Effect of growth hormone on protein phosphorylation in isolated rat hepatocytes

Hepatocytes from male rats were incubated with [32P]Pi for 40 min at 37 degrees C, thereby equilibrating the cellular ATP pool with 32P. Subsequent exposure to bovine growth hormone for 10 additional min did not change the specific activity of cellular [gamma-32P]ATP. Two-dimensional gel electrophoresis or chromatofocusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to fractionate phosphoproteins solubilized from control or hormone-stimulated cells. Stimulation of hepatocytes with 5 nM growth hormone for 10 min at 37 degrees C affected the phosphorylation of a number of proteins including an Mr 46,000 species of pI 4.7 whose phosphorylation was augmented (2.65 +/- 0.50)-fold. A significant fraction of the maximal effect of growth hormone on phosphorylation of the Mr 46,000 species was elicited by 1-5% receptor occupancy. Bovine growth hormone, which binds to somatogenic receptors with great specificity, or recombinant human growth hormone, which is not contaminated with other hormones, affected phosphorylation of hepatic proteins similarly. The Mr 46,000 phosphoprotein was isolated in a fraction enriched in cytosol after centrifugation of cellular homogenates. Phosphorylation of the Mr 46,000 phosphoprotein was also increased (1.75 +/- 0.35)-fold and (2.15 +/- 0.50)-fold by insulin and glucagon, respectively. These observations are consistent with the possibility that selective changes in the phosphorylation state of cellular proteins may mediate growth hormone actions in cells.     

Generation of H2O2 is regulated by cytoplasmic free calcium in cultured porcine thyroid cells

Effects of calcium ionophore A23187 and BAY-K-8644, a calcium channel agonist, on cytoplasmic free calcium ([Ca2+]i) and H2O2 generation were studied in cultured porcine thyroid cells. We monitored continuously the effects of A23187 and BAY-K-8644 on [Ca2+]i and H2O2 generation, using the intracellularly trapped fluorescent Ca2+ indicator, fura-2, and homovanillic acid, respectively. A23187 and BAY-K-8644 induce an immediate increase in [Ca2+]i and H2O2 generation. The A23187- and BAY-K-8644-induced [Ca2+]i responses and H2O2 generation occur immediately, reach a maximum within several seconds, and then slowly decline. The minimum doses of A23187 or BAY-K-8644 to increase [Ca2+]i stimulate H2O2 generation. H2O2 generation is regulated by [Ca2+]i.     

Nitrile hydratase of Pseudomonas chlororaphis B23. Purification and characterization

Nitrile hydratase of Pseudomonas chlororaphis B23 was completely stabilized by the addition of 22 mM n-butyric acid. The enzyme was purified from extracts of methacrylamide-induced cells of P. chlororaphis B23 in eight steps. At the last step, the enzyme was crystallized by adding ammonium sulfate. The crystallized enzyme appeared to be homogeneous from analysis by polyacrylamide gel electrophoresis, analytical ultracentrifuge, and double diffusion in agarose. The enzyme has a molecular mass of about 100 kDa and consists of four subunits identical in molecular mass (approximately 25 kDa). The enzyme contained approximately 4 mol iron/mol enzyme. The concentrated solution of highly purified nitrile hydratase had a pronounced greyish green color and exhibited a broad absorption in visible range with a absorption maxima at 720 nm. A loss of enzyme activity occurred in parallel with the disappearance of the absorption in the visible range under a variety of conditions. The enzyme catalyzed stoichiometrically the hydration of nitrile to amide, and no formation of acid and ammonia were detected. The enzyme was active toward various aliphatic nitriles, particularly, nitriles with 3-6 carbon atoms, e.g. propionitrile, n-butyronitrile, acrylonitrile and cyclopropyl cyanide, served as the most suitable substrates.     

