Germination of phaseolus vulgaris I. Resumption of axis growth

Growth of the excised axis of Phaseolus vulgaris L. (var. White Marrowfat) begins after a 7-hour incubation in buffer or water at 26°. Growth, as measured by axis elongation or fresh weight increase, is linear for at least 8 hours with a resultant fresh weight increase of approximately 65%. Cell elongation begins 4 or 5 hours prior to cell division and 5 or 6 hours prior to radicle protrusion in the intact seed.

The initiation of axis elongation is apparently dependent on synthesis of RNA and protein. Both actinomycin D and puromycin inhibit the initiation of elongation. Actinomycin I) inhibits the incorporation of ATP-8-C¹⁴ into axis RNA and C¹⁴-leucine into protein, while puromycin inhibits the incorporation of C¹⁴-leucine into axis protein.

The respiratory rate of the axes increases sharply at about the time of initiation of cell elongation. Dinitrophenol initially increases O₂ uptake by the axes, but at the end of 15 hours the rates of O₂ uptake by control or dinitrophenol-treated axes are approximately the same.

     

Changes in protein synthesis in rats in response to endurance training

Experiments were conducted to investigate the influence of endurance exercise training on protein synthesis in skeletal muscle, heart, and liver. Training decreased incorporation of [¹⁴C]-leucine into proteins of the stromal fraction of muscle but there was no change in amino acid incorporation into proteins of the sarcoplasmic and myofibrillar fractions. Incorporation of [¹⁴C]-leucine into the protein of heart, liver, and plasma was depressed in trained rats compared to untrained rats. The specific radioactivity of [¹⁴C]-leucine was similar in tissues of trained and untrained rats and thus the depressed amino acid incorporation represents a decrease in the rate of protein synthesis. These observations demonstrate that the adaptation of muscle protein metabolism to endurance training is quite different than the alterations during work-induced hypertrophy of muscle. The difference in adaptation probably relates to the functional differences between the types of exercise. However depression of protein synthesis in trained rats is a general effect in several tissues and not an effect localized in muscle tissue.     

Differential effect of chloramphenicol and actinomycin D on protein synthesis in the decapod crustacean upogebia littoralis

Summary Chloramphenicol and actinomycin D produced opposite effects on the in vivo protein synthesis by the hepatopancreas of the crustacean Upogebia littoralis. Inhibition of protein synthesis was noted within 6 hours after administration of chloramphenicol; maximum inhibition occurred 8 hours after treatment. No inhibition was noted after administration of actinomycin D; on the contrary, this antibiotic appeared to slightly stimulate protein synthesis as measured by rate of C¹⁴-leucine incorporation, within 6 to 8 hours after treatment.     

The incorporation of amino acids and nucleic acid bases into the seedling,reproductive stage and young ear portion of rice plants

Summary The incorporation of C¹⁴-amino acids (aspartic acid, glutamic acid, threonine and proline) and C¹⁴-nucleic acid bases (adenine, guanine, cytosine and uracil) into the seedling, reproductive stage and young ear portion of rice plant was investigated. It was found that C¹⁴-aspartic acid was incorporated into the rice seedling more rapidly than C¹⁴-threonine or C¹⁴-proline; on the other hand C¹⁴-proline was found to be more rapidly incorporated than C¹⁴-aspartic acid into reproductive stage plant and young ear portion. Similarly C¹⁴-adenine was incorporated into the rice seedling more rapidly than other C¹⁴-labelled bases. On the other hand C¹⁴-uracil was preferentially incorporated to C¹⁴-adenine or C¹⁴-guanine into the reproductive stage plant and young ear portion. It is suggested from the results obtained that proline is polymerized into polypeptide or protein in the rice plant more rapidly at the reproductive stage than at the seedling stage and that a higher proportion of pyrimidine bases might be involved into the metabolic process at the reproductive stage of rice plant.     

