or a protein-free diet. The incorporation of ¹⁴C into liver glycogen was stimulated shortly after feeding, but it was reduced at 7 hr, when a large amount of glycogen was still in the liver. On the contrary, the specific activity of blood sugar increased with the lapse of time after feeding. The conversion of ¹⁴C-amino acids into lipids in the liver and carcass was stimulated shortly after feeding.

The incorporation of ¹⁴C into protein was higher in the rats fed the protein-free diet than in those fed the 25% casein diet, and the higher incorporation was partly counterbalanced by the lower incorporation of ¹⁴C into lipids and glycogen in the rats fed the protein-free diet.     

Intervarietal differences in some metabolic functions associated with protein accumulation in rice grains

Summary Total nitrogen, amino nitrogen, glutamic acid dehydrogenase (GDH) activity and incorporation of ³H-uridine and ¹⁴C-amino acids into RNA and proteins, respectively, were compared in the developing grains of three high-protein stocks (IR-480-5-9, GMPR-51 and Erythroceros) and a high-yielding, medium-protein cultivar IR-8. The above parameters were also independently studied in the developing grains of IR-8 grown at 0, 60 and 120 kgN/ha. In addition, mobilization of nitrogen from flag leaf during kernel development was compared in a separate experiment. Higher protein concentration, both in high-protein stocks and in IR-8 grown at 120 kgN/ha, was associated with increased levels of: soluble amino nitrogen, GDH activity, ³H-uridine and ¹⁴C-amino acid incorporation. Significant variation was found among the high protein stocks in mobilization of nitrogen from flag leaf.Research partly supported by International Atomic Energy Agency Research Contract 1035     

Chromosomal proteins of conifers

During imbibition and germination of jack pine seeds, the composition of the total extractable chromatin varied. Relative to DNA, the histone levels decreased as the nonhistone chromosomal proteins (NHCP) increased. New chromosomal proteins were synthesized after 2 days of imbibition as judged by recovery of ¹⁴C-amino acids from the major protein fractions. Phosphorylation of histones from ³²P-phosphoric acid was detected before the incorporation of ¹⁴C-amino acids. In the seed the synthesis and relative changes of chromatin coincided with a fall in total soluble protein and free arginine N. By contrast, adenylate energy charge, free glutamine N and in vitro template activity of chromatin increased during chromatin protein synthesis. When seeds had germinated for 4 days after the start of imbibition more radioactivity, derived from free ¹⁴C-amino acids, was recovered from the NHCP than from the histones. The percentage amino acid composition of most histone fractions remained stable, whereas the composition of NHCP changed more with time. The phosphorylation of NHCP was 8- to 41-fold greater than that of the histones. Phosphorylation of histone H4 was not detected at any stage of germination. Correlations between recovery of radioactivity (³²P and ¹⁴C) from chromosomal proteins and higher adenylate energy charge were positive.     

Synthesis of Terpene Alcohol Esters by Lipase

The metabolic fates of the carbon skeletons of L-(U-¹⁴C)arginine, proline and glutamic acid were investigated in growing rats fed with diets containing different percentages of protein calories (0, 5, 10, 15 and 30 PC%) at 4100 kcal of metabolizable energy per kg of diet.

The incorporation of ¹⁴C into body protein at 12hr after the injection of ¹⁴C-arginine was more than 50% of the dose in all dietary groups, showing a high efficiency of utilization of this amino acid for protein synthesis. The incorporation of ¹⁴C into body protein from ¹⁴C-proline was most increased in the 15 PC% group, and the values were reduced in rats fed with lower and higher PC% diets. The carbon skeleton of ¹⁴C-glutamic acid was extensively oxidized to expired carbon dioxide, and the ¹⁴C incorporation into body protein was markedly less. The pattern of expired ¹⁴C0₂ production from each ¹⁴C-amino acid was in inverse proportion to that of ¹⁴C incorporation into body protein. The results indicate that the metabolic responses of arginine, proline and glutamic acid to dietary protein change at 10 to 15 PC%, where the growth rate reached its approximate maximum.     

