In Vitro Protein Synthesis by Plastids of Phaseolus vulgaris: IV. Amino Acid Incorporation by Etioplasts and Effect of Illumination of Leaves on Incorporation by Plastids 1

Protein synthesis in vitro by etioplasts and chloroplasts from Phaseolus vulgaris was examined to study the factors regulating the development of etioplasts into chloroplasts. The properties of incorporation of (14)C-leucine into protein by etioplasts from plants grown 6.5 days in darkness are similar to those of chloroplasts from plants of the same age that were illuminated for 12 hours. However, the rate of incorporation per plastid by chloroplasts is 4 times higher than the rate of amino acid incorporation by etioplasts. When 6-day-old plants are placed in light, this 4-fold increase occurs within 6 hours and is maintained up to 36 hours. The difference in rate of amino acid incorporation into protein between etioplasts and chloroplasts represents a real difference in the ability of etioplasts and chloroplasts to synthesize protein. A difference in pool size of leucine between etioplasts and chloroplasts does not account for the difference in amino acid incorporation between etioplasts and chloroplasts. Also the difference in photosynthetic capabilities of etioplasts and chloroplasts does not account for the difference in the ability to incorporate amino acid into protein. Furthermore, there are no factors in homogenates of etiolated leaves which inactivate amino acid incorporation into protein by chloroplasts. The difference in rates of amino acid incorporation between etioplasts and chloroplasts is correlated with the state of development of the plastids. The plastids have increased ability to incorporate amino acid into protein when the plastids are undergoing growth and differentiation.     

In Vitro Protein Synthesis by Plastids of Phaseolus vulgaris. I. Localization of Activity in the Chloroplasts of a Chloroplast Containing Fraction from Developing Leaves

The incorporation of uniformly labeled leucine-¹⁴C into protein by a chloroplast containing fraction from developing primary leaves of bean is reported. Chloroplasts, obtained from week old plants grown in darkness, and then illuminated with white light for 12 hours, were shown to be the principal sites of incorporating activity. Incorporation may continue for 2 hours. Rates of up to 50 μμmole leucine incorporated per mg protein per hour are observed when a 1 hour assay period is used. Incorporation is only partially sensitive to ribonuclease.     

Light-induced de Novo Synthesis of Ribulose 1,5-Diphosphate Carboxylase in Greening Leaves of Barley

An antibody specific for ribulose 1,5-diphosphate carboxylase was used to isolate the enzyme from greening barley (Hordeum vulgare L.) leaves. The increase in enzymatic activity during greening was due to de novo synthesis of the enzyme. Increases in enzymatic activity were accompanied by corresponding increases in enzyme protein and by incorporation of radioactive leucine, all of which were inhibited by low concentrations of cycloheximide. ¹⁴C-Labeled amino acids were incorporated into the enzyme by covalent peptide bonding.     

In vitro Protein Synthesis by Plastids of Phaseolus vulgaris. III. Formation of Lamellar and Soluble Chloroplast Protein

Chloroplasts from leaves of plants which had been grown in the dark, and then illuminated for 12 hours were isolated, and allowed to incorporate ¹⁴C-leucine into protein, and the products of this incorporation were studied. Lamellar and soluble proteins are the principal products, and are formed in about equal amounts. Only some of the soluble proteins become heavily labeled. Those with highest specific activity have a molecular weight of the order of 140,000, while the higher molecular weight Fraction I protein has a much lower specific activity. The soluble protein as a whole does not serve as a precursor for the lamellar protein, and vice-versa, although a precursor-product relationship between a minor component of the soluble fraction and the lamellar fraction has not been ruled out. The relative protein synthesizing capabilities of chloroplasts and mitochondria are discussed with reference to the data presented.     

Isotope Discrimination by Ribulose 1,5-Diphosphate Carboxylase: No Effect of Temperature or HCO(3) Concentration

Carbon 13 isotope discrimination by ribulose 1,5-diphosphate carboxylase from soybean (Glycine max [Merr.] cv. Amsoy) was studied as a function of temperature, bicarbonate concentration, and pH. None of these factors affected the degree of discrimination against ¹³C. The average δ¹³C was −28.3%, a value close to that found for whole C₃ plants. The zero temperature response observed here with ribulose 1,5-diphosphate carboxylase corroborates data from whole plants. The lack of effect of bicarbonate concentration on discrimination is consistent with both current theories of alternate forms of carboxylase.     