Plasma membrane ectoglycosyltransferase activity of L1210 murine leukemic cells

L1210 murine leukemia cells after treatment with Cl. perfringens neuraminidase at pH 7.0 incorporated six times more N-acetylneuraminic acid-[C¹⁴] than control cells when incubated for 30 minutes with cytidine 5′-monophosphate N-acetylneuraminic-[C¹⁴] acid and three times more galactose-[C¹⁴] when incubated with uridine diphosphate galactose-[C¹⁴]. These sugars were incorporated in a 10% trichloracetic acid insoluble fraction and more than 75% of the incorporated N-acetylneuraminic acid- [C¹⁴] could be removed by further treatment of these cells with neuraminidase. The incorporation of N-acetylneuraminic acid- [C¹⁴] as a function of time was divided into two rates: a rapid one, active during the first 30 minutes followed by a slower one, similar to the rate observed with untreated cells. The addition of Ba⁺⁺ and Ca⁺⁺ ions at 8.3 mM increased the incorporation of N-acetylneuraminic acid- [C¹⁴] by 25% while 8.3 mM EDTA decreased activity by 58% . The addition of Zn⁺⁺ or Hg⁺⁺ at similar concentrations abolished the incorporation almost completely. The optimal pH for the incorporation of N-acetylneuraminic acid- [C¹⁴] by these neuraminidase treated cells was 6.5. These data suggest that ectoglycosyltransferases are present on the outer surface of the plasma membrane of L1210 cells and are able to catalyze the addition of radiolabeled nucleotide sugars onto macromolecular acceptors (cell surface glycoproteins and glycolipids) prepared by prior incubation of the cells with neuraminidase. Use of these procedures for labeling outer cell surfaces may also prove to be valuable for the study of plasma membrane glycoprotein and glycolipid structure, synthesis, and turnover.     

Biosynthesis of ribulose-1,5-bisphosphate carboxylase in spinach leaf protoplasts

Spinach leaf (Spinacia oleracea L. var. Kyoho) protoplasts sustain protein-synthesizing activity as measured by the incorporation of [¹⁴C]-leucine into the protein fraction both in the light and in the dark. By the immunoprecipitation of ribulose-1,5-bisphosphate (RuP₂) carboxylase with rabbit antibody raised against the purified spinach enzyme preparation, it was found that approximately 7% of the total radiocarbon incorporated into the protein fraction in the light was in the carboxylase molecules. However, there was no measurable net increase observed in the content of the enzyme protein in the experimental conditions employed. It was found that both chloramphenicol and cycloheximide inhibited the incorporation of [¹⁴C]leucine into RuP₂ carboxylase and its constituent subunits, as measured by the immunoprecipitation of the enzyme molecule and its subunits, A and B.     

Does urinary taurine reflect changes in protein metabolism? A study with cycloheximide in rats

Abstract

We have previously reported on the changes in urinary taurine levels in rats following treatment with some hepatotoxic agents and compounds reported to affect protein synthesis. This study follows the time course of the elevation of urinary taurine after treatment of rats with cycloheximide which was maximal 8–12 h alter dosing and was dose related. [³H]-leucine incorporation into proteins was used as an indicator of protein synthesis. There was a significant reduction in [³H]-leucine incorporation into acid precipitable proteins 8 h but not 24 h after dosing. The reduction in incorporation was negatively correlated with the raised levels of both serum and urinary taurine 8 h after dosing. Liver glutathione was raised both 8 and 24 h after dosing rats and liver taurine was significantly reduced at 8 h. It is suggested that measuring urinary taurine in collections made continuously might provide a simple, non-invasive biomarker for monitoring the effects of xenobiotics or other external stimuli on the status of protein synthesis.     

Hormonal control of testicular protein synthesis in developing Tenebrio molitor

During differentiation, the testes of Tenebrio molitor were found to exhibit increases in biosynthetic capacity reflected by changes in the testicular protein content. A gradual increase in testicular protein content was observed during the prepupal stage. The observed increase was more dramatic in the pupal stage and reached its maximum level between days 4 and 7 of pupal development. During the adult stage, the biosynthetic processes for producing protein were apparently reduced following the first few days after adult emergence.The incorporation of radioactive leucine into TCA-precipitable testicular protein was not affected by the administration of exogenous juvenile hormone alone (JH I, 1 μg/animal) during the pupal stage. However, the administration of exogenous ecdysterone (0.5 μg/animal) to pupal T. molitor resulted in an increase in radioactive leucine incorporated into TCA-precipitable testicular proteins, particularly during the first five days after pupal ecdysis.Simultaneous administration of both JH and ecdysterone to mealworm pupae at specific ages indicated that no apparent interaction, synergistic or antagonistic, occurred between these two hormones with respect to [³H]-leucine incorporation. Furthermore, the amount of [³H]-leucine incorporated approximated to that obtained following injection of ecdysterone alone in all the pupal ages studied.     