De-novo synthesis and release of peroxidases in cell suspension cultures of Nicotiana tabacum L.

De-novo synthesis of acid and basic peroxidases has been studied in cell suspension cultures of tobacco by incorporation of ³H- and ¹⁴C-amino acids. Incorporation rates were found to be high for acid peroxidases and low for basic peroxidases. Synthesis of all peroxidases was inhibited by cycloheximide and actinomycin D. Subculturing of the cells increased the rates of radioactive amino-acid incorporation into all peroxidases within the first 24 h. This rise in peroxidase synthesis was correlated with the age of the transferred cells. The older the cells were the more pronounced was the effect. During the culture cycle the high rates of peroxidase synthesis at the second day dropped back to initial values. Peroxidase synthesis was thus inversely related to peroxidase accumulation which was very low at the beginning and increased continuously. By pulse-chase experiments it has been shown that newly synthesized acid peroxidases accumulated in the medium. This process was inhibited by monensin. Only the acid peroxidases were secreted into the cell wall and from there released. The basic peroxidases were not detectable in the medium.Abbreviations AA^* radioactive amino-acid mixture - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Effects of methylglyoxal-bis (guanylhydrazone) and abscisic acid on polyamine metabolism in embryonectomized barley seeds

Incorporation of L-[U-¹⁴C] arginine or L-[U-¹⁴C] ornithine into putrescine (Put), spermidine (Spd) and spermine (Spm) in embryonectomized barley seeds (Hordeum vulgare L. cv. Himalaya) was studied following imbition with methylglyoxal-bis (guanylhydrazone) (MGBG) and abscisic acid (ABA). Both radiolabeled amino acids were incorporated into the amines as a result of active polyamine biosynthesis in the seed during imbibition. In the aleurone layer, the Spd and Spn existed mainly in the free form (acid soluble). However about 50% of Put was recovered in conjugated form(s) (acid insoluble). Imbibition with 5 and 10M ABA for 3 days increased the accumulation of the free form of ¹⁴C-Put, probably as a result of inhibition (70%) of ¹⁴C-Spd accumulation. The ABA treatment showed no significant effect on levels of the conjugated form of Put and Spd. Imbibition with millimolar concentrations of MGBG resulted in (i) abnormal accumulation of the free form of Put and incorporation of ¹⁴C-amino acids into the diamine, (ii) progressive inhibition of the accumulation of the free forms of ¹⁴C-Spd and Spm, and (iii) reduction of the ¹⁴C incorporation into the conjugated forms of Put and Spd. Uptake of ¹⁴C-amino acids was not affected by MGBG treatment. The results indicate that MGBG may inhibit not only the synthesis of Spd and Spm, but the catabolism (e.g. oxidation) of Put in the aleurone layer.This paper is published with the approval of the director of the Kentucky Agricultural Experiment Station.     

Amino acid pools and protein synthesis in germinating maize embryos

Amino acid pools were analyzed in maize (Zea mays L.) axes germinated for 0, 6 and 24 h. Proteins synthesized at early (0–6 h) and late (18–24 h) stages were characterized by gel electrophoresis and fluorography after either ¹⁴C-leucine or ¹⁴C-lysine pulses. An increase of amino acid incorporation after 18–24 h was observed, as well as changes in the protein patterns of the corresponding fluorographs. Analysis of the endogenous amino acid pools showed major changes in contents of proline, alanine, isoleucine, valine, leucine and lysine. A selective increase of lysine incorporation into proteins during the late stage was detected.Under the Academic Colaboration Program between the Universidad Nacional Autónoma de México and the Colegio de Postgraduados, Chapingo, México.     

