3',5'-Cyclic Adenosine Monophosphate- and Ca2+-Calmodulin-Dependent Endogenous Protein Phosphorylation Activity in Membranes of the Bovine Chromaffin Secretory Vesicles: Identification of Two Phosphorylated Components as Tyrosine Hydroxylase and Protein Kinase Regulatory Subunit Type II

Abstract: Membranes of the secretory vesicles from bovine adrenal medulla were investigated for the presence of the endogenous protein phosphorylation activity. Seven phosphoprotein bands in the molecular weight range of 250,000 to 30,000 were observed by means of the sodium dodecyl sulphate electrophoresis and autoradiography. On the basis of the criteria of molecular weight, selective stimulation of the phosphorylation by cyclic AMP (as compared with cyclic GMP) and immunoprecipitation by specific antibodies, band 5 (molecular weight 60,300) was found to represent the phosphorylated form of the secretory vesicle-bound tyrosine hydroxylase. The electrophoretic mobility, the stimulatory and inhibitory effects of cyclic AMP in presence of Mg²⁺ and Zn,²⁺ respectively, and immunoreactivity toward antibodies showed band 6 to contain two forms of the regulatory subunits of the type II cyclic AMP-dependent protein kinase, distinguishable by their molecular weights (56,000 and 52,000, respectively). Phosphorylation of band 7 (molecular weight 29,800) was stimulated about 2 to 3 times by Ca²⁺ and calmodulin in the concentration range of both agents believed to occur in the secretory tissues under physiological conditions.     

Study of Glial Fibrillary Acidic Protein in a Human Glioma Cell Line Grown in Culture and as a Solid Tumor

Abstract: A continuous human glioma cell line grown in culture and as a solid tumor was analyzed for glial fibrillary acidic (GFA) protein. This material provided a rich source for GFA protein that could also be manipulated and controlled. Immunoperoxidase staining at the light and electron microscopic levels revealed that the cell culture and tumor specimens were strongly positive for GFA protein. When aqueous soluble fractions of the cell culture and tumor were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, electroblotted onto nitrocellulose and stained immunochemically, they contained exclusively low molecular weight (41–43 K-dalton) GFA peptides. SDS (0.15%)-soluble fractions contained either low molecular weight only (culture) or a mixture of peptides ranging from 41 to 49K daltons. SDS (1%) extracts of either cell culture or tumor contained only 49K dalton GFA protein. Two-dimensional gel separation revealed that the GFA protein extracted from either the culture or tumor with 1% SDS resolved to two or three spots at pH 5.8. Low molecular weight GFA peptides (<49K daltons) in aqueous and 0.15% SDS-soluble extracts became increasingly more acidic with decreasing molecular weight. The extremely rapid degradation seen suggests that this cell line may be a valuable system for further study of intermediate filament protein turnover.     

A stimulatory subunit in the polypeptide elongation factor-1 of the chick brain

Abstract: The higher-molecular-weight elongation factor-1 (EF-1_H) of the chick brain was observed to contain three subunits (denominated α, β, and γ), contrary to a previous report that the brain EF-1_H consisted of aggregates of low-molecular-weight elongation factor- 1 (EF-1_L). Crude EF-1_H, obtained from 20-day embryonic brain, was treated with 0.4 M ammonium chloride and 0.1 mM GTP, and EF-1_βγ, was obtained using a DEAE-Sephadex column equilibrated in 0.025 mM GTP. Both EF-1_β, and EF-1_γ, were isolated by means of a DE-52 column equilibrated in 6 M urea and were found to have molecular weights of 2.8 and 4.8 × 10⁴, respectively. EF-1_β and EF-1_γ were also obtained from young rat and calf brains by the same procedures. The molecular weight of the isolated EF-1_α was 5 × 10⁴. It was found that EF-1_β stimulated the two EF-1_α-dependent reactions, i.e., phenylalanyl-tRNA binding (reaction 1) and polyphenylalanine synthesis (reaction 2), and also stimulated the nucleotide exchange reaction in the EF- 1_α-guanine nucleotide binary complex (reaction 3). The degrees of stimulation of reactions 1, 2, and 3 by the addition of EF-1_β were 2 to 3 times, about 18 times, and 2 to 3 times as much as with EF-1_α alone, respectively. The amino acid compositions of EF-1_α -1_β, and -1_γ and EF-2 were very similar to those of other eukaryotic tissues. Thus the constituents and properties of EFs of the brain were found to be basically similar to those of other tissues of eukaryotes, although EF-1_β, and EF-1, had not been reported in the brain. A possible physiological significance of EF-1_β during brain development is also discussed.     