Rate of Glycolate Formation During Photosynthesis at High pH

The products of C¹⁴O₂ fixation by Chlamydomonas and Chlorella were studied under conditions most favorable for glycolate synthesis. The highest percentage of the C¹⁴ was incorporated into glycolate in the pH range of 8 to 9. After 1 to 2 minutes as much as 40% of the C¹⁴ was found in glycolate products and only a trace of C¹⁴ was present as phosphoglycerate. Below pH 8 the rate of photosynthesis was much faster, but only a small percent of the C¹⁴ was incorporated into glycolate in 1 or 2 minutes, while a high percent of the C¹⁴ accumulated in phosphoglycerate. C¹⁴ labeling of glycolate even at pH 8 or above did not occur at times shorter than 10 seconds. During the first seconds of photosynthesis, nearly all of the C¹⁴ was found in phosphoglycerate and sugar phosphates. Thus glycolate appears to be formed after the phosphate esters of the photosynthetic carbon cycle.

Washing Chlamydomonas with water 2 or 3 times resulted in the loss of most of their free phosphate. When a small aliquot of NaHC¹⁴O₃ was added to washed algae in the absence of this buffering capacity, the pH of the algal medium became 8 or above and much of the fixed C¹⁴ accumulated in glycolate.

     

A COMPARATIVE STUDY OF THE METABOLISM OF DE NOVO SYNTHESIZED FATTY ACIDS FROM ACETATE AND GLUCOSE, AND EXOGENOUS FATTY ACIDS, IN SLICES OF RABBIT CEREBRAL CORTEX DURING DEVELOPMENT

Slices of rabbit cerebral cortex, from the foetal stage to the adult have been used to compare lipid synthesis from fatty acids synthesized de novo from [U-¹⁴C]glucose and [1-¹⁴C]acetate, with lipid synthesis from exogenous albumin-bound [1-¹⁴C]palmitate. Incorporation into cellular lipid has been determined in terms of DNA, protein, wet wt. of tissue and wet weight of whole brain. On a wet wt. basis, maximum incorporation of glucose carbon into lipid occurred in the foetal brain while lipid synthesis from acetate and palmitate was maximum at 4–14 days after birth. Glucose and acetate were incorporated into a diversity of lipids (with increasing amounts of phosphatidylcholine synthesized during maturation), while palmitate was incorporated into the free fatty acid and triglyceride fractions. A greater proportion of acetate was incorporated into fatty acids of chain-length longer than C16 compared with the incorporation of palmitate. However, on a molar basis de novo synthesized and exogenous palmitate were elongated, desaturated and incorporated into phospholipids at a similar rate, while exogenous palmitate was incorporated to a greater extent than de nova synthesized fatty acid into the triglyceride fraction. This difference in metabolism may be due to the different size of the non-esterified fatty acid pool in the two situations. At the period of their most active formation, the very long-chain fatty acids may be synthesized from a pool of the C18 series of fatty acids (saturated and monoenoic) not in equilibrium with the bulk of C₁₈ acids in cerebral lipids. This could be a pool of acyl groups derived from ethanolamine phospholipids.     

Abnormal expression of normal transferrin alleles in cattle

Six subclones were derived from a single clone isolated from a strain of chromosomally stable rat fibroblast-like cells. Three of the subclones were diploid, three were tetraploid, and all were derived from a single somatic cell. Abnormal chromosomes or total chromosome numbers differing from multiples of haploid were not found. Measurements of rate of formation of hydroxyproline in protein from H³-proline, the accumulation of hydroxyproline in medium and cell layer with time, and the rate of incorporation of C¹⁴-leucine into protein that coprecipitates with collagen were employed to determine the rate of synthesis of collagen. By each of these measurements the rate of synthesis was found to respond directly to presumed ploidy of genes for collagen.This work was supported by Grant HE 08072 from the National Institutes of Health.     

