cDNA cloning of an mRNA encoding a sulfur-rich 10 kDa prolamin polypeptide in rice seeds

Using a rice maturing seed pUC9 expression library, we isolated a cDNA clone corresponding to 10 kDa sulfurrich prolamin by immunoscreening. A longer cDNA clone was obtained from a gtll library by plaque hybridization using this ³²P-labeled cDNA as a probe. A polypeptide sequence composed of 134 amino acids was deduced from the nucleotide sequence. A 24 amino acid signal peptide was assigned by computer calculation for the membrane spanning region and Edman sequencing of the purified mature polypeptide. Remarkably, 20% of methionine and 10% of cysteine were found in the mature polypeptide as well as high contents of glutamine, and hydrophobic amino acids. Part of the amino acid sequence was homologous with a conserved cysteine-rich region found in other plant prolamins. Two repeats of amino acid sequence were found in the polypeptide.     

Kinetic analysis of the binding of guanine nucleotide to bovine brain smg p25A

Bovine brain smg p25A, a guanine nucleotide-binding protein with a Mr of about 25,000, bound specifically GTP, guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) and GDP. The initial velocities of the binding of GTP gamma S to GDP-bound smg p25A and the dissociation of GDP from this protein increased by decreasing Mg2+ concentrations or increasing NaCl concentrations. The initial velocity of the binding of GTP gamma S to GDP-free smg p25A was not affected by changing Mg2+ concentrations. These results indicate that the dissociation of GDP from smg p25A limits the binding of GTP to this protein, and suggest that there is a protein stimulating the dissociation of GDP from smg p25A and thereby stimulating the binding of GTP to this protein in mammalian tissues. In fact, the protein stimulating the dissociation of GDP, but not of GTP gamma S, from smg p25A was detected in bovine brain cytosol.     

Respiration-Dependent Proton and Sodium Pumps in a Psychrophilic Bacterium, Vibrio sp. Strain ABE-1

Respiration-dependent proton and sodium flows in a psychrophilicbacterium, Vibrio sp. strain ABE-1, were examined. At alkalinepH, this bacterium grew without being affected by a proton conductor,carbonylcyanide m-chlorophenylhydrazone (CCCP). O₂-pulse intoanaerobic cell suspensions prepared with Na^$-free buffers inducedtransient alkalization in the presence of CCCP and acidificationat pH 8.5 and 6.5, respectively. However, using cells preparedwith Na^$-containing buffer, the transient pH changes of thecell suspension could be simultanously detected at both pHs.Several inhibitory experiments suggested that the acidificationand alkalization should be attributed to a respiration-dependentprimary H^$ pump and Na^$ pump, respectively, and that the latterwas similar to that first reported in a marine bacterium, Vibrioalginolyticus. This Na^$ pump may have supported the CCCP-resistantgrowth at alkaline pH. The H^$ and Na^$ pumps operated very actively at low temperatures,such as 5?C, and should markedly help sustain bacterial growthat low temperatures. (Received May 30, 1987; Accepted November 13, 1987)     

Colchicine-binding activity of carrot tubulin at different culture age

Tubulin contents in the extract from cultured carrot cells at different growth phases were investigated by measuring colchicine-binding activity. The addition of vinblastine and dithiothreitol to the reaction mixture appreciably improved the stability of both free and colchicine-bound tubulins. Colchicine-binding activity in the cell extract obtained from stationary phase was more labile than that from log phase though the extract showed higher affinity to colchicine. After purification, however, tubulin from the cells at different growth phases showed the same affinity and its colchicine-binding activity was much more stable than in crude extract. The colchicine-binding activity in the crude extract was corrected for the decay during measurement and apparent difference in the affinity so that the activity in the cells containing different kind and amount of interefering substances could be compared. The corrected amount of colchicine that binds to the 100,000×g extract was 46 pmol/10⁵ cells at log phase. It decreased with the progression of culture age from linear to stationary phase. Combining the data with the morphological observation, it was suggested that the log phase cells contained larger free tubulin pool than the linear or stationary phase cells.     

Thioridazine enhances lysosomal accumulation of epidermal growth factor and toxicity of conjugates of epidermal growth factor with Pseudomonas exotoxin

Thioridazine, a phenothiazine calmodulin inhibitor, aggravated the cytotoxic effect of a conjugate (EGF-PE) of epidermal growth factor (EGF) coupled with Pseudomonas exotoxin against cultured HeLa cells. Other phenothiazine calmodulin inhibitors, trifluoperazine and chlorpromazine, also intensified the cytotoxic effect of EGF-PE, whereas N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7) had no such effect. By using iodinated epidermal growth factor ( [125I]EGF), the effect of thioridazine on intracellular transport of EGF was examined. The release of radioactivity associated with [125I]EGF into medium was slow in the presence of thioridazine. The Percoll gradient centrifugation pattern showed that thioridazine delayed both the appearance of [125I]EGF in lysosomes and the disappearance of [125I]EGF from the lysosomes. The pH value in lysosomes was 5.28 in thioridazine-treated HeLa cells, while that in untreated cells was 5.15. Thioridazine was found to inhibit lysosomal enzyme activities of cathepsin B and acid phosphatase, but not beta-hexosaminidase when cell extracts were treated with the drug. Electron microscopy showed an increased number of electron-dense bodies, possibly autophagosomes/lysosomes in HeLa cells grown for 48 h with 3 micrograms/ml thioridazine. The potentiating action of EGF-PE by thioridazine is discussed in relation to the altered lysosomal function in treated cells.     

