Studies on the mechanism of the conversion of cytosol δ-aminolevulinic acid synthase to the mitochondrial enzyme in the liver of the allylisopropylacetamide-treated rat

δ-Aminolevulinic acid (ALA) synthase was partially purified from liver cytosol fraction of rats treated with allylisopropylacetamide (AIA). The cytosol ALA synthase showed an apparent molecular weight of 320,000. The cytosol ALA synthase of this size dissociates into at least three protein components when subjected to sucrose density gradient centrifugation in the presence of 0.25 m NaCl: one is the catalytically active protein with an s value of about 6.4 or a molecular weight of 110,000, and the other two are catalytically inactive binding proteins showing s values of about 4 and 8, respectively. Recombination of the 6.4 S protein and the 4 S protein yielded a protein complex with an apparent molecular weight of 170,000 and recombination of all three protein components resulted in formation of the original cytosol ALA synthase. The cytosol ALA synthase also loses its binding proteins when treated with various proteases; thus, the enzyme-active protein obtained after papain digestion was very similar, if not identical, to mitochondrial ALA synthase. When treated with trypsin, however, the cytosol ALA synthase was converted to an enzyme showing an apparent molecular weight of 170,000, which probably represents the complex of the mitochondria-type enzyme and the 4 S binding protein. The cytosol ALA synthase tends to aggregate to form a dimer with an apparent molecular weight of 650,000–700,000. The aggregated form of the cytosol ALA synthase was less susceptible to trypsin digestion. Hemin strongly stimulated dimer formation of the cytosol ALA synthase and the aggregate produced by contact with hemin was very tight and did not easily dissociate into its respective protein components by sucrose gradient centrifugation or even after treatment with trypsin. The possible mechanisms of the conversion of cytosol ALA synthase to the mitochondrial enzyme and also of the inhibition by hemin of the intracellular translocation of ALA synthase are discussed.     

Cultivation of mosquito cell lines in serum-free media and their effects on dengue virus replication

Summary Seven mosquito cell lines from five species (Aedes aegypti, Ae. albopictus, Ae. pseudoscutellaris, Culex tarsalis, andToxorhynchites amboinensis) were adapted to three kinds of serum-free media (SEM), which were composed of equal volumes of tryptose phosphate broth and of either Leibovitz (L15) medium, Eagle’s minimum essential medium, or Medium 199 with Hanks’ salts. Population growth rates of the cells cultivated in the SMFs were generally slower than those of original cell cultures maintained in conventional media containing bovine sera. A karyological study showed a significant shift to heteroploidy in two of the four cell lines examined. Four SMF-adapted sublines were compared with parental cultures for replication of dengue viruses.Ae. aegypti RML-12,Ae. albopictus C6/36,Ae. pseudoscutellaris AP-61, andTx. amboinensis TRA-171 demonstrated different levels of alteration in virus replication ranging from lower titers (as inAe. albopictus C6/36) to comparable or higher titers (as inAe. aegypti RML-12) when they were simultaneously inoculated with four dengue serotypes. Use of trade names and commercial sources is for identification only and does not constitute endorsement by the Public Health Service or by the U.S. Department of Health and Human Services.     

A microsomal endopeptidase from liver with substrate specificity for processing proproteins such as the vitamin K-dependent proteins of plasma.

The microsomal fraction of rabbit liver contains an endopeptidase that cleaves synthetic peptides that mimic the amino acid sequences of the processing sites of many proproteins, including the vitamin K-dependent proteins. The endopeptidase (M(r) 69,000) was extracted from liver microsomes with 1% Lubrol and purified about 2,700-fold. The substrate employed for isolation and characterization of the enzyme was the decapeptide acetyl-Ala-Arg-Val-Arg-Arg-Ala-Asn-Ser-Phe-Leu (prothrombin peptide), in which hydrolysis occurred on the carboxyl side of the paired Arg-Arg residues. The purified enzyme, whose activity was enhanced 1.8-fold by 0.1 mM CoCl2, has a Km = 80 microM and Vmax = 21,000 nmol.min-1.mg-1 and a pH optimum of 8.7. Proteolytic cleavage of decapeptide substrates was dependent on an arginine residue at positions P1 and P4. The enzyme was completely inhibited by EDTA and 1,10-phenanthroline as well as by p-chloromercuriphenylsulfonic acid and Hg2+. Inhibitors of serine proteases and cysteine proteases had no effect. Based on the substrate preference, the endopeptidase appears to be a good candidate for the enzyme responsible for the precursor processing of the vitamin K-dependent proteins and a number of other proproteins that are synthesized via the secretory pathway in liver and other tissues.     