In Vitro Chick Embryo Cell Response to Strain MC29 Avian Leukosis Virus

Strain MC29 avian leukosis (myelocytomatosis) virus induced infection, elaboration of virus, and morphological alteration in chick embryo cells in vitro. Virus liberation began within 18 hr, morphological change was detectable at about 40 hr, and the cultures could be completely altered within 80 hr after infection. Altered cells were about half the volume and grew at approximately twice the rate of uninfected elements. The output of virus estimated by electron microscopy was about 140 particles per cell per hr. Deoxyribonucleic acid remained constant, but ribonucleic acid increased in both infected and control cells in adjustment to culture environment. The rates of uptake and incorporation of ³H-uridine and the incorporation of ³H-thymidine increased in the infected cells with onset of morphological change but were unaffected by processes of infection and virus elaboration per se. Incorporation of a ¹⁴C-amino acid mixture was slightly greater in the infected than in control cells. The speed of continuity of infection and massive morphological alteration constitute a unique response to avian tumor viruses, and the system gives promise of singular value for detailed studies of the processes of infection and morphological change.     

Amino Acid incorporation in developing fucus embryos

The incorporation of ¹⁴C-leucine and ¹⁴C-amino acid mixture into protein in unfertilized eggs and developing embryos of the brown alga Fucus vesiculosus L. was studied. Bacterial contamination was initially a problem, but it was found that the addition of 40 μg/ml chloramphenicol to the incubation medium would inhibit bacterial protein synthesis without affecting early development of the Fucus embryos. The kinetics of uptake and incorporation of ¹⁴C-leucine into the trichloroacetic acid-soluble and -insoluble fractions indicated that the exogenous precursor did not equilibrate with the main soluble leucine pool before incorporation into protein. Uptake and incorporation of leucine by embryos 90 to 175 minutes old were proportional to exogenous leucine concentration over the range 5 × 10⁻⁶ m to 5 × 10⁻³ m. Unfertilized eggs will incorporate ¹⁴C-leucine into protein. The rate of this incorporation increases dramatically in newly fertilized eggs with a maximum rate at 3.5 hours, a period of cell wall formation and increasing metabolic rates. Thereafter, the rate of incorporation declines until approximately 15 to 17 hours when it increases again concurrently with the onset of rhizoid initiation and cell division.     

Ethanol inhibits phosphatidylcholine and phosphatidylethanolamine biosynthesis in human leukemic monocyte-like U937 cells

The effect of ethanol (ETOH) on the incorporation of [¹⁴C]oleic acid (18:1) into lipid in human monocyte-like U937 cells was investigated. With increasing time of exposure to ETOH, the percentage of the label distributed into neutral lipid (NL) declined from 35 per cent (3 h) to 10 per cent (24 h) accompanied by increased incorporation into phospholipid (PL). [¹⁴C] 18 : 1 was preferentially incorporated into triglyceride (TG) and phosphatidylcholine (PC), comprising over 65 per cent and 50 per cent of the label associated with NL and PL, respectively. Low concentrations of ETOH (⩽ 1·0 per cent; v/v) had no effect. At concentrations greater than 1·5 per cent, there was enhanced incorporation into TG and diacylglycerol (DAG) in a 24-h incubation period, while at 16 h the label in phosphatidylethanolamine (PE) was decreased. The effect of ETOH on the CDP-choline or ethanolamine pathway was examined by monitoring the incorporation of [³H]choline or [¹⁴C]ethanolamine into PC or PE, respectively. At low concentrations ETOH had no effect on either choline uptake or the incorporation into PC. Higher concentrations (≥ 1·5 per cent) for 3 and 6 h resulted in a slightly decreased choline uptake, and the reduction (40–50 per cent) of incorporation into PC suggests that the CDP-choline pathway was inhibited. There was a similar inhibition of the incorporation of [¹⁴C]ethanolamine into PE. When the cells were incubated for 3 h in the presence of 2 per cent ETOH and with labelled 18 : 1 and PL-base, the ratios of incorporation (base/18 : 1) into PC and PE fractions decreased, indicating that the major inhibition lay in blockage of the availability of the base moiety for PL formation. Analysis of the distribution of the label into metabolites revealed that ETOH inhibited the conversion of [¹⁴C] ethanolamine into [¹⁴C]phosphorylethanolamine. The reduction in incorporation was not due to the enhanced breakdown of base-labelled PL. Our results indicate that ETOH has an inhibitory effect on the CDP-choline or ethanolamine pathway.     

