Stability of the hexagonal lattice structure formed by an R-form lipopolysaccharide of Klebsiella: decrease in the stability by electrodialysis and recovery by addition of the magnesium

The R-form lipopolysaccharide (LPS) from Klebsiella strain LEN-111 (O3-:K1-) forms a hexagonal lattice structure with a lattice constant of 14 to 15 nm when it is precipitated by addition of two volumes of 10 mM MgCl2-ethanol. When the LPS was suspended in various buffers (50 mM) at pH 2 to 12 for 24 hr at 4 C, at pH 2 and 3 pits of the hexagonal lattice structure markedly disappeared, at pH 4 to 8.5 the lattice structure was stable, and at pH 9 to 12 it tended to loosen somewhat. The LPS from which cations were removed by electrodialysis retained the ability of hexagonal assembly, although the lattice constant of the hexagonal lattice of the electrodialyzed LPS was large. The lattice structure of the electrodialyzed LPS was much more labile than that of the non-electrodialyzed LPS at alkaline pH levels and the former was completely disintegrated into ribbon-like structures when the LPS was suspended in 50 mM Tris buffer at pH 7.7 or higher. However, the electrodialyzed LPS formed a hexagonal lattice structure in Tris buffer at pH 8.5 containing 0.1 to 100 mM MgCl2. The lattice constants of the hexagonal lattice formed by the electrodialyzed LPS at 10 or 100 mM MgCl2 were very similar to that of the lattice of the non-electrodialyzed LPS. From these results it is concluded that the lability of the hexagonal lattice structure of the electrodialyzed LPS at alkaline conditions is due to removal of Mg2+ by electrodialysis.     

Biological functions of serine proteases in mast cells in allergic inflammation

Serine proteases in mast cell granules, such as chymase, atypical chymase, and tryptase, which are major proteins in the granules, may play important roles in the process of immunoglobulin E (IgE)-mediated degranulation and in pathobiological alterations in tissues. Indeed, inhibitors of chymase, substrate analogs, and antichymase F(ab')2, but not inhibitors of tryptase, markedly inhibited histamine release induced by IgE-receptor bridging but not that induced by Ca ionophore. In contrast, inhibitors of metalloprotease inhibited histamine release induced not only by IgE-receptor bridging but also by Ca ionophore. These results suggest that chymase and metalloprotease are involved at different steps in the process of degranulation. The extents of inhibition of histamine release were closely correlated with the amounts of the inhibitors of chymase accumulated in the granules. After degranulation, the released proteases may in part contribute to pathobiological alterations in allergic disorders through generations of C3a anaphylatoxin and thrombin by human and rat tryptase, respectively, and those of angiotensin II and a chemotactic factor of neutrophils by human and rat chymase, respectively. Moreover, chymase and atypical chymase from rat were shown to destroy type IV collagen, and human tryptase was found to hydrolyze various plasma proteins, such as fibrinogen and high-molecular-weight kininogen. The biological activities of tryptase and chymase from rat may be regulated by their dissociation from and association with trypstatin, an endogenous inhibitor of these proteases.     

Purification and Characterization of Kynurenine Aminotransferase I from Human Brain

Abstract: Two kynurenine aminotransferases (KATs), arbitrarily termed KAT I and KAT II, are capable of producing the neuroinhibitory brain metabolite kynurenic acid from l -kynurenine in human brain tissue. Here we describe the purification of KAT I to homogeneity and the subsequent characterization of the enzyme using physicochemical, biochemical, and immunological methods. KAT I was purified from human brain ∼2,000-fold with a yield of 2%. Assessed by polyacrylamide gel electrophoresis, KAT I migrated toward the anode as a single protein with a mobility of 0.5. The pure enzyme was found to be a dimer consisting of two identical subunits of ∼60 kDa. Among several oxo acids tested, KAT I showed highest activity with 2-oxoisocaproate. Kinetic analyses of the pure enzyme revealed an absolute K _m of 2.0 m M and 10.0 m M for l -kynurenine and pyruvate, respectively. KAT I activity was substantially inhibited by l -glutamine, l -phenylalanine, and l -tryptophan, using either pyruvate (1 m M ) or 2-oxoisocaproate (1 m M ) as a cosubstrate. l -Tryptophan inhibited enzyme activity noncompetitively with regard to pyruvate ( K _i = 480 µ M ) and competitively with regard to l -kynurenine ( K _i = 200 µ M ). Anti-KAT I antibodies were produced against pure KAT I and were partially purified by conventional techniques. Immunotitration and immunoblotting analyses confirmed that KAT I is clearly distinct from both human KAT II and rat kynurenine-pyruvate aminotransferase. Pure human KAT I and its antibody will serve as valuable tools in future studies of kynurenic acid production in the human brain under physiological and pathological conditions.     