In vitro Protein Synthesis by Plastids of Phaseolus vulgaris. II. The Probable Relation Between Ribonuclease Insensitive Amino Acid Incorporation and the Presence of Intact Chloroplasts

Amino acid incorporation into protein by chloroplasts from primary leaves of Phaseolus vulgaris L., var. Black Valentine is only partially inhibited by 400 μg/ml ribonuclease. The rate of incorporation, in the presence of ribonuclease, is progressively inhibited with time, and ceases after about half an hour. Preincubation of chloroplasts at 25°, in the absence of ribonuclease, increases the inhibitory effect of ribonuclease on the initial rate of incorporation of amino acid into protein. Examination of electron micrographs of freshly prepared chloroplast suspensions shows that chloroplasts are largely intact. However, after incubation at 25° for 1 hour the chloroplasts are disrupted, as indicated by loss of their stroma contents. It is concluded that the intact chloroplast membrane is relatively impermeable to ribonuclease. Amino acid incorporating activity probably becomes inhibited as the inside of the chloroplast is made accessible to ribonuclease by breakage of membranes during incubation at 25°.     

Incorporation of C-Leucine into Apple Leaf Protein and Its Inhibition by Protein Synthesis Inhibitors during Growth and Senescence

Of the total ¹⁴C-leucine taken up by intact apple (Pyrus malus L., Golden Delicious) leaf discs, 44 to 62% is incorporated into protein from June to early October. Of this amount, an average of 35% is released by mild, room temperature acid hydrolysis. Prior to mid-August when leaf protein begins to decline, 15 to 20% of the ¹⁴C-leucine incorporated into protein occurs in water-(buffer) soluble protein, of which only 3% is released by mild acid hydrolysis. After mid-August, 40% of the label in protein occurs in soluble protein. The specific radio-activity of the soluble protein increases by 4- to 5-fold after mid-August, while that of total protein increases by less than 2-fold. In presenescent leaves (before the decline of protein in August) 20 micrograms per milliliter cycloheximide inhibits the incorporation of ¹⁴C-leucine into protein by 71%, and 20 micrograms per milliliter chloramphenicol inhibits it by 30%. In senescing leaves, cycloheximide inhibits ¹⁴C-leucine by 85% or more, while chloramphenicol inhibits it by less than 15%. Coincident to the initial decline of leaf protein, chloramphenicol greatly loses its ability to inhibit the incorporation of ¹⁴C-leucine into apple leaf protein. At all leaf ages, chloramphenicol increases the loss of chlorophyll from apple leaf discs. The effect of cycloheximide on leaf disc senescence changes with leaf age: in early season samples, it increases the loss of chlorophyll; in mid-season samples, it has no effect; and in late season samples, it retards the loss of chlorophyll.     

In Vitro Incorporation of Selenomethionine into Protein by Vigna radiata Polysomes

Vigna radiata polysomes efficiently incorporated [⁷⁵Se]selenomethionine, [¹⁴C]methionine, and [¹⁴C]leucine in vitro. The optimal conditions for translation were determined to be 4.8 millimolar Mg²⁺, 182 millimolar K⁺, and pH 7.4. The rates of incorporation of [⁷⁵Se]selenomethionine and [¹⁴C]methionine were similar when measured separately, but [⁷⁵Se]selenomethionine incorporation was 35% less than [¹⁴C]methionine incorporation when both amino acids were present in equal molar concentrations. Polyacrylamide gel electrophoresis of the hot trichloroacetic acid precipitable translation products demonstrated synthesis of high molecular weight labeled proteins in the presence of [⁷⁵Se]selenomethionine or [³⁵S]methionine. No major differences in molecular weights could be detected in the electrophoretic profiles. Utilization of selenomethionine during translation by Vigna radiata polysomes establishes a route for the assimilation of selenomethionine by plants susceptible to selenium toxicity.     