Effect of Ethrel and Ceratocystis fimbriata on the Synthesis of Fatty Acids and 6-Methoxy Mellein in Carrot Root

The rate of incorporation of ¹⁴C from acetate-1-¹⁴C into fatty acids by carrot root discs, 18 hours after inoculation with Ceratocystis fimbriata, was 9-fold greater than that in freshly cut discs. The rate in discs treated with water or Ethrel was 3-fold greater. The rate of incorporation of ¹⁴C from glucose-U-¹³C into fatty acids was 3-fold greater 18 hours after any of the above treatments. The rate of ¹⁴C incorporation from malonate-2-¹⁴C into fatty acids 24 hours after inoculation with C. fimbriata or treatment with water was 25 and 60%, respectively, of that in freshly cut discs. Linoleic acid was the principal fatty acid in carrot root, but incorporation of ¹⁴C from acetate-1-¹⁴C into the acid was low until 18 hours after inoculation with C. fimbriata or treatment with Ethrel. Turnover rates of the fatty acids appeared low and were similar for all treatments.     

Synthesis and assembly of ribulosebisphosphate carboxylase enzyme during greening of barley plants

The synthesis and activation of ribulosebisphosphate carboxylase was studied in etiolated barley leaves during increasing periods of light irradiation. Comparisons were made among enzymatic activity, ¹⁴C-amino acid incorporation into anti-ribulosebisphosphate carboxylase precipitable and 16S protein, and total mass of enzyme. A major portion of newly synthesized anti-ribulosebisphosphate carboxylase specific protein preceded light-induced increase in enzyme activity by a significant period of time. These findings are consistent with a model in which both subunits of ribulosebisphosphate carboxylase are synthesized in response to an early event in greening and subsequently become associated to active oligomeric carboxylase species.     

Protein synthesis patterns : relevance of old and new messenger RNA in germinating maize embryos

The proteins synthesized during the first hours of seed imbibition were studied in axes and scutellum of maize embryos separately. Increase in fresh weight was followed in the embryonic axes through the germination period. Pulse labeling experiments with ¹⁴C-amino acids were carried out at two stages of development: 0 to 6 and 18 to 24 hours in the presence and absence of α-amanitin. The proteins were analyzed by two-dimensional gel electrophoresis and fluorography. Results showed a major pattern of proteins common to both tissues, axes and scutellum (`house keeping' proteins), besides the specific proteins synthesized by each tissue. In the axes, the changes in proteins observed between the periods of 0 to 6 and 18 to 24 hours of development seem to be due both to newly synthesized mRNA as well as to delayed translation of stored mRNA species.     

Lipid biosynthesis in warm- and cold-acclimatized sea anemones, Metridium senile (L.)

Sea anemones, Metridium senile (L.), naturally acclimatized to warm (18°C) and cold (0°C) conditions, were exposed to either [2-¹⁴C]acetate, [16-¹⁴C]palmitate, or [4-¹⁴C]cholesterol for periods up to 24 h. Isotope incorporation into triglyceride (TG), wax esters (WE), and polar lipid (PL) was recorded. Compared to warm-acclimatized groups, incorporation of [2-¹⁴C]acetate into WE of cold anemones was dramatically reduced, while TG incorporation remained at about the same levels. Highest values were recorded for PL in both groups. Using radiolabeled palmitate, incorporation values for WE were very low in both acclimatization groups though TG uptake remained comparatively high. Also noteworthy was a significant decrease in PL activity in cold anemones. Fatty acid analysis of total lipid, wax ester, triglyceride and phospholipid fractions showed a general shift towards increased chain length and unsaturation in cold-acclimatized anemones.     

