Purification and characterization of cytosol phosphoenolpyruvate carboxykinase from bullfrog (Rana catesbeiana) liver.

Cytosol PEP carboxykinase has been purified to electrophoretic homogeneity from bullfrog liver homogenate. The enzyme is a single polypeptide chain with a molecular weight of approximately 72,000-75,000. The purified enzyme catalyzed oxaloacetate decarboxylation (nucleoside triphosphate-supported), phosphoenolpyruvate carboxylation, and an exchange reaction between oxaloacetate and [14C]HCO3-in the presence of ITP or CTP. Manganese is absolutely required for the enzyme-catalyzed phosphoenolpyruvate carboxylation, whereas it can be replaced by Mg2+ for the oxaloacetate decarboxylation and the exchange reaction. The optimal pH of each reaction is dependent on the divalent metal ion used. The dependence of the enzyme activity on Mn2+ is markedly different in the phosphoenolpyuvate carboxylation and the oxaloacetate decarboxylation reactions.     

Distribution of AMP-deaminase isozymes in rat tissues

1. The distribution of AMP deaminase isozymes in rat tissues was analyzed by electrophoresis on cellulose acetate membrane, by chromatography on phosphocellulose column, and by the application of immunological technique employing specific antisera against three parental AMP deaminases (isozymes A, B and C). Skeletal muscle extracts and diaphragm extracts contain a single identical isozyme, isozyme A. The major isozyme species of liver, kidney and testes are also identical and they are isozyme B. Heart extracts contains isozyme C exclusively. Extracts of brain, lung and spleen contain five isozymes, presumably a complete set of five B-C hybrids. 2. Developmental patterns of AMP deaminase isozyme were studied. In early postnatal life, extracts of heart, liver, kidney and lung contain five isozymes similar to those observed in adult brain. During postnatal development, a shift to isozyme C occurs in heart, whereas a shift to isozyme B occurs in liver and kidney. Five isozymes in lung remain throughout development. In brain a shift of B to five isozymes is observed during development. Isozyme A is the predominant form in muscle throughout postnatal development. 3. AMP deaminase in the regenerating liver was analyzed, but the data indicated that there was no change of isozyme distribution during hepatic regeneration.     

Enhancing effects of CO2 on chloroplast regeneration in glucose-bleached cells of Chlorella protothecoides I. Role of non-photosynthetic CO2-fixation in chloroplast regeneration

Carbon dioxide enhanced chloroplast regeneration in glucose-bleachedcells of Chlorella protothecoides in the presence of CMU inthe light. Both the formation of chlorophyll and the synthesesof RNA and protein were considerably enhanced. The CO₂ metabolism of algal cells during greening was investigatedusing ¹⁴C-bicarbonate as the tracer. Radiocarbon was largelyincorporated into purine and pyrimidine bases in nucleic acidand the arginine in protein, specifically at the crabon atomsderived from carbamylphosphate. ¹Part of this investigation was reported at the conference onthe "Autonomy and biogenesis of mitochondria and chloroplasts"held at Canberra in 1969 (4). (Received August 19, 1975; )     

Optimal control of a semibatch fermentation

This work is concerned with the optimization study of the semibatch fermentation by which an amino acid is produced. The particular fermentation studied is the synthesis of lysine by the auxotrophic mutant. Applying Green's theorem to the maximization problem was proposed, and it succeeded in determining the feed rate of the substrate that maximized the production rate of the desired product.     

An endonuclease activity in Bacillus subtilis specific for UV-irradiated DNA

An enzyme activity specific for UV-DNA¹ was found in the extract of $\text{Bacillus subtilis}$(Marburg 168). The enzyme preparation obtained from the extract by ammonium sulfate precipitation acts on UV-DNA endonucleolytically and induces single strand breaks. The number of single strand breaks introduced in DNA is proportional to UV dose.     