Arabinogalactan Protein from a Crude Cell Organelle Fraction of Phaseolus vulgaris L

Sonication of a crude cell organelle fraction from hypocotyl tissue of dark-grown bean seedlings, and from suspension-cultured cells released a hydroxyproline-containing protein. The purification of this protein is described. It was found to be an arabinogalactan protein composed of 90% carbohydrate and 10% protein. The major sugars are galactose, arabinose, and uronic acids, and the major amino acids are hydroxyproline, serine, and alanine. Its molecular weight was estimated at 1.4 × 10⁵ daltons and the isoelectric point at pH 2.3. The molecule is soluble in 5% trichloroacetic acid and can be precipitated with β-galactosyl Yariv antigen. Pulse-chase experiments indicated that it was a secretory protein. The biosynthesis of arabinogalactan proteins is discussed.     

The Effects of Shade Treatment and Light Intensity on Rihulose-1,5-Diphosphate Carboxylase Activity and Fraction I Protein Level in the First Leaf of Barley

It has been confirmed that shading leaves from day 5 onwardslowers the rate of CO₂ fixation when they are placed in saturatingirradiances. The reduction due to shade treatment is about 46per cent and a similar reduction in maximum chlorophyll contentof the leaf follows shading. Maximum amounts of total solubleprotein and of Fraction I protein are less in shaded leavesthan in control leaves and prolonged treatment leads to a declinein leaf protein content. The relative amounts of different proteinare also affected by treatment; in control leaves Fraction Iprotein accounts for about 45 per cent of the total but in shadedleaves the value is about 30 per cent. Increases and decreasesin leaf protein amount, with concomitant changes in the ratioof Fraction I to total protein can be brought about by removingshades and re-applying them. Such changes can be induced evenin fully expanded leaves in which net protein synthesis is notusually found. Maximal amounts of leaf protein are found in irradiances of60 W m^–2 or more, with lower values at lower light intensities.Where the first leaf is held in a stream of CO₂-free air a lowerlevel of protein is found. This, and the ratio of Fraction Ito total protein, are similar to values for shaded leaves, andsuggest the involvement of photosynthetic carbon fixation indetermining leaf protein amount. A 1:1 linear correlation between amount of Fraction I proteinand RuDP carboxylase activity is shown but the rate of CO₂ incorporationby leaf extracts is 2–3 times greater than that of theintact leaf. The significance of this and the effect of irradianceon leaf protein amount are discussed.     

Incorporation of Large Subunits into Ribulose Bisphosphate Carboxylase in Chloroplast Extracts : Influence of Added Small Subunits and of Conditions during Synthesis

The incorporation of newly synthesized large subunits into ribulose bisphosphate carboxylase/oxygenase (RuBisCO) in pea chloroplast extracts occurs at the expense of intermediate forms of the large subunit which are complexed with a binding protein. Most subunits of this binding protein are found in dodecameric complexes in chloroplast extracts. Addition of small subunits to these extracts results in approximately 40 to 60% increased incorporation of newly made large subunits into RuBisCO at low or zero concentrations of ATP, but is without significant effect at high concentrations of ATP, a condition in which the dodecameric binding protein complex is dissociated into subunits. Overall, these data support the assumption that the incorporation of large subunits into RuBisCO in chloroplast extracts reflects de novo assembly rather than `mere' exchange of subunits. The in vitro assembly of large subunits into RuBisCO is a function of the conditions under which the large subunits are synthesized in organello. When the large subunits are made in chloroplasts suspended in 188 millimolar sorbitol, they are approximately 2- to 3-fold better able to assemble into RuBisCO when subsequently incubated in vitro than when they are synthesized in chloroplasts suspended in 375 millimolar sorbitol. This observation indicates that mere synthesis of large subunits is not sufficient to confer maximal assembly competence on large subunits.     