Reversible Inactivation of Ferredoxin-Nitrate Reductase from the Cyanobacterium Plectonema boryanum. The Role of Superoxide Anion and Cyanide

Assimilatory nitrate reductase (NR) from the cyanobacteriumPlectonema boryanum exhibits both ferredoxin (Fd)- and methylviologen (MV)-linked activities. Native (Fd-linked) activitywas reversibly inactivated by the exposure of a dithionite solutionof the enzyme to air, whereas MV-linked activity remained unaffected.Cyanate and azide, competitive inhibitors of NR, suppressedthe dithionite-induced inactivation, and the inactivation wasspecifically prevented by superoxide dismutase (SOD). Xanthineoxidase reaction inactivated Fd-linked activity but not MV-linkedactivity, and the native activity was protected by SOD and catalase.Photoactivated FAD/ EDTA irreversibly inactivated both Fd- andMV-linked activities, and the former activity was protectedby ascorbic acid. Dithionite-inactivated enzyme was restoredto its Fd-linked activity when incubated with cyanate or azidein the presence of dithionite or reduced Fd. Cyanide inactivatedboth Fd- and MV-linked activities when the enzyme was incubatedunder reducing conditions. The cyanide-inactivated enzyme wasalso reactivated by cyanate and azide under reducing conditions.It is suggested that superoxide and cyanide act as ligands tothe molybdenum center in the reversible inactivation of thenative activity of cyanobacterial Fd-NR. (Received March 3, 1986; Accepted May 30, 1986)     

Changes in Lipoxygenase Components of Rice Seedlings during Germination

Changes in lipoxygenase (LOX) activity were followed duringthe germination of rice seeds. The enzyme activity of 3-day-oldseedlings was 20 times higher than that of ungerminated seeds.Sixty per cent of the increased activity was found in shoots.The increase in LOX activity was mainly due to an increase inlipoxygenase-2 (LOX-2), a minor component in ungerminated seeds;this increase was inhibited by cycloheximide. LOX-2 was isolatedfrom the 3-day-old seedlings and compared for its enzymologicalproperties with rice lipoxygenase-3 (LOX-3), a major componentin ungerminated seeds. Both LOX-2 and LOX-3 were stable at pH5 to 8, but LOX-2 was more heatstable than LOX-3. Apparent K_mvalues of LOX-2 and LOX-3 for linoleic acid were 170 and 59µM, and those for linolenic acid were 5,300 and 88 µM,respectively. Both LOXs were inhibited by some metal ions andantioxidants. (Received February 5, 1986; Accepted May 9, 1986)     

Saponins can cause the agglutination of phospholipid vesicles

The interaction of saponins with phospholipid vesicles was investigated by means of liposomal agglutination or a precipitation assay. Ginsenoside-Rc, which has an α-l-arabinofuranose residue at the non-reducing terminus, exhibited remarkable agglutinability toward egg yolk phosphatidylcholine vesicles, while other saponins lacking this characteristic sugar residue showed less or no agglutinability. The molar ratio of ginsenoside-Rc to egg phosphatidylcholine in the aggregates was estimated to be 0.4–0.5 by a precipitation assay using ¹⁴C-labeled egg phosphatidylcholine vesicles. The agglutination was inhibited by p-nitrophenyl α-l-arabinofuranoside but not by p-nitrophenyl β-d-glucopyranoside or arabinogalactan. The results indicated that the α-l-arabinofuranose residue in ginsenoside-Rc should be important for the expression of the agglutinability. The agglutinability of ginsenoside-Rc toward lipid vesicles depended on both the polar head groups and fatty acyl chains of phospholipids. Egg yolk phosphatidylcholine vesicles were strongly agglutinated by ginsenoside-Rc, although sphingomyelin, phosphatidylethanolamine, phosphatidic acid and phosphatidylserine were less agglutinated. The agglutinability of ginsenoside-Rc was effective for phosphatidylcholines with short or unsaturated fatty acyl chains. The results suggested that the interaction of ginsenoside-Rc with phospholipid membranes should be affected not only by the chemical structure of the phospholipid but also by the membrane fluidity.