Biliary and urinary excretion rates and serum concentration changes of four bilirubin photoproducts in Gunn rats during total darkness and low or high illumination.

On cycled exposure of Gunn rats to total darkness and low and high illumination, biliary excretion rates of (EZ)- and (ZE)-bilirubin and (EZ)-cyclobilirubin increased up to approx. 10-fold from the mean basal values of 1.2 and 0.2 microgram/h to the mean maximum values of 25.2 and 4.2 micrograms/h respectively, and at the same time those of (EE)-bilirubin and (EE)-cyclobilirubin also increased, but at very much lower rates than those of the first-mentioned two. During the low illumination only (EZ)- and (ZE)-bilirubin and (EZ)-cyclobilirubin appeared in the urine; during the high illumination (EE)-bilirubin and (EE)-cyclobilirubin also appeared, showing a similar excretion pattern to that observed in the bile, but the total urinary excretion rates were lower than the total biliary excretion rates. The serum bilirubin concentrations fell gradually to lower values, accompanied by an increment in (EZ)- and (ZE)-bilirubin, but (EZ)-cyclobilirubin was not detected. It is concluded that during phototherapy the predominant pathway for the removal of bilirubin from the body in the Gunn rat is by biliary excretion of the geometric photoisomers (EZ)- and (ZE)-bilirubin, derived from Z----E isomerization, and the structural photoisomer (EZ)-cyclobilirubin, formed from intramolecular endo-vinyl cyclization.     

Evidence that the decarboxylation reaction occurs before the first methylation in ubiquinone biosynthesis in rat liver mitochondria

Biosynthesis of ubiquinone-9 was studied by incubating rat liver mitochondria with p-hydroxy[U-14C]benzoate, solanesyl diphosphate and S-adenosyl-L-methionine. When methylation reactions were inhibited by replacing S-adenosyl-L-methionine with S-adenosyl-L-homocysteine, nonaprenyl p-hydroxybenzoate and three other labeled peaks, designated as P1, P2 and P3 according to their retention times on HPLC, were observed. No carboxyl group was present in P1, P2 or P3 because the radioactivities disappeared when p-hydroxy[U-14C]benzoate was replaced by p-hydroxy[carboxyl-14C]benzoate. Compound P2 seemed to be hydroxylated but not methylated because its radioactivity markedly diminished under anaerobic conditions and the radioactivity was not incorporated into the compound from S-adenosyl-L-[methyl-3H]methionine, suggesting that P2 is 6-hydroxynonaprenylphenol. The complete correspondence of the retention times of P2 and chemically synthesized 6-hydroxynonaprenylphenol on HPLC further confirmed this possibility. P2 was a precursor of ubiquinone-9 because the radioactivity of the compound was incorporated into ubiquinone when incubated with mitochondria. The results suggest that the decarboxylation may occur prior to the first methylation in the ubiquinone biosynthesis in rat liver mitochondria, though it has been generally considered that in eukaryotes the first methylation precedes the decarboxylation.     

Construction of a non-support bioreactor: optical resolution of 2-(4-chlorophenoxy)propanoic acid in an organic solvent system

Summary A non-support bioreactor, a novel column reactor packed with a free non-supported enzyme was constructed by applying the insolubility of the enzyme in organic solvents. Stereoselective esterification of 2-(4-chlorophenoxy)propanoic acid by lipase OF 360 from Candida cylindracea with n-tetradecanol was selected as a model reaction. Non-supported lipase revealed threefold higher activity than Celite-adsorbed lipase by maintaining high stereoselectivity in a batch reaction. In continuous operation, a non-support bioreactor produced the ester with fourfold higher productivity to that of a column reactor packed with Celite-adsorbed lipase (an adsorbed bioreactor). However, the optical purity of the remaining (S)-acid was low even when the conversion ration was kept at approximately 50%. Lipase recovered from the non-support bioreactor after continuous operation retained the original stereoselectivity in a batch reaction. Therefore, semi-continuous operation was conducted by recycling the substrate solution at a high flow rate. The non-support reactor showed high stereoselectivity and ten times the productivity compared with the adsorbed bioreactor. The reason for this high performance is discussed. Offprint requests to: A. Tanaka     