Chaperonin-Repairable Subtle Incompleteness of Protein Assembly Induced by a Substitution of Hydrogen with Deuterium: Effect of GroE on Deuterated Ribulose 1,5-Bisphosphate Carboxylase

For investigating the effect of slight modification of proteinson their higher-ordered structure, and that of chaperonin onthe functional assembly of proteins, we prepared partially deuteratedribulose 1,5-bisphosphate carboxylase (Rubisco) by cultivatingChlorella ellipsoidea in 100 mol% D₂O medium. Chlorella cellsgrown in the D₂O medium (D-Chlorella) contained almost the sameamount of Rubisco (D-Rubisco) as the cells grown in H₂O medium(H-Chlorella) determined by Western blotting using Rubisco-specificantibody, whereas the activity of D-Rubisco determined by carbonfixation was only 28% of that of Rubisco from H-Chlorella (H-Rubisco).D-Rubisco, however, showed similar K_m and pH and temperatureoptima to those of H-Rubisco as well as similar antibody bindingcapability. The enzyme activity of D-Rubisco was recovered to84% of that of H-Rubisco by the addition of GroE proteins (GroEL,chaperonin 60, and GroES, chaperonin 10), members of the chaperoninfamily produced by Escherichia coli. These data suggest thatD-Rubisco has subtle incompleteness in terms of functional assembly,a situation that is correctable by chaperonin. (Received August 8, 1994; Accepted January 9, 1995)     

Effects of Mitochondrial Protein Synthesis Inhibitors on the Incorporation of Isoleucine into Plasmodium falciparum In Vitro1

Plasmodium falciparum was grown in human erythrocytes in vitro and the effect of chloramphenicol, erythromycin, and tetracycline on growth and maturation of the parasites and on their ability to incorporate [³H]isoleucine into protein was observed. Exposure of rings to high concentrations of chloramphenicol had little effect on subsequent maturation of the rings whereas brief (4 h) exposure of trophozoites caused a dose-dependent inhibition of subsequent ring formation. Incorporation of [³H]isoleucine into protein was not affected during at least 6 h of exposure to high concentration of the three drugs examined, but appreciable inhibition was observed after 21 h, with chloramphenicol being the least effective inhibitor. These results suggest that there is a stage-specific effect of inhibition of mitochondrial protein synthesis on subsequent development and that the mitochondria are essential for growth and development even though they lack a functional Krebs cycle.     

Regulation of Soybean Net Photosynthetic CO(2) Fixation by the Interaction of CO(2), O(2), and Ribulose 1,5-Diphosphate Carboxylase

Kinetic properties of soybean net photosynthetic CO₂ fixation and of the carboxylase and oxygenase activities of purified soybean (Glycine max [L.] Merr.) ribulose 1, 5-diphosphate carboxylase (EC 4.1.1.39) were examined as functions of temperature, CO₂ concentration, and O₂ concentration. With leaves, O₂ inhibition of net photosynthetic CO₂ fixation increased when the ambient leaf temperature was increased. The increased inhibition of CO₂ fixation at higher temperatures was caused by a reduced affinity of the leaf for CO₂ and an increased affinity of the leaf for O₂. With purified ribulose 1,5-diphosphate carboxylase, O₂ inhibition of CO₂ incorporation and the ratio of oxygenase activity to carboxylase activity increased with increased temperature. The increased O₂ sensitivity of the enzyme at higher temperature was caused by a reduced affinity of the enzyme for CO₂ and a slightly increased affinity of the enzyme for O₂. The similarity of the effect of temperature on the affinity of intact leaves and of ribulose 1,5-diphosphate carboxylase for CO₂ and O₂ provides further evidence that the carboxylase regulates the O₂ response of photosynthetic CO₂ fixation in soybean leaves. Based on results reported here and in the literature, a scheme outlining the stoichiometry between CO₂ and O₂ fixation in vivo is proposed.     

Synthesis of the Small Subunit of Ribulose-1,5-bisphosphate Carboxylase by Soluble Fraction Polyribosomes of Pea Leaves

The products of amino acid incorporation by pea (Pisum sativum L.) leaf soluble fraction polyribosomes in the wheat germ system were examined by two-dimensional electrophoresis and fluorography.     

