On the synthesis and degradation of the multiple forms of catalase in mouse liver: effects of aminotriazole and p-chlorophenoxyisobutric acid ethyl ester

Aspects of the synthesis and degradation of the multiple forms of catalase in mouse liver have been investigated. The kinetics of return of catalase after aminotriazole inhibition indicated a product-precursor relationship between the granular and supernatant pools of the enzyme, and electrophoretic resolution of the individual heteromorphs of catalase during this treatment served to substantiate this relationship and indicated, in addition, that aminotriazole-inhibited catalase may be partially reactivated in the cytosol. Changes in the activity of mouse liver and kidney catalases after the administration of chlorophenoxyisobutyric acid ethyl ester (CPIB) were also monitored. After an initial decline in activity, a rapid increase to an elevated steady state occurred, with an approximately threefold increase in the liver and twofold increase in the kidney. Subcellular fractionation of the livers of CPIB-treated mice showed a massive initial increase in the supernatant pool of catalase, accompanied by a steady decrease in the activity of the peroxisomal pools. Activity increased in the peroxisomal pools at later stages of treatment, but even after a new CPIB-induced steady state was achieved, the supernatant pool of catalase remained grossly elevated. Electrophoresis of the individual heteromorphs of catalase in the supernatant after CPIB treatment showed a predominance of the most cathodal migrating forms, and turnover studies demonstrated that catalase in CPIB-treated animals exhibited a substantially lowered rate of degradation by comparison with normal animals. These results have been discussed in relation to the intracellular sequestration and turnover characteristics of catalase.     

Is the cytosolic catalase induced by peroxisome proliferators in mouse liver on its way to the peroxisomes?

Dietary treatment of male C57B1/6 mice with clofibrate, nafenopin or WY-14.643 resulted in a modest (at most 2-fold) increase in the total catalase activity in the whole homogenate and mitochondrial fraction prepared from the livers of these animals. On the other hand, the catalase activity recovered in the cytosolic fraction was increased 12- to 18-fold, i.e. 30-35% of the total catalase activity in the hepatic homogenate was present in the high-speed supernatant fraction after treatment with these peroxisome proliferators. A study of the time course of the changes in peroxisomal and cytosolic catalase activities demonstrated that the peroxisomal activity both increased upon initiation of exposure and decreased after termination of treatment several days after the increase and decrease, respectively, in the corresponding cytosolic activity. This finding suggests that the cytosolic catalase may be on its way to incorporation into peroxisomes.     

Immunogold labelling indicates high catalase concentrations in amorphous and crystalline inclusions of sunflower (Helianthus annuus L.) peroxisomes

Summary Immunogold labelling and electron microscopy were used to investigate whether catalase was present in peroxisomal inclusions, the composition of which has not yet been determined in plant cells. In the mesophyll cells of sunflower (Helianthus annuus L.) cotyledons, the catalase gold label was confined to peroxisomes. At day 2 of postgerminative growth in darkness, peroxisomes were free of inclusions, and the matrix was homogeneously labelled with gold particles. Thereafter, amorphous inclusions appeared, but by day 5 of growth, conspicuous crystalline inclusions (cores) were the predominant type. This developmental change, first observed in cotyledons grown in continuous light between day 2.5 and 5, also took place in cotyledons kept in permanent darkness. Both amorphous and crystalline inclusions showed a much higher immunogold label than did the peroxisomal matrix, indicating that catalase was a component of both types of peroxisomal inclusions. In contrast to catalase, the immunogold label of glycolate oxidase was almost completely absent from cores and was confined to the peroxisomal matrix. Together with reports on the absence of other enzymes from peroxisomal inclusions in sunflower and other species (Vaughn, 1989) our results suggest that catalase is a major constituent of amorphous and crystalline peroxisomal inclusions in plants.     