In vivo inhibition of aspartate aminotransferase in mice by L-hydrazinosuccinate

L-Hydrazinosuccinate, which has been shown to be a slow-, tight-binding inhibitor of aspartate aminotransferase (EC 2.6.1.1) in vitro, was tested as an inhibitor in vivo of the enzyme as well as other pyridoxal enzymes. Intraperitoneal administration to mice at a dose of 0.6 mmol/kg rapidly decreased aspartate aminotransferase activities in liver and kidney cytosols to a minimal level lower than 10% of the original, and no appreciable reversal of the inhibition was observed after 24 h; at lower doses the activities were significantly recovered during the same period following an initial marked decrease. Of the other pyridoxal enzymes tested, alanine aminotransferase in liver was the most sensitive to the inhibitor. It was initially inhibited as severely as aspartate aminotransferase, but the inhibition was reversed considerably faster. Aspartate aminotransferase activities in brain and heart were less severely affected than those in liver and kidney; they were less markedly lowered initially and were substantially recovered after 24 h. Consistent with the observed organ specificity, heated extracts from brain and heart in the mice administered with the inhibitor showed relatively weak inhibitory activities in vitro to aspartate aminotransferase purified from pig heart, while the extracts from liver and kidney were strongly inhibitory.     

A new solid-phase synthesis of oligoribonucleotides by the phosphoro-p-anisidate method using tetrahydrofuranyl protection of 2''-hydroxyl groups.

Six nonaribonucleotides containing the 5'-splice site, one complementary nonamer and an octadecamer containing the 3'-splice site have been synthesized on a polymer support using the phosphoro-p-anisidate method. A 5'-linked 2'-O-tetrahydrofuranyl-N-protected nucleoside 3'-(o-chlorophenyl)phosphoro-p-anisidate was used as the starting nucleotide, and the chain elongated in the 3'-direction by removing the p-anisidate protecting group with isoamyl nitrite under neutral conditions. The octadecamer has been synthesized using dinucleotide blocks and a 3'-terminal trinucleotide.     

Photosynthetic Characteristics of Photoautotrophically Cultured Cells of Tobacco

The photosynthetic characteristics of photoautotrophically culturedcells of tobacco (Nicotiana tabacum cv. Samsun NN) as well asthose of photomixotrophically cultured cells and green leaveswere investigated. Analyses revealed that on a fresh weightbasis cultured tobacco cells had lower chlorophyll contentsthan cells of green leaves. The chlorophyll content per chloro-plast,however, was almost the same in both types of cell, and thechloroplast number per cell accounted for only small differencesin the cellular chlorophyll content. This indicates that thelarger cell volume of cultured cells is the main factor in thedifference in the chlorophyll content of these cells. Photosynthetic activity measurements also showed differencesin the chloroplasts of cultured and leaf cells. The maximumactivities of photosystem I and the Hill reaction for the culturedcells were about half those for leaf cells on a per unit chlorophyllbasis. Moreover, photo-autotrophic cells had relatively constantphotosystem I and Hill reaction activities during growth; whereas,on a fresh weight basis these activities in leaf cells reflecteddevelopmental changes in the chlorophyll content. Lithium dodecyl sulfate-polyacrylamide gel electrophoresis showedqualitatively similar thylakoid polypeptide compositions forcultured and leaf cells at all stages of growth even thoughthere were quantitative decreases in the contents of severalpolypeptides in the cultured green cells (especially in photomixotrophiccells) in comparison to the polypeptide contents of tobaccoleaves. We speculate that the lower photosynthetic activityof the cultured cells may be caused by this reduction in thecontents of certain thylakoid polypeptides. (Received November 14, 1988; Accepted June 19, 1989)     

Cytosolic adenylate kinase catalyzes the synthesis of thiamin triphosphate from thiamin diphosphate

An attempt was made to purify a porcine skeletal muscle enzyme catalyzing the formation of thiamin triphosphate (TTP) from thiamin diphosphate (TDP), requiring ATP, Mg2+ and a cofactor (creatine). As the purification proceeded, the reaction requirements for ATP and creatine were lost and then a requirement for ADP was manifested. The activity responsible for TTP synthesis from TDP, ADP, and Mg2+ was found to be copurified with adenylate kinase [EC 2.7.4.3] activity, and was finally purified to a single band on SDS-PAGE. Antiserum obtained against the purified enzyme preparation inhibited both adenylate kinase activity and the TTP-synthesizing activity to exactly the same extent. These results indicate that adenylate kinase catalyzes TTP formation from TDP in vitro.     

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.