Germination of Phaseolus vulgaris: IV. Patterns of Protein Synthesis in Excised Axes

Soluble proteins from excised Phaseolus vulgaris axes incubated for 1 hour in ³H or ¹⁴C- amino acid mixtures at different times during the period leading up to initiation of cell elongation were compared by acrylamide gel electrophoresis. Differences in electrophoretic patterns were found when proteins from axes incubated during the 1st hour of imbibition were compared with proteins from axes incubated during the hour when cell elongation was initiated. These differences greatly diminished by the 2nd hour of imbibition which suggests that they were due primarily to incomplete axis imbibition. A 5-hour actinomycin D treatment which reduced amino acid incorporation by 40% in the 5th hour had no apparent effect on the electrophoretic pattern during that hour.     

In Vitro Stability and Inactivation of Peptide Hydrolases Extracted from Phaseolus vulgaris L.

Endopeptidase activity against azocasein had a higher temperatureoptimum (50°C) in leaf extracts than in cotyledon extracts(37°C). The temperature optima for aminopeptidase (46°C)and for carboxypeptidase (53°C) were similar in leaf andcotyledon extracts. The endopeptidase activity showed an excellentstability in crude extracts from leaves even at 37°C, whilethe endopeptidase in cotyledon extracts was less stable. Carboxypeptidasewas very stable in both leaf and cotyledon extracts. Aminopeptidasewas the least stable of the enzymes investigated and its inactivationrate depended on the source of the extract. A moderate stabilitywas observed in extracts of leaves or of ungerminated seeds,but this enzyme was rapidly inactivated in cotyledon extractsat pH 5.4. At pH 7.5 aminopeptidase remained active longer thanat pH 5.4. From experiments with mixed extracts it could beconcluded that in cotyledons an aminopeptidase inactivatingfactor was formed during germination. This factor was heat sensitive,excluded by Sephadex G-25, precipitated by 75% ammonium sulfateand inhibited by tosyl-L-lysine chloromethyl ketone. These datasuggest that the factor is a protein and considering the similarproperties it appears possible that it is the endopeptidaseformed during germination. (Received May 15, 1981; Accepted July 18, 1981)     

CHLOROPLAST STRUCTURE AND FUNCTION IN ac-20, A MUTANT STRAIN OF CHLAMYDOMONAS REINHARDI : I. CO2 Fixation and Ribulose-1,5-Diphosphate Carboxylase Synthesis

A mutant strain of the green alga Chlamydomonas reinhardi, ac-20, is described in which both the rate of CO₂ fixation by whole cells and the rate of carboxylation of ribulose-1,5-diphosphate in cell-free extracts are reduced, particularly when sodium acetate is present in the growth medium. Of the enzymes of the reductive pentose phosphate cycle tested, only ribulose-1,5-diphosphate carboxylase activity is reduced in the mutant strain, and it appears that the low carboxylase activity limits the strain's rate of photosynthetic carbon metabolism. Evidence is presented to show that the fluctuation in the level of the enzyme activity in the presence or absence of acetate results from the fluctuation in the level of some factor(s) limiting the rate of synthesis of the protein.     

Protein Degradation in Lemna with Particular Reference to Ribulose Bisphosphate Carboxylase: II. The Effect of Nutrient Starvation

The concept of ribulose bisphosphate carboxylase as a storage protein is not supported in the case of Lemna minor, where the enzyme appears to be particularly stable under conditions of nitrogen starvation. Total nutrient starvation in light and in the dark induced the degradation of this enzyme, but not at an enhanced rate compared with other leaf proteins and, surprisingly, darkness inhibited the degradation of chlorophyll which occurs with total nutrient starvation in the light. The data suggest that Lemna is not programmed to senesce in response to nutrient starvation. Differences in the pattern of protein degradation, which occurred under the stress conditions employed, are not consistent with a simple model of protein degradation in which the degradative system is assumed to be located in the vacuole. The data is best explained by a dual system in which cytosolic proteins are degraded by a vacuolar/lysosomal system and chloroplast proteins are degraded within the chloroplast. Whatever the system of degradation, our data do not support the proposed correlation between the rate of protein degradation and either protein charge or size.