Incorporation of Tritium from Thymidine-methyl-H3 into DNA,RNA, Lipids and Protein in Logarithmic and Stationary Phase Tetrahymena pyriformis,Strain W*

SYNOPSIS. Numerous reports may be found in the literature on cytoplasmic and non-DNA utilization of tritium from H³-thymidine. Such reports underscore the need to clarify the metabolic fate of H³-thymidine. This investigation outlines the fate of thymidinemethyl-H³ (TMH³) in logarithmic phase and stationary phase Tetrahymena pyriformis, strain W. Isotope identification by liquid scintillation spectrometry in chemically derived fractions of log phase cultures grown thruout the initial 48 hours of population growth with TMH³ revealed the majority of the radioactivity (90% of intracellular recovery) to be in the DNA fraction. The remainder of the intracellular label was recovered in the acid soluble fraction, lipid fraction, and a small amount in the RNA and cell residue. On chromatographs, tritium appeared only in the thymine moiety of the nucleic acid derivatives. Hence in dividing cells, thymidine-methyl-H³ is “essentially” specific for DNA at the dosage used although some incorporation into other compounds was detected. Fractionation of the lipid extract from the above experiment on a florisil column localized most of the label to the triglyceride and phospholipid fractions with some recovery in the cholesterol-esters. Similar scintillation counting of the various fractions of early stationary phase cells incubated for the last 48 hours of culture with TMH³ revealed limited tritium distribution in all fractions.     

Replication of mitochondrial DNA in the early development of Misgurnus fossilis

Mitochondria isolated from Misgurnus fossilis embryos at various developmental stages were incubated with ³H-dTTP in vitro and the incorporation into mtDNA was determined. It has been found that the rate of mtDNA labeling increases exponentially with a doubling time of 7 hr from 0.01 pmole of ³H-dTMP/mg protein/hr in mitochondria from unfertilized eggs to 0.4 pmoles of ³H-dTMP/mg protein/hr in mitochondria of 35 hr embryos. The pool of intramitochondrial dTTP decreases 2.5 times during the first 10 hr after fertilization, then remains practically constant up to 35 hr of development. The rate of exogenous ³H-dTTP incorporation into the acid soluble pool of isolated mitochondria at two stages is approximately proportional to the pool size. Thus identical specific activities of ³H-dTTP inside mitochondria would be obtained even with pools of different sizes. We conclude that the increase of ³H-dTMP incorporation into mtDNA in development reflects genuine activation of mtDNA synthesis. As early as 6 hr after fertilization the bulk of the label incorporated into mtDNA is found in the fraction associated with covalently closed molecules. This pattern of labeling characteristic for replicating mtDNA is maintained throughout early development. In contrast such preferential label incorporation into the closed circular fraction was not found with mitochondria of unfertilized eggs. Closed mtDNA from unfertilized eggs contains not more than 1% of molecules with D-loops. In 35 hr embryos the corresponding value is equal to about 4%. Activation of mtDNA replication in embryogenesis is probably due to the activation of mechanisms responsible for the generation of primers for replication. DNA polymerase activity solubilized from mitochondria remains unchanged in the course of embryogenesis.     

Interaction with deoxycholate of rat liver peroxisomal and cytosolic catalase

Rat liver catalase was found to interact with deoxycholate (DOC). When purified, the peroxisomal catalase was precipitated at pH 6 in the presence of DOC, whereas in the peroxisomal extract (with DOC) it was unsedimentable at pH 6. The membrane fraction in the extract interacted with the catalase instead of DOC, and prevented the precipitation of catalase with DOC at pH 6. The peroxisomal catalase seemed to be easily modified by lysosomal protease during manipulation, and this proteolytic cleavage rendered the molecule able to interact with the membrane. On the other hand, the cytosolic catalase, both in the cytosol fraction and in the purified preparation, sedimented at pH 6 in the presence of DOC. The cytosolic catalase was far more resistant to proteolytic modification than the peroxisomal catalase. The molecule of peroxisomal catalase is assumed to have a site for recognizing the membrane, whereas such a structure may be absent in the cytosolic catalase or may not be easily exposed by proteolytic cleavage.     

