Effect of abscisic acid and auxin on ribonuclease during ageing of bean endocarp tissue sections

The activity of RNase increases rapidly upon cutting sectionsof bean (Phascolus vulgaris L. var. Kentucky Wonder) endocarp,peaks within 4 to 8 hr and then declines. This rapid developmentof RNase activity is inhibited by cycloheximide. Auxin (naphthaleneaceticacid, NAA) accelerates the rate of decline of RNase. Abscisicacid (ABA) enhances the level of RNase between 4 and 24 hr,associated with a decline in RNA, and this effect of ABA isobscured in the presence of auxin. ¹ This work was supported by National Science Foundation Grant(GB-8316) to J. A. Sacher. ² On leave from Laboratorio di Radiobiochimica ed EcofisiologiaVegetale, C. N. R., Roma, (Italy), with a Fellowship supportedby North Atlantic Threaty Organization. ³ Present address: Instituto di Botanica, Universita di Ban,Bari, 70126, Italy. (Received April 1, 1971; )     

Properties of auxin binding sites in different subcellular fractions from maize coleoptiles

In-vitro binding of labeled auxins to sedimentable particles was tested in subcellular fractions from homogenates of maize (Zea mays L.) coleoptiles. The material was fractionated by differential centrifugation or on sucrose density gradients. It was confirmed that the major saturable binding activity (site I) for 1-naphthyl[1-¹⁴C]acetic acid is associated with vesicles derived from the endoplasmatic reticulum. A second type of specific auxin binding (site II) could be distinguished by several criteria, e.g. by the low affinity towards phenylacetic acid. The particles carrying site II could be clearly separated from markers of the endoplasmatic reticulum, the plasmalemma, the mitochondria and the nuclei, while their density as well as sedimentation velocity correlated with particle-bound acid phosphatase, indicating a localization at the tonoplast. In contrast to site I, binding at site II was hardly affected by a supernatant factor and by sulfhydryl groups. However, the specificity pattern of site II towards auxins and auxin analogs was very similar to that of site I tested in the presence of supernatant factor. The existence of a third auxin receptor localized in plasma membrane-rich gradient fractions was indicated by a preferential in-vitro binding of 2,4-dichlorophenoxyacetic acid.Abbreviations 1-NAA 1-naphthyl acetic acid - 2-NAA 2-naphthyl acetic acid - IAA 3-indolyl acetic acid - PAA phenyl acetic acid - 2,4-D 2,4-D-dichlorophenoxy acetic acid - D-2,4-DP dichlorophenoxy isopropionic acid - NPA 1-N-naphthyl phthalamic acid - ER endoplasmatic reticulum - SF supernatant factor     

RNA metabolism in rabbit brain: study with neuron-glia and subcellular fractions

—Uridine incorporation into RNA of rabbit brain was studied by using an in vitro system for incubation of brain slices for up to 180 min. Neuron-enriched and glia-enriched fractions were prepared by ficoll density gradient centrifugation, and various subcellular fractions were prepared by sucrose density gradient centrifugation. Although the difference was not as great as in the case of l -leucine incorporation into protein, the neuron-enriched fraction consistently showed a higher specific radioactivity than the glia-enriched fraction. The specific radioactivity of the nuclei increased promptly and remained high at 180 min; the increase in the microsomes was gradual. Comparison of these data suggests that both neuron-enriched and glia-enriched fractions retain high radioactivities in their nuclei at 180 min when a considerable portion of the ribosomal RNA in these fractions is not labeled. The sharp diffusion gradient of nucleotides is discussed in relation to the acid-soluble radioactivity.     

Effects of inhibitors of RNA and protein synthesis on bean hypocotyl hook opening and their implications regarding phytochrome action

Summary Inhibitors of protein and RNA synthesis (cycloheximide, puromycin, chloramphenicol, and actinomycin D), as well as Co⁺⁺, induce opening of the hypocotyl hook of bean seedlings during the early stage of the opening period both in the darkness and red light. The response is transitory, however, complete straightening of a hook can not be achieved in the presence of these agents. These agents abolish the response of hooks to red illumination. They also block the suppression of hook opening caused by IAA and ethylene. The response and sensitivity to GA are not affected by the inhibitors. Inhibitors of DNA synthesis (FUDR and mitomycin C) have no effect on hook opening. It appears that in this growth response RNA and protein synthesis are more immediately involved in ethylene action than they are in the cell elongation process or the action of GA thereon.The results indicate that phytochrome does not induce hook opening simply by activating genes whose products directly promote growth. It is suggested that the regulation of ethylene formation by light and auxins may be exerted by way of influences on tissue levels of phenolic inhibitors of ethylene biosynthesis.     