Purification and characterization of membrane-bound alcohol dehydrogenase from Acetobacter polyoxogenes sp. nov.

Summary Membrane-bound alcohol dehydrogenase (ADH) was purified from the membrane fraction of an industrial-vinegar-producing strain, A. polyoxogenes sp. nov. NBI1028 by solubilization using Triton X-100 and subsequent column chromatography on DEAE-Sepharose CL-6B and hydroxyapatite. The purified enzyme was homogeneous on polyacrylamide disc gel electrophoresis (PAGE). Upon sodium dodecyl sulphate-PAGE, the enzyme showed the presence of two subunits with a molecular mass of 72 000 daltons and 44 000 daltons, respectively. The small subunit was identified as cytochrome c. In addition, absorption and fluorescence spectra showed the the presence of pyrroloquinoline quinone in the purified ADH. The ADH preferentially oxidized aliphatic alcohols with a straight carbon chain except for methanol. Formaldehyde and acetaldehyde were also oxidizable substrates. The apparent K _m for ethanol was 1.2 mM. The optimum pH and temperature were 5.0–6.0 and 40°C, respectively. p-Chloromercuribenzoic acid and heavy metals such as CuSO₄ were inhibitory to the enzyme activity. Ferricyanide was effective as an electron acceptor.Offprint requests to: M. Fukaya     

Protective effect of sulfhydryl compounds on acute toxicity of morphinone

The ability of sulfhydryl compounds to provide protection against the acute toxicity of morphinone was investigated in mice. Subcutaneous administration of morphinone produced a reduction of hepatic non-protein sulfhydryl concentration. Pretreatments of mice with glutathione or cysteine significantly increased the survival rate of mice given a lethal dose of morphinone, whereas morphinone lethality was markedly potentiated by diethyl maleate. On the other hand, the administration of morphine produced a dose dependent reduction of hepatic non-protein sulfhydryl contents. However, neither glutathione nor cysteine protected mice from the acute toxicity of morphine. A possible explanation for these observations was proposed as follows: morphine is oxidized by morphine 6-dehydrogenase to morphinone, and the morphinone thus produced decreases the sulfhydryl contents in the liver. This mechanism is supported by the fact that morphinone reacts easily with glutathione and cysteine $\text{in vitro}$.     

Isolation and characterization of a novel trypsin-like protease found in rat bronchiolar epithelial Clara cells. A possible activator of the viral fusion glycoprotein.

A novel trypsin-like protease associated with rat bronchiolar epithelial Clara cells, named Tryptase Clara, was purified to homogeneity from rat lung by a series of standard chromatographic procedures. The enzyme has apparent molecular masses of 180 +/- 16 kDa on gel filtration and 30 +/- 1.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Its isoelectric point is pH 4.75. Studies with model peptide substrates showed that the enzyme preferentially recognizes a single arginine cleavage site, cleaving Boc-Gln-Ala-Arg-4-methylcoumaryl-7-amide most efficiently and having a pH optimum of 7.5 with this substrate. The enzyme is strongly inhibited by aprotinin, diisopropylfluorophosphate, antipain, leupeptin, and Kunitz-type soybean trypsin inhibitor, but inhibited only slightly by Bowman-Birk soybean trypsin inhibitor, benzamidine, and alpha 1-antitrypsin. Immunohistochemical studies indicated that the enzyme is located exclusively in the bronchiolar epithelial Clara cells and colocalized with surfactant. An immunoreactive protein with a molecular mass of 28.5 kDa was also detected in airway secretions by Western blotting analyses, suggesting that the 30-kDa protease in Clara cells is processed before or after its secretion. Proteolytic cleavage of the hemagglutinin of influenza virus is a prerequisite for the virus to become infectious. Tryptase Clara was shown to cleave the hemagglutinin and activate infectivity of influenza A virus in a dose-dependent way. These results suggest that the enzyme is a possible activator of inactive viral fusion glycoprotein in the respiratory tract and thus responsible for pneumopathogenicity of the virus.