Metabolism of arginine by aging and 7 day old pumpkin seedlings

The metabolism of arginine by etiolated pumpkin (Cucurbita moschata) seedlings was studied over various time and age intervals by injecting arginine-U-¹⁴C into the cotyledons. At most, 25% of the ¹⁴C was transported from the cotyledon to the axis tissue and the amount of this transport decreased with increasing age of the seedlings. The cotyledons of 25 day old plants contained 60% of the administered ¹⁴C as unmetabolized arginine. Little ¹⁴C was in sugars and it appeared that arginine was the primary translocation product. Time course studies showed that arginine was extensively metabolized and the labeling patterns suggest that different pathways were in operation in the axis and cotyledons. The amount of arginine incorporated into cotyledonary protein show that synthesis and turnover were occurring at rapid rate. Only 25% of the label incorporated into protein by 1.5 hr remained after 96 hr. The label in protein was stable in the axis tissue. By 96 hr 50% of the administered label occurred as ¹⁴CO₂ and it appeared that arginine was metabolized, through glutamate, by the citrio acid cycle in the cotyledons. The experiments showed that an extensive conversion of arginine carbon into other amino acids did not occur.     

Intracellular localization of phosphodiesterase induced by cyclic adenosine 3',5'-monophosphate in Dictyostelium discoideum

The intracellular distribution of phosphodiesterase [EC 3.1.4.17] induced by cyclic adenosine 3',5'-monophosphate (cAMP) in Dictyostelium discoideum was studied. When cAMP-treated cells were homogenized and fractionated according to the method of de Duve et al. ((1955) Biochem, J. 60, 604), the specific activity of phosphodiesterase was highest in the light mitochondrial fraction. Peaks of specific activities of alkaline phosphatase (marker enzyme of membrane) and catalase (marker enzyme of peroxisomes) also appeared in the same fraction as phosphodiesterase. However, after centrifugation of the light mitochondrial fraction in a sucrose density gradient, the activity of phosphodiesterase was clearly separated with that of catalase (density 1.19 g/ml) and showed three peaks at lower density (1.10, 1.13, 1.17 g/ml) with good reproducibility. Some parts (1.13, 1.17 g/ml) of the activity in the gradient overlapped with alkaline phosphatase activity, but in the density fraction of 1.10 g/ml the activity of alkaline phosphatase was hardly detectable. When the light mitochondrial fraction was treated with Emulgen 108, or sonicated, phosphodiesterase was more easily solubilized than alkaline phosphatase and catalase, and was found in supernate after centrifugation at 20,000 X g for 30 min. In order to distinguish the locations of the three enzymes, the supernatant of the light mitochondrial fraction treated with Emulgen 108 was subjected to charge shift electrophoresis. The electrophoretic mobilities of phosphodiesterase and catalase were unaffected by ionic detergent. However, alkaline phosphatase shifted towards the anode in the presence of anionic detergent (sodium deoxycholate), and shifted towards the cathode in cationic detergent (cetyltrimethylammonium bromide), relative to nonionic detergent (Emulgen 108) alone. Thus, some part of the phosphodiesterase induced by cAMP may be associated with the plasma membrane, but the remainder is localized in some kind of intracellular particle of lower density. Moreover, the association with the membrane or particle is more easily dissociated than that of alkaline phosphatase, and the liberated phosphodiesterase is rather hydrophilic.     

Relationships between the parasitoid Hyposoter exiguae and Trichoplusia ni: Prevention of host pupation at the endocrine level

RNA synthesis in normal Trichoplusia ni fifth instars and hosts parasitized at ca. 12 hr post-ecdysis was followed by measuring ³H-uridine incorporation with an autoradiographic technique.Uptake of ³H-uridine was high in control prothoracic glands at 6 and 30 hr and their cytology indicated an active secretory phase which was most pronounced at 30 hr. At the same time, glands of parasitized larvae decreased incorporation and appeared less active than controls. At > 75 hr, control fat body cells incorporated almost no label but were filled with RNA-protein granules apparently sequestered from the haemolymph preparatory to pupation. With respect to incorporation and cytology, fat body of parasitized larvae was unchanged from earlier in the instar, which indicates that the changeover to pupal preparations had not taken place. Imaginal wing disks incorporated label and grew appreciably in control larvae but abruptly decreased uptake and showed no size increase in parasitized larvae. Incorporation of Malpighian tubule, midgut epithelium, and certain muscles at > 75 hr showed little change in parasitized larvae, but in controls activity was reduced and histolysis occasionally was evident in muscles.The parasitoid, Hyposoter exiguae, apparently prevented host larvae from pupating by preventing activation of host prothoracic glands in the fifth instar. Other tissues which are normally activated for metamorphosis by the prothoracic glands continued normal larval activities until the end of the association.     