Incorporation of a kinin, N, 6-benzyladenine into soluble RNA

Kinin requiring tobacco and soybean tissues incubated on a medium containing N,6-benzyladenine-8-C¹⁴ incorporated C¹⁴ into several RNA components including adenylic and guanylic acids. About 15% of the label taken up by the tissues appeared in RNA while the remainder was distributed among several metabolites in the soluble, nonpolynucleotide fraction. Tissue grown on a kinin labeled in the side chain (N,6-benzyladenine-benzyl-C¹⁴) also incorporated a small, but nevertheless repeatable, amount of radioactivity into minor RNA components.

Ultracentrifugation studies and methylated albumin chromatography indicated that the bulk of the label from benzyladenine-benzyl-C¹⁴ is in soluble RNA. Approximately 50% of the C¹⁴ in soluble RNA is in a component which has chromatographic properties like that of benzyladenine.

It is suggested that the biological action of the kinins may hinge on their providing substituted bases in RNA in tissues which through differentiation no longer synthesize RNA-methylating enzymes. As an alternative it was hypothesized that a small amount of benzyladenine was incorporated into a m-RNA, acting there as a derepressing agent, perhaps by preventing its normal repressing function.

     

Pituitaries of rats neonatally treated with oestrogen

Summary Pituitaries of male and female rats neonatally treated with oestradiol dipropionate were examined with light and electron microscopy, disc electrophoresis and in vitro C¹⁴-leucine incorporation.Single dose of oestradiol administered shortly after birth to male rats has an prolonged stimulative effect on LTH synthesis and release. This is expressed in the cell hypertrophy, proliferation of ER, increase of corresponding band density and higher rate of ¹⁴C-leucine incorporation. In females this effect was less pronounced.STH cell in the treated male pituitaries were degranulated, corresponding hormone band density significantly lowered and incorporation of ¹⁴C-leucine was reduced. This could be considered as an inhibitory effect of oestradiol on STH synthesis and release. Such effect was more expressed two than four months after treatment and is more pronounced in male than in female rats.The technical assistence of Slobodanka Selimovi is gratefully acknowledged.     

The stimulation of amino acid incorporation into the protein of unfertilized sea urchin eggs by gamma-irradiation

Control and γ-irradiated sea urchin (Arbacia punctulata) eggs were incubated with H³-leucine. No incorporation of the amino acid into the proteins of most unirradiated eggs was detectable by autoradiography. All irradiated eggs incorporated the amino acid and the amount of autoradiographic labelling varied greatly from cell to cell.     

EFFECT OF DIMETHYL SULFOXIDE ON ZINC65 UPTAKE,RESPIRATION, AND RNA AND PROTEIN METABOLISM IN BEAN (PHASEOLUS VULGARIS) TISSUES

The effect of dimethyl sulfoxide (DMSO) on zinc⁶⁵ uptake, respiration, RNA, and protein metabolism in various tissues of two bean (Phaseolus vulgaris L.) cultivars showing differential growth responses to zinc has been studied. At a concentration of 1%, DMSO stimulated zinc uptake in excised roots, stem-callus tissue, leaf disks, and enzymically isolated leaf cells, but did not significantly alter the uptake and incorporation of C¹⁴-uracil into RNA and C¹⁴-methionine into protein, although a slight inhibition was discernible in some tissues. At a higher concentration (10%) DMSO increased Zn⁶⁵ uptake in excise roots incubated for 2 hr; however, at the same concentration, C¹⁴-uracil and C¹⁴-methionine uptake and incorporation were considerably inhibited in all the tissues. Oxygen uptake as measured with Warburg manometers was impaired, and the inhibition showed a time and concentration dependency. The fact that DMSO inhibited respiration and RNA and protein metabolism, while at the same concentration zinc uptake was increased, suggests that zinc uptake in beans is primarily a non-metabolic process. The possible mechanisms of DMSO action are discussed in the light of its reported effects on membrane permeability and cell metabolism.     