Unfolding and refolding of the constant fragment of the immunoglobulin light chain containing an intramolecular mercury bridge

The conformation of the constant fragment of the immunoglobulin light chain in which the intrachain disulfide bond is replaced by the bond S-Hg-S (CL-Hg fragment), was as compact as that of the intact CL fragment, but its stability to guanidine hydrochloride was lower than that of the intact CL fragment [Goto, Y. & Hamaguchi, K. (1986) Biochemistry in press]. The kinetics of reversible unfolding and refolding of the CL-Hg fragment by guanidine hydrochloride were studied and compared with those for the intact CL and reduced CL fragments [Goto, Y. & Hamaguchi, K. (1982) J. Mol. Biol. 156, 891-910, 911-926]. The unfolding kinetics were explained on the basis of a three-species mechanism, U1----U2----F, where U1 and U2 are respectively slow-folding and fast-folding species of unfolded protein, and F is folded protein. However, an additional isomerization, though its contribution to the overall reaction process is small, had to be taken into account to explain the refolding kinetics. The kinetic properties of interconversion between U1 and U2 were similar to those for the intact CL and reduced CL fragments. This suggested that the same prolyl residue is involved in the isomerization reactions in the unfolded states of the intact CL, reduced CL, and CL-Hg fragments. The rate constant for the unfolding process, F to U2, was about 20 times greater than those for the intact CL and reduced CL fragments, while the rate constant for the refolding process, U2 to F, lay between the values for the intact CL and the reduced CL fragment. The free energy profiles of unfolding and refolding of the intact CL, reduced CL, and CL-Hg fragments were compared.     

Interactions between the Nucleus and Cytoplasmic Organelles during the Cell Cycle of Euglena gracilis in Synchronized Cultures: II. Associations between the Nucleus and Chloroplasts at an Early Stage of the Cell Cycle under Photoautotrophic Conditions

Chloroplast-nucleus interactions were examined in cells of Euglenagracilis Z synchronized under photoautotrophic conditions. Thechloroplasts were localized near the cell periphery. At an earlystage of the cell cycle, however, some chloroplasts were transientlylocated in the inner space close to the nucleus. Electron microscopyusing serial cell sections revealed that the chloroplast formedprotrusions at several sites, which became associated with thenucleus. The outer membrane of the chloroplast envelope wasin contact, or at least continuous in part, with the outer membraneof the nuclear envelope at the sites of association, and densematerial was present in the chloroplast membrane. A chromosomewas close to each site of the association between these twoorganelles. Most of the chloroplasts including those in associationwith the nucleus were connected by fine bridges. The 4',6-diamidino-2-phenylindole-stainednucleoids in the chloroplast associated with the nucleus appearedto have a thread-like shape. There was another type of chloroplast-nucleusconnection, in which an intervening membranous body was in contactwith the outer part of the nuclear envelope on one side andwith the chloroplast envelope on the other side. ¹ This work was reported at the 48th Annual Meeting of the BotanicalSociety of Japan, Kyoto, October, 1983. (Received June 5, 1984; Accepted November 20, 1984)     

Purification and properties of diamine oxidase from pea epicotyls

Diamine oxidase (DAO) (EC 1.4.3.6) was purified from pea epicotyls to homogeneity by the criterion of polyacrylamide gel electrophoresis (PAGE). The pu     

Characterization of hepatitis B virus DNA polymerase

Hepatitis B virus (HBV) particles were separated from the blood-plasma containing HBe and HBs antigens (subtype adr) and the nature of the endogenous DNA polymerase in the HBV core particles was studied. The HBV endogenous DNA polymerase activity was examined under the conditions used for preparation of HBV vaccine. The endogenous DNA polymerase activity was reduced slowly upon the heat treatment or the formalin treatment. The reductions of the activity were 65% and 70% upon the heat treatment at 60 C for 10 hr and the formalin treatment at 37 C for 90 hr, respectively. Properties of the HBV endogenous DNA polymerase were studied by utilizing specific inhibitors against the eukaryotic DNA polymerases. Our results showed that the HBV endogenous DNA polymerase is resistant to aphidicolin and N-ethylmaleimide, and sensitive to 2',3'-dideoxythymidine 5'-triphosphate, phosphonoformic acid and 9-beta-D-arabinofuranosyladenosine 5'-triphosphate.