Occurrence and biosynthesis of catalase at different stages of seed maturation

It was to be shown whether during the biogenesis of microbodies some of their components were already present in the cell prior to the organelle's assembly. To this end, the occurrence and properties of catalase in soluble and particular fractions of ripening cucumber seeds were examined. Homogenates of seeds from ripening fruits were fractionated by isopycnic density gradient centrifugation, and thus catalase was found in three different fractions: as a soluble enzyme in the gradient supernatant, as a membrane fraction at density d=1.18 kg l^-1, and in association with microbodies. In the early steps of seed formation, catalase was detected at density d=1.18 kg l^-1 and in the gradient supernatant. At a later stage of seed maturation, however, catalase was primarily associated with microbodies which exhibited an equilibrium density of d=1.23 kg l^-1. M _r as well as subunit M _r of catalase were determined, and their close immunological relationship to leaf peroxisomal catalase and glyoxysomal catalase was demonstrated. Biosynthesis of catalase at different stages of seed maturation was investigated by in vivo labeling with l-[³⁵S]methionine, l-[¹⁴C]leucine and -[³H]aminolaevulinic acid. Electrophoretic analysis of de novo synthesized catalase subunits revealed the occurrence of a heavy form (M _r 57,500) in the soluble fraction; this form was preferentially labeled. A light form, M _r 53,500, was detected in microbodies and also in the soluble fraction. The findings lend support to the hypothesis that the rate of catalase synthesis is highest in an early stage of seed formation, when globulins have already been formed, but before de novo synthesis of malate synthase has commenced. Prior to microbody assembling, a cytoplasmic pool of catalase was labeled.Abbreviations EDTA Na₂-ethylenediaminotetraacetate - Hepes 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid - M _r molecular weight     

An Apparent Paradox in the Occurrence, and the In Vivo Turnover, of C-Terminal Tyrosine in Membrane-Bound Tubulin of Brain

: Tubulin tyrosine ligase catalyzes the reversible addition of tyrosine to the C-terminus of tubulin α chains. By using ligase and carboxypeptidase A in conjunction, we have previously shown that brain cytoplasmic tubulin exists in three forms: 15–40% already has C-terminal tyrosine, another 10-30% can accept additional tyrosine, and about one-half is an uncharacterized species which is not a ligase substrate. A membrane-bound fraction of brain tubulin, purified by vinblastine precipitation from a detergent extract, has been found to differ by the complete absence of preexisting tyrosine. The membrane fraction from which tubulin was extracted also contained masked forms of both ligase and a distinct detyrosylating enzyme, which can be released by detergent extraction. The turnover of α-chain C-terminal tyrosine in vivo was studied by incubating brain mince with labeled tyrosine, or injecting it intracerebrally, under conditions where protein synthesis was inhibited. Tyrosine appeared to turn over to about the same extent in membrane-bound, as in soluble, tubulin. This apparently paradoxical result was not due to ATPase in the membrane fraction, which might have allowed ligase-catalyzed exchange between free and fixed tyrosine. Authentic [¹⁴C]tyrosylated tubulin added to the brain membrane fraction was not detyrosylated or subject to endoprotease digestion during subsequent procedures to isolate tubulin. The unexpected finding that tubulin tyrosylated at the C-terminal in vivo appears to be in the “non-substrate” fraction points toward a possible resolution of the paradox.     