Biochemical effects of miconazole on fungi. II. Inhibition of ergosterol biosynthesis in Candida albicans.

The effects of the antifungal agent miconazole nitrate on the ergosterol biosynthesis in Candida albicans were investigated after in vitro contact with the drug for 1, 4, 16 and 24 h. A time- and dose-(2.10^?10–10^?4 M) dependent inhibition of [¹⁴C]acetate incorporation into ergosterol was observed. Fifty percent inhibition of the acetate incorporation into ergosterol was found after 1 h incubation in the presence of 10^?9 M miconazole. Simultaneously 24-methylenedihydrolanosterol, lanosterol, obtusifoliol, 4,14-dimethylzymosterol and 14-methylfecosterol accumulated.The accumulation of 14 α-methyl sterols suggests that this antifungal agent is a potent inhibitor of one of the metabolic steps involved in the demethylation at C-14. The absence of 24-methyl sterols and of sterols with a C-22 [23] double bond in miconazole treated C. albicans indicates that miconazole also inteferes with the reduction of the 24(28)-double bond and with the introduction of the 22(23)-double bond.Miconazole also intervenes to a small extent in triglyceride synthesis. However, in all circumstances studied, ergosterol biosynthesis was affected at lower doses than those interfering with the acetate incorporation into triglycerides. 16 and 24 h of incubation in the presence of miconazole (≥ 10^?6 M) also resulted in an increased fatty acid synthesis.It is suggested that the miconazole-induced inhibition of the C-14 demethylation may be at the origin of the previously observed permeability changes in miconazole treated C. albicans.     

Collagen and elastin synthesis in the developing chick aorta

Thoracic aortas from 8- to 18-day embryonic chicks were incubated in vitro for 30 min with [³H]glycine and the newly synthesized, labeled proteins were subjected to polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The gels were fractionated and the incorporation of label into procollagen (125,000 M_r) and tropoelastin (70,000 M_r) was estimated by summation of the radioactivity found in the appropriate regions of the gel. The analyses showed that at Day 8 approximately 14% of the incorporated [³H]glycine was found in procollagen and 22% in tropoelastin. In the following 6 days of development, there was a significant decline in the relative incorporation into procollagen and an increase into tropoelastin so that at Days 14–18 less than 10% of the label was found in collagen and 40% was now found in tropoelastin. Since glucocorticoids have been shown to alter the rate of synthesis of other proteins in the developing chick, 150 μg of hydrocortisone was injected into 8-day eggs and 24 h later the aortas were incubated and treated as described above. The pattern of protein synthesis exhibited by the hormone-treated aortas resembled that of 14- to 18-day embryos. Furthermore, incubation of 8-day aortas with 10^?8m hydrocortisone for 24 h produced a significant increase in the rate of elastin synthesis relative to that of other proteins. These results demonstrate that collagen and elastin synthesis vary during development of the chick aorta and they suggest that glucocorticoids may be involved in the control of their synthesis.     

Monotertiarybutylhydroquinone effects on Tetrahymena pyriformis.

The molecular toxicity of monotertiarybutylhydroquionone (TBHQ) was studied using $\text{Tetrahymena}$$\text{pyriformis}$ as a model cell system. TBHQ at 26 ppm in the media inhibited cell growth by 50%. TBHQ inhibited the oxidation of ¹⁴C-acetate to ¹⁴CO₂. In addition, increasing concentrations of TBHQ decreased the incorporation of ¹⁴C-acetate into lipids and protein, ¹⁴C-amino acids into protein, ³H-uridine into RNA and ³H-thymidine into DNA. The incorporation of ¹⁴C-acetate into glycogen increased with concentrations up to 20 ppm TBHQ in the media while glycogen synthesis decreased with 40 ppm TBHQ.