Differential Effects of Nitrate and Light on the Expression of Glutamine Synthetases and Ferredoxin-Dependent Glutamate Synthase in Maize

The effects of nitrate and light on the expression of genesfor glutamine synthetase (GS) isoproteins and ferredoxin-dependentglutamate synthase (Fd-GOGAT) were studied in different organsof maize seedlings by analyzing the levels of the respectivepolypeptides and mRNAs. In roots, the levels of plastidic GSand of a novel, root-specific GS molecule localized in the extraplastidiccompartment were increased markedly by nitrate, whereas Fd-GOGATand cytosolic GS remained at their initial levels. Ammonia wasnot effective in inducing the plastidic GS and Fd-GOGAT butit did induce the novel GS isoprotein. In leaves, cytosolicand plastidic GSs and Fd-GOGAT were present in both mesophyllcells (MC) and bundle sheath cells (BSC). Upon addition of nitrate,the level of plastidic GS increased preferentially in MC, andupon exposure of etiolated seedlings to light, the levels ofplastidic GS and Fd-GOGAT increased in BSC in a coordinatedmanner. The relationship between the expression of genes forGSs and Fd-GOGAT and the physiological role of the GS/GOGATcycle is discussed in terms of the characteristics of nitrogenmetabolism in roots, MC, and BSC. (Received August 11, 1992; Accepted September 21, 1992)     

Prevalence of spotted fever group rickettsia antibody in Apodemus speciosus captured in an endemic focus in Miyazaki Prefecture, Japan

Fourteen of Apodemus speciosus (large Japanese field mouse) were captured near the place where one of the patients with spotted fever group rickettsiosis had been infected, in Takaoka town, Miyazaki Prefecture. In the town, four human cases were reported. All of the mice had antibodies against Rickettsia japonica and R. montana. The incidence of the antibody was significantly higher in Apodemus mice in the area than in those from nonendemic area.     

Molecular cloning and sequence analysis of cDNA coding for rat liver hemoprotein H-450

cDNA clones coding for hemoprotein H-450 were isolated from a rat liver cDNA library using anti-H-450 antibody. The molecular weight calculated from the deduced amino acid sequence comprising 547 amino acid residues was 60,085. The N-terminal sequence and a partial internal amino acid sequence of purified H-450, which were determined chemically, were both found in the amino acid sequence of H-450 deduced from the nucleotide sequence. H-450 mRNA is expressed in liver, kidney, and brain. A homology search of amino acid sequences indicated that H-450 shows no homology with cytochrome P-450, but shows significant homology with bacterial O-acetylserine (thiol)-lyases. However, H-450 has no O-acetylserine (thiol)-lyase activity.     

Biological characterization of human brain natriuretic peptide (BNP) and rat BNP: species-specific actions of BNP

We examined the diuretic-natriuretic activities of rat BNP and human BNP in anesthetized rats in vivo and their vasorelaxant activities for rat thoracic aorta and porcine coronary artery in vitro. Rat BNP was almost equipotent to rat ANP in diuresis and natriuresis with relative potencies of 1.6 and 2.5, respectively, while human BNP exerted no significant activity. Rat ANP, rat BNP and human BNP relaxed PGF2 alpha-contracted rat aortic strips with IC50 values of 0.62, 0.64 and 12.1 nM, respectively, while they relaxed PGF2 alpha-contracted porcine coronary arteries with IC50 values of 0.04, 1.10 and 0.02 nM, respectively. These results strongly suggest that the biological action of BNP is species-specific.     

New method of immobilization of microbial cells by capture on the surface of insoluble pyridinium-type resin

A new method for the immobilization of microbial cells has been developed. Whole cells of Escherichia coli with aspartase activity were immobilized by capture on the surface of cross-linked poly(N-benzyl-4-vinylpyridinium bromide) containing styrene (BVPS resin), an insoluble pyridinium-type resin. When a suspension of the bacterial cells in buffer solution was passed through a glass column containing beads of BVPS resin, the cells were captured on the resin surface and formed an immobilized cell system. A fixed-bed column reactor containing 300 mg of the bacterial cells immobilized by capture on 10 g of BVPS resin beads was used for the preparation of L-aspartic acid from ammonium fumarate. Continuous operation of tne bioreactor produced L-aspartic acid in a quantitative yield when the influent substrate concentration was 0.1M and the flow rate was 0.41-0.83 bed volumes per hour at pH 7.4-7.7 at 30 degrees C.