Sulfation of fucoidan in Fucus embryos. I. Possible role in localization

Zygotes of the brown alga Fucus distichus L. Powell divide into two cells which are structurally and biochemically different from each other. Cytochemical staining and autoradiography indicate that a sulfated polysaccharide is localized in only one of the two cells. Up to 10 hr after fertilization, no localization of sulfated polysaccharides is detectable in zygotes, and little ³⁵S (Na₂³⁵SO₄) is incorporated into an acid-soluble carbohydrate fraction. Between 10 and 16 hr, during rhizoid initiation and several hours before the first cell division, there is a large increase in the amount of ³⁵S incorporated into this fraction. The label is found associated with the sulfated fucose polymer fucoidan. Various extraction techniques and labeling experiments demonstrate that fucoidan is unsulfated at fertilization and undergoes little metabolic activity or turnover during the first 24 hr. Thus, the incorporation of sulfate into this carbohydrate fraction appears to involve a sulfation of a preexisting, unsulfated fucan polymer. The degree of sulfation achieved at this time in vivo is sufficient for migration of fucoidan through an electric field in agarose or acrylamide gels. The possible role of sulfation as a mechanism for the localization of fucoidan in the rhizoid cell by means of an intracellular electrical gradient is discussed.     

The intracellular distribution of calcium in the mucosa of the avian shell gland

The intracellular distribution of calcium has been studied in the mucosa of the avian shell gland, a tissue which transports large quantities of calcium during discrete time intervals. Ca⁴⁵ was administered to hens either in a single dose followed by sacrifice 5 min later or in repeated doses over an extended period followed by sacrifice 2 hr or 24 hr after the last injection. Subcellular fractions were isolated by differential centrifugation and analyzed for Ca⁴⁵. The Ca⁴⁵ was located principally in the particulate fractions; the concentration (CPM Ca⁴⁵/mg N) was highest in the mitochondrial fraction. Comparisons of (1) the Ca⁴⁵ distribution in shell gland cells with that of liver cells, (2) the alterations which occur due to the phase of the egg laying cycle, (3) the effects due to the time elapsed since the last injection of Ca⁴⁵, and (4) the Ca⁴⁵ distribution of the short term experiments with that of the long term experiments revealed that the mitochondrial fraction of the shell gland appeared to be active in the movement of calcium. The microsomal fraction showed increased values in CPM Ca⁴⁵/mg N when calcification was occurring, which may indicate that the subcellular components of this fraction have a role in calcium transport. The nuclear and supernatant fractions did not seem to be involved in the transport process. The implications of these results concerning the manner by which calcium may be controlled on a cellular level in this system are discussed.     

Postnatal development of peroxisomal and mitochondrial enzymes in rat liver.

Subcellular organellles from livers of rats three days prenatal to 50 weeks postnatal were separated on sucrose gradients. The peroxisomes had a constant density of 1.243 g/ml throughout the life of the animal. The density of the mitochondria changed from about 1.236 g/ml at birth to a constant value of 1.200 g/ml after two weeks. The peroxisomal and mitochondrial fatty acid beta-oxidation and the peroxisomal and supernatant activities of catalase and glycerol-3-phosphate dehydrogenase were measured at each age, as well as the peroxisomal core enzyme, urate oxidase, and the mitochondrial matrix enzyme, glutamate dehydrogenase. All of these activities were very low or undetectable before birth. Mitochondrial glutamate dehydrogenase and peroxisomal urate oxidase reached maximal activities per g of liver at two and five weeks of age, respectively. Fatty acid beta-oxidation in both peroxisomes and mitochondria and peroxisomal glycerol-3-phosphate dehydrogenase exhibited maximum activities per g of liver between one and two weeks of age before weaning and then decreased to steady state levels in the adult. Peroxisomal beta-oxidation accounted for at least 10% of the total beta-oxidation activity in the young rat liver, but became 30% of the total in the liver of the adult female and 20% in the adult male due to a decrease in mitochondrial beta-oxidation after two weeks of age. The greatest change in beta-oxidation was in the mitochondrial fraction rather than in the peroxisomes. At two weeks of age, four times as much beta-oxidation activity was in the mitochondria as in the peroxisomal fraction. Peroxisomal glycerol-3-phosphate dehydrogenase activity accounted for 5% to 7% of the total activity in animals younger than one week, but only 1% to 2% in animals older than one week. Up to three weeks of age, 85% to 90% of the liver catalase was recovered in the peroxisomes. The activity of peroxisomal catalase per g of rat liver remained constant after three weeks of age, but the total activity of catalase further increased 2.5- to 3-fold, and all of the increased activity was in the supernatant fraction.     

