Salt-tolerance in plants. I. Ions, compatible organic solutes and the stability of plant ribosomes

Abstract Polysomes and ribosomes recovered from a number of plant species were tested for stability when incubated at 25°C in salt solutions in the absence of ATP and initiation factors. Stability was assessed by sucrose density gradient analysis. The stability was inversely proportional to salt concentrations above 125 mol m⁻³ KCl. Polysomes were less stable in the presence of Na⁺ than K⁺ salts, and were much less stable in Cl⁻ than in acetate salts. Polysomes from Triticum aestivum. Hordeum vulgare, Capsicum annuum, Helianthus annuus. Pisum sativum, Atriplex nummularia, Beta vulgaris, Cladophora sp., Enteromorpha sp. and Corallina cuvieri were similarly sensitive to KCl. Polysomes from Ulva lactuca were more sensitive than the other species. Cytoplasmic and plastid polysomes from T. aestivum were similarly unstable in 500 mol m⁻³ KCl. Unprogrammed ribosomal subunit couples from T. aestivum, B. vulgaris and U. lactuca showed Mg²⁺-dependent conformational instability and dissociation in KCl. Slight differences in ribosomal stability were observed between species, but these were unrelated to the salt tolerances of the plants. The ‘compatible’ organic solutes, glycinebetaine and proline, failed to reduce ion-induced instability. Ribosome yield and polysome profiles were similar in leaves of B. vulgaris containing significantly different levels of both Na⁺ and Cl⁻ after growth in media containing 50 or 200 mol m⁻³ NaCl. The results are consistent with the hypothesis that plants maintain a cytoplasmic solute environment that is compatible with ribosomal stability.     

Streptomycin retards the phenotypic maturation of chick myogenic cells

Summary As part of an effort to optimize conditions required for the complete maturation of muscle cells in vitro, we have investigated the effects of the antibiotics penicillin, streptomycin, and Fungizone (amphotericin B) on the development of cultured chick embryo skeletal muscle. It is shown that even low dosages of streptomycin, but not penicillin or Fungizone, retard protein synthesis and accumulation in these cultures. Myosin accumulation was also reduced and the appearance of striations in fused cells was delayed in myotubes formed in medium containing streptomycin. Additional data suggest that this overall retardation of myogenesis is due to the influence of streptomycin on maturing myotubes rather than early proliferation and cell fusion. These results are discussed with regard to recent efforts to promote the full maturation of muscle cells grown in culture. This research was supported by National Institutes of Health Grant NS 155882 and a Task Force on Drug Development Research Contract from The Muscular Dystrophy Association.     

Radiation inactivation of membrane proteins: Molecular weight estimates in situ and after Triton X-100 solubilization

Target size analysis by radiation inactivation is widely used for molecular weight determination of membrane enzymes and receptors in situ without the need for prior solubilization or purification. However, since most molecular weight data available in the literature on membrane proteins involve the use of detergents for solubilization, the target sizes of membrane proteins in situ and after solubilization by detergent treatment have been compared. Using data from the literature and personal results, three different types of behavior of membrane proteins in presence of detergents were found: (i) uncoupling of subunits (electric eel acetylcholinesterase, placental steroid sulfatase, and human nonspecific β-glucosidase); (ii) coupling of protein molecules (mouse liver neuraminidase, and rat liver insulin receptor regulatory component); and (iii) no major change in quaternary structure (rat liver insulin receptor, kidney γ-glutamyltransferase, asialoglycoprotein receptor, insulin degrading enzyme, and human leucocyte neuraminidase). For all these proteins, there is a statistically significant increase in target size of about 24% over the value obtained in situ without detergent. A relatively large body of literature data involving a variety of membrane proteins, membrane types, and irradiation conditions (electron accelerators or ⁶⁰Co sources, and proteins irradiated in lyophilized form or frozen solution) was examined, and it was concluded that target sizes of membrane proteins, irradiated in the presence of Triton X-100, should be diminished by a factor of about 24% to obtain the molecular weight value.     

Changes in intracellular pH and inorganic phosphate concentration during and after muscle contraction as studied by time-resolved 31P-NMR. Alkalinization by contraction

The morula and the mesenchyme blastula nuclei contained approx. 30 nuclear proteins which were preferentially released by limited digestion with DNase I, but no proteins were released from sperm nuclei. While most of the proteins released by DNase I digestion were common to the two embryonic stages, 2 and 6 proteins were specific or enriched in morulae and mesenchyme blastulae, respectively.     