Glutathione dependent control of protein disulfide-sulfhydryl content by subcellular fractions of hepatic tissue

The disulfide-sulfhydryl (SS/SH) ratios of subcellular fractions of rat hepatic tissue were found to vary diurnally with the ratio lowest in the early morning and highest in the early evening. These changes were found in the nuclear, microsomal and cytosol fractions. The primary reaction is the reversible formation of mixed disulfides of glutathione with proteins. This formation is controlled by the activity of thiol transferase and the level of oxidized glutathione (GSSG) as substrate. Several enzymes including mitochondrial and microsomal oxidases, glutathione reductase and peroxidase and glucose-6-phosphate dehydrogenase were found to control the levels of GSSG. An NADPH-dependent microsomal oxidase system, inhibited by GSSG, was found to produce activated oxygen which served as substrate for flutathione peroxidase. Evidence is presented for the concept that the formation of mixed disulfides of proteins with glutathione is a mechanism for maintenance of a disulfide-sulfhydryl ratio such that the integrity of particulate membranes is maintaine during oxidative and reductive stresses on the hepatic cells.     

Comparative studies of effect of auxin and ethylene on permeability and synthesis of RNA and protein

The effects of ethylene on permeability and RNA and protein synthesis were assayed over a 6 to 26 hr period in tissue sections from avocado (Persea gratissima Gaertn. F., var. Fuerte), both pulp and peel of banana (Musa sapientum L., var. Gros Michel), bean endocarp (Phaseolus vulgaris L., var. Kentucky Wonder Pole beans) and leaves of Rhoeo discolor. Ethylene had no effect on permeability in 4 of the 5 tissues, but sometimes enhanced solute uptake in banana peel; it had either no effect or an inhibitory effect on synthesis of RNA and protein in sections from fruits of avocado and banana. Auxin (α-naphthalene acetic acid) stimulated synthesis of RNA and protein in bean endocarp and Rhoeo leaf sections, whereas ethylene inhibited both basal and auxin-induced synthesis. It is concluded that in these tissues the auxin effect is not an ethylene effect.     

RNA synthesis in germinating red bean seeds

Nucleic acids from ³²P-labelled germinating red bean seeds wereinvestigated by means of MAK column chromatography. 1) In cotyledons,synthesis of D-RNA occurred in the early stages of germination,3 to 24 hr after the onset of imbibition. ³²P was also incorporatedinto rRNA continuously at rather a moderate rate. DNA-RNA hybridizationexperiments revealed that the proportion of heterogeneous RNA(D-RNA) to rRNA decreased gradually. Nucleotide analysis suggestedthat tRNA was synthesized de novo, and that its CCA-end exchangewas remarkable at early stages of germination. 2) In embryos,however, the incorporation of ³²P into rRNA was very much greaterthan into D-RNA, and the exchange reaction at CCA-end of tRNAwas not detected. The role of D-RNA, found in cotyledons inthe initial stages of germination, was discussed. ¹Present address: Research Institute for Biochemical Regulation,Faculty of Agriculture, Nagoya University, Chikusa-ku, Nagoya,Japan. (Received May 10, 1972; )     

Mechanism of the ATP-dependent phosphatidylserine synthesis in liver subcellular fractions

It has been shown that the ATP-dependent incorporation of [14C]serine into phosphatidylserine in rat liver mitochondrial and microsomal fractions is prevented by EGTA. On the other hand, at low (microM) Ca2+ concentrations, serine incorporation is strongly stimulated by ATP and Mg2+. This stimulatory effect is reduced by calcium ionophore A23187. It is therefore suggested that the ATP-dependent process is that of serine base-exchange reaction, stimulated by endogenous Ca2+ accumulated inside the microsomal vesicles by Ca2+,Mg2+-ATPase. The mitochondrial activity can be accounted for by contamination by the endoplasmic reticulum.     

Effect of undernutrition on DNA and RNA synthesis in subcellular fractions from different regions of the developing rat brain

The effect of undernutrition on the incorporation of [methyl-³H]thymidine into DNA and of 5-[³H]uridine into RNA of cerebral hemispheres, cerebellum, and brain stem was studied in vivo and in vitro in rats. The labeling of DNA from nuclei and mitochondria and of RNA from nuclei, mitochondria, microsomes, and soluble fractions, was also measured in vitro. The results demonstrate that nucleic acid synthesis is impaired and delayed during undernutrition. Specific effects were observed for the different brain regions and subcellular fractions: at 10 days nuclear and mitochondrial DNA and RNA synthesis was impaired, whereas at 30 days only the mitochondrial nucleic acid synthesis was affected.The delay of DNA and RNA labeling, caused by undernutrition, was most evident in the cerebellum, probably due to its intense cell proliferation during postnatal development. The specific sensitivity of mitochondria as compared to other subcellular fractions, may be due to the intense biogenesis and/or turnover of nucleic acids in brain mitochondria not only during postnatal development, but also in the adult animal.     

Triacylglycerol synthesis by subcellular fractions of maturing safflower seeds

A subcellular particulate fraction (10³ g, precipitate) prepared from maturing safflower seeds catalysed triacylglycerol synthesis from oleoyl-CoA.     