On the nature and characteristics of the multiple forms of catalase in mouse liver.

In order to clarify the nature of the heterogeneity of mouse liver catalase, the enzyme was purified and characterized by several criteria. Absorption and sedimentation properties provided little indication of significant differences between the mouse liver enzyme and catalases from other mammalian sources which do not display multiplicity. A denotement of the nature of the variformity in mouse liver catalase was provided, however, by the demonstration that the heteromorphs may be interconverted under conditions which favor the addition or removal of sialic acid residues. It was also observed that CMP-sialic acid, together with microsomal extract, protected the supernatant (desialated) pool of catalase from inactivation upon storage; and that the pattern of multiplicity which was exhibited by the purified enzyme on isoelectric focusing, was considerably altered by incubation with neuraminidase. With regard to the individual characteristics of the separate forms of purified mouse liver catalase, significant differences were noticeable in relation to their isoelectric points, specific activities, heme content, and specific binding of [¹⁴C]aminotriazole.     

The biosynthesis of gramicidin S in a cell-free system

1. A cell-free system prepared from Bacillus brevis cells, harvested in the late phase of growth and consisting of the 11000g supernatant, has been shown to incorporate into gramicidin S the five constituent amino acids added in labelled form. The results are consistent with complete synthesis and not merely a completion of pre-existing intermediate peptides. 2. The incorporation of ¹⁴C-labelled amino acids by the 11000g supernatant into gramicidin S requires an energy source. Omission of phosphoenolpyruvate and pyruvate kinase from the incubation mixture prevents incorporation into gramicidin S. The cell-free system incorporates [¹⁴C]-leucine, -proline and -phenylalanine over a period of 4hr. With [¹⁴C]leucine, incorporation into gramicidin S takes place in the range pH6–9 with maximum incorporation at pH7·0. High concentrations of chloramphenicol or puromycin decreased the incorporation into gramicidin S by only about 20%. 3. The 50000g supernatant exhibited no decrease in ability of incorporating [¹⁴C]valine into gramicidin S as compared with the 11000g supernatant. About 40% of the incorporating ability remained in the 105000g supernatant after 3hr. centrifugation. When recombining the 105000g sediment with the 105000g supernatant, some increase in incorporation over that obtained with the supernatant alone was obtained. The findings tend to support the view that gramicidin S is synthesized in a different manner from that of proteins.     

Catalase modifies yeast Saccharomyces cerevisiae response towards S-nitrosoglutathione-induced stress

Abstract

Nitric oxide is known to be a messenger in animals and plants. Catalase may regulate the concentration of intracellular ^?NO. In this study, yeast Saccharomyces cerevisiae cells were treated with 1–20 mM S-nitrosoglutathione (GSNO), a nitric oxide donor, which decreased yeast survival in a concentration-dependent manner. In the wild-type strain (YPH250), 20 mM GSNO reduced survival by 32%. The strain defective in peroxisomal catalase behaved like the wild-type strain, while a mutant defective in cytosolic catalase showed 10% lower survival. Surprisingly, survival of the double catalase mutant was significantly higher than that of the other strains used. Incubation of yeast with GSNO increased the activities of both superoxide dismutase (SOD) and catalase. Pre-incubation with cycloheximide prevented the activation of catalase, but not SOD. The concentrations of oxidized glutathione increased in the wild-type strain, as well as in the mutants defective in peroxisomal catalase and an acatalasaemic strain; it failed to do this in the mutant defective in cytosolic catalase. The activity of aconitase was reduced after GSNO treatment in all strains studied, except for the mutant defective in peroxisomal catalase. The content of protein carbonyls and activities of glutathione reductase and S-nitrosoglutathione reductase were unchanged following GSNO treatment. The increase in catalase activity due to incubation with GSNO was not found in a strain defective in Yap1p, a master regulator of yeast adaptive response to oxidative stress. The obtained data demonstrate that exposure of yeast cells to the ^?NO-donor S-nitrosoglutathione induced mild oxidative/nitrosative stress and Yap1p may co-ordinate the up-regulation of antioxidant enzymes under these conditions.     