Identification of specific proteins and glycoproteins associated with membrane fractions isolated from Zea mays L. coleoptiles

The aim of this work was to identify proteins specific for plant cell membranes which could then be used as unique markers. A crude membrane fraction was isolated from corn coleoptiles and separated on non-linear sucrose density gradients. Separation of endoplasmic reticulum (NADH-cytochrome c reductase), mitochondria (cytochrome c oxidase), golgi (inosine diphosphatase), and plasma membranes (N-1-naphthylphthalamic acid-binding) was achieved. The membrane proteins from the gradient fractions were separated using sodium dodecyl sulphate-poly-acrylamide gel electrophoresis and the gels stained with coomassie blue or with concanavalin A/peroxidase to detect glycoproteins. Proteins specific for the various membranes were identified. Five proteins including two glycoproteins were plasma membrane markers. Protoplasts were isolated and iodinated using lactoperoxidase/glucose oxidase covalently attached to beads. Eleven iodinated proteins were found and three of these corresponded to proteins specifically associated with plasma membranes in the density gradients. Two methods for detecting Ca²⁺-binding proteins following sodium dodecylsulphate polyacrylamide gel electrophoresis were employed. The majority of such proteins were found in the endoplasmatic reticulum and one was specific for plasma membranes. In vitro and in vivo phosphorylation of membrane proteins was examined and the majority of proteins phosphorylated were glycoproteins. Two of the phosphorylated proteins (M_r=110,000 and 20,000) were also iodinated on protoplasts and may be part of the plasma membrane ATPases.Abbreviations ER endoplasmic reticulum - IDP inosine diphosphate - NPA N-1-naphthylphthalamic acid     

Plant spacing effect on yield of cotton irrigated with saline waters

Summary The response of the cotton plant (Gossypium hirsutum L.) to 9.0 and 12.5 cm intra- and 75.0 and 96.5 cm inter-row spacing was studied under irrigation with saline water (5.5 dS/m, SAR 18). In general, the dry weight per plant matter, the leaf area, number of bolls and flowers, and yield per plant were all affected significantly by intra-row spacing but, not by inter-row spacing. However, on a unit area basis, they were affected only by the inter-row spacing. This indicates that competition between plants exists when intra-row spacing is reduced, whereas no significant competition occurs due to a decrease in the inter-row spacing. Although the effect of intra-row spacing on yield for a unit area was found to be not significant, the effect of inter-row spacing was significant. Yield in plots with the conventional spacing (96.5 cm between rows and 12.5 cm between plants in the row) was 4863 kg/ha, whereas the yield in plots with 75 cm between rows was about 23% higher (5974 kg/ha). The lint percentage and the plant height were not affected significantly by either intra- or inter-row spacings.Contribution from the Institute of Soils and Water, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel. No.676-E, 1983 series.     

Spectroscopic characterization of extracts from humic and fulvic fractions: IR and1H NMR spectra

Summary Infrared and proton resonance spectra have been used to characterize fraction extracted sequentially from humic and fulvic acids by diethylether, acetone, dioxane, tetrahydrofuran, pyridine and dimethylformamide. The results showed that the same solvents extracted structurally similar components from both humic and fulvic acids. On the other hand, the spectra showed solvent-dependent differences, some being characteristic for a preponderance of aliphatic structures, others for aromatic structures.     

The exchange hypothesis for the formation of chromatid aberrations: an experimental test using bleomycin

The association between bleomycin-induced chromatid aberrations and BUdR-label exchange between sister chromatids was investigated in order to evaluate Revell's exchange hypothesis for the formation of chromatid aberrations. The results of this study indicate that a larger than expected proportion of chromatid breaks can be accounted for by the exchange hypothesis though not all breaks are the result of incomplete exchange.     