Widespread Distribution of the 14-3-3 Protein in Vertebrate Brains and Bovine Tissues: Correlation with the Distributions of Calcium-Dependent Protein Kinases

A highly specific antiserum was prepared against bovine brain 14-3-3 protein, a protein kinase-dependent activator of tyrosine and tryptophan hydroxylases. The immunoassay using this antiserum proved the presence of 14-3-3 protein in various bovine tissues and in brains of various vertebrate species. The quantitative analysis indicated that the tissue distribution of 14-3-3 protein is more closely related to the known distributions of the Ca2(+)-dependent protein kinases, i.e., Ca2+/calmodulin- and Ca2+/phospholipid-dependent protein kinases, rather than those of tyrosine and tryptophan hydroxylases. This result, together with the available data on this protein, suggests potential roles of the 14-3-3 protein in more diverse kinase-mediated processes than the predicted role in monoamine synthesis.     

Specific purification of glyceraldehyde-3-phosphate dehydrogenase by hydrophobic chromatography on immobilized colchicine

Hydrophobic column chromatography of bovine brain extracts (40-80% ammonium sulfate fraction) on immobilized colchicine resulted in the selective elution of one major protein with decreasing ionic strength of medium. This protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.1) on the basis of its biochemical properties, N-terminal amino-acid sequence and enzymatic activity. The present method enabled GAPDH to be isolated with a high recovery (80%; 184 mg/kg brain) and could be of potential use for the purification of GAPDH from various tissues.     

Foetomaternal relationships of serum bile acid pattern estimated by high-pressure liquid chromatography.

The bile acid patterns in the maternal and umbilical vein and artery serum samples were analysed by a two-step chromatographic method involving group separation by piperidinohydroxypropyl-Sephadex LH-20 and high-pressure liquid chromatography using immobilized 3 alpha-hydroxy steroid dehydrogenase. Glycochenodeoxycholate predominates in the maternal blood and taurochenodeoxycholate in the umbilical blood. In cases where a free bile acid was detected in the maternal blood, the same bile acid was also demonstrated in the corresponding cord blood. The concentrations of taurocholate and taurochenodeoxycholate were found to be significantly higher in the umbilical artery than in the corresponding umbilical vein. Our data suggest that there is a bidirectional placental transfer of free bile acids and that there is a transfer of taurine-conjugated primary bile acids from the foetus to the mother.     

Biological activities and spectroscopic properties of chromophoric and fluorescent analogs of adenine nucleoside and nucleotides, 2′,3′-O-(2,4,6-trinitrocyclohexadienylidene) adenosine derivatives

The ribose-modified chromophoric and fluorescent analog of ATP 2′,3′-O-(2,4,6-trinitrocyclohexadienylidene) adenosine 5′-triphosphate (TNP-ATP) has been synthesized previously (Hiratsuka, T., and Uchida, K. (1973) Biochim. Biophys. Acta 320, 635–647 and Hiratsuka, T. (1976) Biochim. Biophys. Acta 453, 293–297). In the present study, four TNP-derivatives of ATP, ADP, AMP and adenosine were synthesized and compared for several chemical, spectral and enzymatic properties. Their visible absorption and fluorescent properties were found to be quite similar. Visible absorption and fluorescence spectra of TNP-derivatives were sensitive to solvent polarity. TNP-adenosine and TNP-AMP showed considerable substrate activities with adenosine deaminase and alkaline phosphatase, respectively. TNP-ATP proved to be an excellent substitute for ATP in adenylate kinase and myosin ATPase systems. The results indicate that these analogs are useful as chromophoric and fluorescent probes for hydrophobic regions in adenine nucleoside and nucleotide requiring enzymes.     