Characterization of Labeled Residues from 5-Diazouracil-2-C Incorporated into Ribonucleic Acids of Filamentous Escherichia coli

E. coli B, filamented with 5-diazouracil (DZU)-2-¹⁴C, yielded ribonucleic acid (RNA)-(DZU-2-¹⁴C) which was converted by pancreatic ribonuclease to ¹⁴C-mono-and oligo-nucleotides. The mixed ¹⁴C-mononucleotides isolated by diethylaminoethyl-cellulose fractionation were identified as cytidylic, uridylic, and hydroxyuridylic acids, by using a combination of paper chromatography and treatment with alkaline phosphatase and cytidine deaminase. Rifampin blocked incorporation of DZU-2-¹⁴C under conditions which inhibit RNA synthesis. Division inhibition by DZU-2-¹⁴C and the incorporation into Escherichia coli B were retarded by uracil but not by other RNA bases. In a pyrimidine-requiring E. coli, DZU substituted for uracil or cytosine to an extent limited by toxic effects. Cytosine and uracil retarded these effects and retarded the incorporation of DZU-2-¹⁴C into the pyrimidineless strain. A small proportion of DZU-2-¹⁴C was converted by the latter strain into hydroxyuridylic acid, but the bulk of the incorporated label was in cytidylic and uridylic acid, as in the wild strain.     

A cotyledon slice system for the electron autoradiographic study of the synthesis and intracellular transport of the seed storage protein of Vicia faba

Summary Cultured slices of cotyledon tissue from 60-day broad bean have been shown to synthesise globulin protein extensively. About 80% of the ³H-leucine incorporated into protein was incorporated into globulin protein; ³⁵S-sulphate was also incorporated by the slices.The rate and dependence of the incorporation on the quantity of radiochemical added and the volume of culture medium were also demonstrated. It was concluded that the cotyledon slices maintained the in vivo behaviour during the period of the experiments.The slice system was used in an electron autoradiographic experiment. It was shown that grain counts were associated initially with the endoplasmic reticulum then moved to the protein bodies. It was concluded that globulin protein is synthesised by the ribosomes of the endoplasmic reticulum and then moves to the protein bodies; it was suggested that the process takes 25 min.     

The Effect of Kinetin on Protein Level of Brassica Leaf Disks

The senescent leaf disks from Brassica rapa L. were floated on a medium containing ¹⁴C-L.-leucine in the presence or absence of kinetin. The increase in radioactivity in protein fraction of kinetin-treated disks was almost linear with time, while the increase in untreated disks incubated for 3 days started to slow down. The leaf disks were first incubated on the solution of ¹⁴C-L-leucine and then transferred to either kinetin solution or water in order to study whether the slower incorporation is due to the decreased rate of protein synthesis or that of protein retention. The decrease in radioactivity of disks floated on kinetin was slower than that of disks floated on water. The slower decrease in radioactivity caused by kinetin was not due to an increased turnover rate, since the same phenomena were observed in the presence of cold leucine or casein hydrolysate solution. These results suggest that kinetin retards the decomposition of protein in leaf disks and does not stimulate the synthesis of the bulk of protein.     

Enzymatic Modification of Protein Functionality: Implantation of Potent Amphiphilicity to Succinylated Proteins by Covalent Attachment of Leucine Alkyl Esters

This work is a new attempt to modify the functionality of food proteins by the use of a potential activity of papain. Fish protein concentrate, soy protein isolate, casein, ovalbumin and gelatin were succinylated prior to using them as substrates. Each of the L-leucine n-alkyl esters with different alkyl-chain lengths was covalently incorporated into each substrate by applying the unusual papain-catalyzed reaction [Agric. Biol. Chem., 43, 1065 (1979)]. The resulting products posessed high whippability and/or emulsifying activity. The highest whippability was observed when leucine butyl ester or hexyl ester was incorporated, while the incorporation of higher alkyl esters generally caused an increase in emulsifying activity.