Correlative changes in sucrose uptake, ATPase activity and membrane fluidity in carnation petals during senescence

Apparent sucrose uptake. ATPase activity and membrane fluidity changes were studied during the development and senescence of carnation flowers ( Dianthus caryophyllus L., cv. Cerise Royallette). Typical changes associated with senescence of a cut flower, such as respiration, ethylene production and fresh weight, were measured. Concomitant with a rise in respiration and ethylene production and a decline in fresh weight, a sharp decrease in apparent sucrose uptake was observed. Sucrose uptake was pH dependent (pH optimum, 5.5) and influenced by membrane integrity. Apparently, the activity of ATPase is related to sucrose uptake, because similar changes occurred during flower development. In addition, the activity of ATPase was well correlated with membrane fluidity.
It is suggested that sucrose uptake is controlled by ATPase activity, which in turn is modulated by membrane lipid fluidity. The decline in membrane fluidity associated with senescence leads to a corresponding reduction in ATPase activity and sucrose uptake. Further evidence supporting this view comes from experiments in which senescence was enhanced by 1-aminocyclopropane-l-carboxylic acid. It shortened the time to decline in fresh weight, rise in respiration and ethylene production. In parallel, reduction in membrane fluidity, ATPase activity and sucrose uptake were observed.     

Role of aminocyclopropane-l-carboxylic acid in ethylene release by distal tissues following localized application of ethephon in Cucurbita pepo

The application of ethephon to a single leaf of Cucurbita pepo L. cv. Trailing Marrow plants caused a huge increase in ethylene production from the treated organ and an increased rate of ethylene production from other parts of the plant. These increases were particularly marked in the shoot apex and expanding leaf. Prior treatment with aminoethoxyvinylglycine (AVG), an ethylene biosynthesis inhibitor, blocked the increased production of ethylene at sites distant from the point of ethephon application. This strongly suggests that the increased ethylene production at these distant sites is due to ethylene biosynthesis and not a result of the translocation of ethylene released by the breakdown of ethephon at the site of application. Assays of 1-aminocyclopropane-l-carboxylic acid (ACC), an ethylene precursor, showed that it increased substantially after ethephon application but was at undetectable levels in the presence of AVG. It is proposed that the application of ethephon stimulates ethylene biosynthesis, but that transport through the plants is effected by ACC which is then converted to ethylene at the shoot apex and leaves.     

Some variations in the composition of suberin from the cork layers of higher plants

The monomeric composition of the suberins from 16 species of higher plants was determined by chromatographic methods following depolymerization of the isolated extractive-free cork layers with sodium methoxide-methanol. 1-Alkanols (mainly C₁₈C₂₈), alkanoic (mainly C₁₆C₃₀), α,ω-alkanedioic (mainly C₁₆C24), ω-hydroxyalkanoic (mainly C₁₆C₂₁), dihydroxyhexadecanoic (mainly 10,16-dihydroxy- and 16-dihydroxyhexadecanoic), monohydroxyepoxyalkanoic (9,10-epoxy-18-hydroxyoctadecanoic), trihydroxyalkanoic (9,10, 18-trihydroxyoctadecanoic), epoxyalkanedioic (9,10-epoxyoctadecane-1,18-dioic) and dihydroxyalkanedioic (9,10-dihydroxyoctadecane-1 18-dioic) acids were detected in all species. The suberins differed from one another mainly in the relative proportions of these monomer classes and in the homologue content of their 1-alkanol, alkanoic, α,ω-alkanedioic and ω-hydroxyalkanoic acid fractions. C₁₈ epoxy and vic-diol monomers were major components (32–59%) of half of the suberins examined (Quercus robur, Q. ilex, Q. suber, Fagus sylvatica, Castanea sativa, Betula pendula, Acer griseum, Fraxinus excelsior) where as ω-hydroxyalkanoic and α,ω-alkanedioic acids predominated in those that contained smaller quantities of such polar C₁₈ monomers (Acer pseudoplatanus, Ribes nigrum, Euonymus alatus, Populus tremula, Solanum tuberosum, Sambucus nigra, Laburnum anagyroides, Cupressus leylandii). All species, however, contained substantial amounts (14–55 %) of ω-hydroxyalkanoic acids, the most common homologues being 18:1 (9) and 22: 0. The dominant α,ω-alkanedioic acid homologues were 16: 0 and 18: 1 (9) whereas 22: 0, 24: 0 and 26: 0, and 20: 0, 22: 0 and 24: 0 were usually the principal homologues in the 1-alkanol and alkanoic acid fractions, respectively. The most diagnostic feature of the suberins examined was the presence of monomers greater than C₁₈ in chain length; most of the C₁₆ and C₁₈ monomers identified in the suberins also occur in plant cutins emphasizing the close chemical similarity between the two anatomical groups of lipid biopolymer.