Hydrogen evolution by co-immobilized chloroplasts and Clostridium butyricum

Spinach chloroplasts and Clostridium butyricum cells were immobilized in 2% agar gel. Crude ferredoxin isolated from spinach and benzyl viologen were used as electron carriers. The optimum pH for both NADP reduction by immobilized chloroplasts and for hydrogen evolution by immobilized Cl. butyricum was 8.0. The optimum temperature was between 25 and 30°C for NADP reduction by immobilized chloroplasts, and 37°C for hydrogen evolution by immobilized cells. The total amount of hydrogen evolved in 6 h was 41 μmol/mg Chl for the immobilized chloroplast-benzyl viologen-immobilized Cl. butyricum system, and 11 μmol/mg Chl for the immobilized chloroplast-ferredoxin-Cl. butyricum system. The systems evolved only a trace amount of hydrogen when dichlorophenyldimethylurea was added. The immobilized chloroplast-benzyl viologen-immobilized Cl. butyricum system evolved hydrogen continuously for 6 h, and immobilized Cl. butyricum retained the initial hydrogenase activity. However, the photoreduction activity of chloroplasts decreased to 30% of the initial activity after 6 h of reaction.     

The effect of clinorotation on vestibular compensation in upside-down swimming catfish.

Upside-down swimming catfish Synodontis nigriventris can keep upside-down swimming posture stably under pseudo-microgravity generated by clinostat. When the vestibular organ is unilaterally ablated, the operated S. nigriventris shows disturbed swimming postures under the clinorotation condition. However, about 1 month after the operation, unilateral vestibular organ-ablated S. nigriventris shows stable upside-down swimming posture under the condition (vestibular compensation). In contrast, a closely related upside-up swimming catfish Synodontis multipunctatus belonging to same Synodontis family can not keep stable swimming postures under the clinorotation conditions. In this study, we examined the effect of continuous clinorotation on vestibular compensation in intact and unilateral vestibular organ-ablated Synodontis nigriventris and Synodontis multipunctatus. After the exposure to continuous clinorotation, the postures of the catfish were observed under microgravity provided by parabolic flights of an aircraft. Unilateral vestibular organ-ablated S. nigriventris which had been exposed to continuous clinorotation showed stable swimming postures and did not show dorsal light reaction (DLR) under microgravity. This postural control pattern of the operated catfish was similar to that of intact catfish. Intact and unilateral vestibular organ-ablated S. multipunctatus showed DLR during microgravity. Our results confirmed that S. nigriventris has a novel balance sensation which is not affected by microgravity. DLR seems not to play an important role in postural control. It remains unclear that the continuous clinorotation effects on vestibular compensation because we could not keep used unilateral vestibular organ-ablated fish alive under continuous clinorotation for uninterrupted 25 days. This study suggests that space flight experiments are required to explore whether gravity information is essential for vestibular compensation.     

Inorganic pyrophosphatase in the roundworm Ascaris and its role in the development and molting process of the larval stage parasites.

Inorganic pyrophosphatase (PPase) is an important enzyme that catalyzes the hydrolysis of inorganic pyrophosphate (PPi) into ortho-phosphate (Pi). We report here the molecular cloning and characterization of a gene encoding the soluble PPase of the roundworm Ascaris suum. The predicted A. suum PPase consists of 360 amino acids with a molecular mass of 40.6 kDa and a pI of 7.1. Amino acid sequence alignment and phylogenetic analysis indicates that the gene encodes a functional Family I soluble PPase containing features identical to those of prokaryotic, plant and animal/fungal soluble PPases. The Escherichia coli-expressed recombinant enzyme has a specific activity of 937 micro mol Pi.min-1.mg-1 protein corresponding to a kcat value of 638 s-1 at 55 degrees C. Its activity was strongly dependent on Mg2+ and was inhibited by Ca2+. Native PPases were expressed in all developmental stages of A. suum. A homolog was also detected in the most closely related human and dog roundworms A. lumbricoides and Toxocara canis, respectively. The enzyme was intensely localized in the body wall, gut epithelium, ovary and uterus of adult female worms. We observed that native PPase activity together with development and molting in vitro of A. suum L3 to L4 were efficiently inhibited in a dose-dependent manner by imidodiphosphate and sodium fluoride, which are potent inhibitor of both soluble- and membrane-bound H+-PPases. The studies provide evidence that the PPases are novel enzymes in the roundworm Ascaris, and may have crucial role in the development and molting process.