Nature of the phytochrome effect on the binding of glycolate oxidase to peroxisomes in vitro

The attachment of glycolate oxidase to the peroxisomal fraction derived from etiolated barley leaves (Hordeum vulgare L. cr. Dvir) is affected by light. The effect of red irradiation is reversed by subsequent far-red irradiation, indicating the involvement of phytochrome. This phytochrome effect is assumed to be related to phytochrome binding. Indeed, prevention by filipin (1.2·10^-6 mol g^-1 f wt) or cholesterol of phytochrome binding to membranes abolishes the effect of light on the interaction between glycolate oxidase and the peroxisomal fraction. Glycolate oxidase binding is affected by addition of quasi-ionophores such as gramicidin and filipin at a concentration of 0.6·10^-3 mol g^-1 f wt. This fact indicates that peroxisome-glycolate oxidase interaction may be affected by membrane potential. Since both ion transport and membrane potential are known to be affected by phytochrome, it is proposed that phytochrome acts in the light-induced modulation of glycolate oxidase attachment as a quasi-ionophore.Abbreviations GO glycolate oxidase - Pr and Pfr phytochrome forms absorbing in red and far-red, respectively - R and F red and far-red irradiation - Cumulative 20 Kp 20,000 g pellet obtained by centrifugation of the crude extract - 1 Kp 1,000 g pellet - 20 Kp 20,000 g pellet, obtained by centrifugation of 1 Kp supernatant - 1 Kp, 20 Kp and cumulative 20 Kp pellets obtained after density centrifugation through a sucrose cushion     

Phytochrome-controlled Leaf Unrolling and Protein Synthesis

In the primary leaf sections of etiolated wheat (Triticum aestivum L.) seedlings, red light-induced unrolling is accompanied by an increase in incorporation of ¹⁴C-leucine into protein. By differential centrifugation, the unrolling response was found to be closely related to incorporation of the amino acid into the supernatant fraction (105,000g). Cycloheximide and chloramphenicol inhibit both leaf unrolling and synthesis of the supernatant protein, although chloramphenicol exerts its effect more strongly on the fraction which presumably contains the plastids. In a barley (Hordeum vulgare L.) albino mutant completely devoid of ribulose diphosphate carboxylase activity, only incorporation of ¹⁴C-leucine into the supernatant fraction is substantially promoted by red light. This mutant exhibits the photoresponse of leaf unrolling.     

The Kinetics of the Synthesis of Ribosomal RNA in E. coli

The kinetics of the synthesis of ribosomal RNA in E. coli has been studied using C¹⁴-uracil as tracer. Two fractions of RNA having sedimentation constants between 4 and 8S have kinetic behavior consistent with roles of precursors. The first consists of a very small proportion of the RNA found in the 100,000 g supernatant after ribosomes have been removed. It has been separated from the soluble RNA present in much larger quantities by chromatography on DEAE-cellulose columns. The size and magnitude of flow through this fraction are consistent with it being precursor to a large part of the ribosomal RNA.

A fraction of ribosomal RNA of similar size is also found in the ribosomes. This fraction is 5 to 10 per cent of the total ribosomal RNA and a much higher proportion of the RNA of the 20S and 30S ribosomes present in the cell extract. The rate of incorporation of label into this fraction and into the main fractions of ribosomal RNA of 18S and 28S suggests that the small molecules are the precursors of the large molecules. Measurements of the rate of labeling of the 20, 30, and 50S ribosomes made at corresponding times indicate that ribosome synthesis occurs by concurrent conversion of small to large molecules of RNA and small to large ribosomes.