Cooperation of various subcellular fractions in protein degradation in vitro

In order to test the possibilities in protein degradation between cell organelles comparatively, [3H]- and [14C]-leucine short-time labelled subcellular fractions from rat liver were incubated with each other at pH 6.9. All fractions tested were able to degrade short-lived proteins from foreign fractions, whereby the lysosomal supernatant fraction showed the highest proteolytic activity, which declines in the sequence: lysosomes--nuclei--mitochondria--cytosol--microsomes. Short-lived cytosolic proteins were especially suited as substrate for neutral proteases from all other fractions, but also microsomal, mitochondrial and nuclear proteins were well degraded by foreign fractions in comparison with the substrate autoproteolysis. Therefore in vivo manyfold cooperations between several organelles in protein catabolism seem to be possible.     

Localization of cap-binding protein in subcellular fractions of HeLa cells

The 26,000-M(r) cap-binding protein was analyzed by a cross-linking assay in cell fractions from uninfected and poliovirus-infected HeLa cells. Cap-binding protein was found in the postribosomal supernatant (S-200) and in the ribosomal salt wash. The cap-binding protein in the S-200 had a sedimentation coefficient of 5 to 7S and lacked the ability to restore translation in extracts of poliovirus-infected cells.     

Auxin binding to subcellular fractions from Cucurbita hypocotyls: In vitro evidence for an auxin transport carrier

An auxin binding sive, with characteristics different from the previously described auxin binding sites I and II in maize coleoptiles, is reported in homogenates of zucchini (Cucurbita pepo L. cv. Black Beauty) hypocotyls. Evidence from differential centrifugation and sucrose and metrizamide density gradients indicates that the site is localized on the plasma membrane. The site has a K_D of 1–2×10^–6 M for indole acetic acid and has a pH optimum of 5.0. Binding specificity measured with several auxins, weak auxins, and anti-auxins generally parallels the activities of the same compounds as inhibitors of auxin transport. 1-N-naphthylphthalamic acid and 2,3,5-triiodobenzoic acid (2,3,5-TIBA), both auxin transport inhibitors in vivo, increase specific auxin binding to this site. 3,4,5-TIBA, which can partially reverse 2,3,5-TIBA's transport inhibition when the two substances are added together in vivo, partially reverses 2,3,5-TIBA's increase in specific auxin binding to the plasma membrane site when added with 2,3,5-TIBA in vitro. Preliminary investigations indicate that a similar plasma membrane site exists in maize (Zea mays L.) coleoptiles. It is suggested that different conformations of this site may function during active auxin transport.Abbreviations IAA indole-3-acetic acid - NPA 1-N-naphthylphthalamie acid - 2,3,5-TIBA 2,3,5-triiodobenzoic acid - 3,4,5-TIBA 3,4,5-triiodobenzoic acid - 1-NAA 1-naphthaleneacetic acid - 2-NAA 2-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - DTE dithioerythritol - MOPS N-morpholino-3-propansulfonic acid - CCO cytochrome c oxidase - CCR NADH: cytochrome c reductase - glu I glucan synthetase I - ER endoplasmic reticulum     

Isolation and characterization of subcellular protein fractions from mouse heart

In this study, we report different protocols used to obtain highly enriched and well-characterized protein fractions that could be used to determine the subcellular localization of proteins. Different protein fractions (total, cytosolic, total membrane, sarcolemmal, and nuclear) were isolated from mouse heart by a combination of either polytron homogenization or liquid nitrogen pulverization followed by density gradient centrifugation. Triton X-100 was used in specific fractions to help in the solubilization of proteins obtained with fractionation protocols. Following the isolation, enzymatic assays and Western blot analysis were used to evaluate the enrichment and/or cross-contamination of these protein fractions. Glucose-6-phosphate dehydrogenase, Na+/K+-ATPase, mitochondrial Ca2+-ATPase, sarco-endoplasmic reticulum Ca2+-ATPase, glucose-regulated protein, and nucleoporin P62 were used as specific markers for the cytosol, sarcolemma, mitochondria, sarco-endoplasmic reticulum, endoplasmic reticulum, and nucleus, respectively. The results show that we obtained enriched protein fractions with little to no cross-contamination. These purification protocols allow us to obtain different protein fractions that could be used in a wide variety of studies.     

Water and salt stresses, kinetin and protein synthesis in tobacco leaves

The capacity of tobacco (Nicotiana rustica) leaf discs to incorporate l-leucine ¹⁴C into proteins was measured. Leaf discs were obtained from plants which experienced soil water depletion, or which were exposed to a saline or osmotic stress in the root medium. The stresses were brief of relatively short duration and water potential did not decrease below 4 bars in the root media. Leaf discs were sampled 2 hours after stress removal, achieved by reirrigation, or replacement of saline and osmotic solutions with normal nutrient solution. Plants were always turgid when leaves were sampled.