Coordinated synthesis of the nuclear protein cyclin and DNA in serum-stimulated quiescent 3T3 cells

Quantitative two-dimensional gel electrophoretic analysis (IEF) of the nuclear polypeptide cyclin together with autoradiographic studies have revealed a coordinate synthesis of cyclin and DNA after serum stimulation of quiescent 3T3 cells. These results strengthen the notion that cyclin may be a central component of the pathway(s) that regulate cell proliferation.     

A spin-labeled derivative of Procion brilliant blue MX-R. Synthesis and interaction with pig heart nucleoside diphosphate kinase

The ¹H-NMR spectrum of cucumber basic blue protein (CBP) has been recorded. Examination of the spectrum of the reduced protein suggests that one or more sidechains exist in conformations which interconvert slowly at ambient temperatures. His 39, His 84 and Met 89 are identified as copper ligands by redox titration and by amino acid sequence homology with plastocyanin and azurin. The importance of a Phe sidechain close to the Met ligand in the potential blue copper site is confirmed. Broadening of His ligand resonances at elevated temperatures reveals an exchange process at the reduced copper centre.     

Two cDNA clones coding for the legumin protein of Pisum sativum L. contain sequence repeats

The sequence of two cDNA clones coding for the whole of the -subunit and most of the -subunit of legumin are presented together with a considerable amount of protein sequence data to confirm the predicted amino acid sequence. A unique feature shown by these cDNAs is the presence of three 56 base pair tandem repeats in the region encoding the C terminal of the polypeptide. The tandem repeats are also exhibited in the predicted polypeptide sequence as three 18 amino acid repeats which contain extremely high proportions of polar, mainly acidic, residues. The new sequences are compared to the previously published sequence of some shorter legumin cDNAs (Nature 295: 76–79). In the region where the sequences overlap, the previous cDNAs differ from the new ones by only a few base substitutions but most of the repeated region is not present though the sequences on either side are. The possibility that the absence of the repeats may reflect the difference between two types of legumin gene, rather than an artefact of the cloning of the cDNAs, is discussed.     

Changes in intracellular proteolysis in Escherichia coli during prolonged growth with a low concentration of dihydrostreptomycin

Abstract Escherichia coli was cultured with a low concentration of dihydrostreptomycin (2.5 μg/ml.). Growth was similar to untreated controls for 10 h after which a slow decline in growth rate occurred; growth ceased after 20 h. Intracellular catabolism of pulse-labelled protein synthesised at various points during the antibiotic treatment increased during the first 10 h, but during the second 10 h proteolysis progressively declined to almost control values. The production of an aberrant proteolytic system is one possible explanation.     

Contribution to the structural characterization of eucaryotic PSI reaction centre—I. Critical analysis of the polypeptidic composition of different P700 enriched fractions

In thylakoid membranes, several peptides of high MW are present which may interfere with the study of CP1's components. Modifying Cleveland's technique [7] for limited proteolysis, we have characterized the polypeptides found in the 60 kD region. Some may result from incomplete washing of the CF1 while others come from the CP1; indeed, this chlorophyll protein complex, which has a higher MW (above 100 kD), very often undergoes a dissociation into smaller components of about 60 KD MW.Analysis of the protein content of different preparations commonly used to obtain PSI reaction centre enriched fractions has been performed. The and subunits of CF1 are among the main contaminants of most of these preparations. A further purification step is described which can be applied to all these preparations, but numerous peptides are still present in the active fractions. It is most unlikely that all these polypeptides are required for the primary photochemical event, and this emphasizes the necessity to find a new simple method to purify PSI reaction centres.     

The preparation of a sarcolemmal fraction from evacuated muscle slices

A novel procedure is described for preparing a plasma membrane fraction from skeletal muscle (i.e., sarcolemma). The procedure entails evacuating the myoplasm from muscle slices as a preliminary step to homogenization and fractionation. The evacuated muscle slices are composed of a stroma-containing sarcolemma, which is then homogenized and fractionated, utilizing a sequence of differential and discontinuous sucrose density gradient centrifugations. On the basis of electron microscopy, selective enzyme markers and α-bungarotoxin binding in innervated and denervated muscles, the fraction most enriched with sarcolemma is recovered from the 0.5/0.7 M interface of a discontinuous sucrose gradient.