A Method for Molecular Analysis of Catalase Gene Diversity in Seawater

Catalase plays an important role in the metabolism of marine bacteria and has potential impact on the marine environment. Four PCR primers were designed to amplify the catalase gene fragments in marine bacteria by applying metagenomic DNA from Yellow Sea surface water as the template. Of the four reproducible target PCR products, the longest one with 900 bp were chosen for catalase gene library construction by the T-vector and the white Escherichia coli colonies in the library was screened through restriction-digesting the reamplified insert fragments by the selected restriction endonuclease MboI, and then the bands of the resulting products were displayed in the agarose gel by electrophoresis. The unique restriction fragment length polymorphism (RFLP) pattern was selected and the corresponding catalase gene fragments were sequenced, which verified that every unique RFLP pattern represented one type of catalase. This PCR–RFLP method above was established to investigate the bacterial catalase diversity in seawater.     

Novel recombinant human lactoferrin: Differential activation of oxidative stress related gene expression

Lactoferrin, an iron-binding protein found in high concentrations in mammalian exocrine secretions, is an important component of the host defense system. It is also a major protein of the secondary granules of neutrophils from which is released upon activation. Due to its potential clinical utility, recombinant human lactoferrin (rhLF) has been produced in various eukaryotic expression systems; however, none of these are fully compatible with humans. Most of the biopharmaceuticals approved by the FDA for use in humans are produced in mammalian expression systems. The Chinese hamster ovary cells (CHO) have become the system of choice for proteins that require post-translational modifications, such as glycoproteins.     

Effect of Carbohydrates and Starvation on Nitrogen Sparing Action of Methionine and Threonine in Rats

Formaldehyde dehydrogenase from Pseudomonas putida C-83 was found to contain 7 halfcystine residues per subunit monomer, as checked by the method of performic acid oxidation. Approximately 7 sulfhydryl groups per subunit monomer were titrated with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) after denaturation with 8 m urea. In the native enzyme, modification of three sulfhydryl groups per subunit with p-chloromercuribenzoate (PCMB) led to the complete loss of enzyme actiyities for both formaldehyde and n-butanol. Hydrogen-peroxide competitively inhibited the enzyme activity for formaldehyde, while it was only slightly inhibitory to the activity for n-butanol. Both formaldehyde and hydrogen-peroxide protected one sulfhydryl group per subunit monomer from modification with PCMB. Moreover, hydrogen-peroxide was hardly reactive to the enzyme which was preincubated with formaldehyde.

From these observations, we conclude that one of three PCMB-reactive sulfhydryl groups is essential for the binding of formaldehyde, and hydrogen-peroxide modifies this sulfhydryl group.     

The Heterogeneity of the 7S Soybean Protein by Sepharose Gel Chromatography and Disc Gel Electrophoresis

Two kinds of N-acetylmuramidase, M-1 and M-2 enzymes, that were isolated from the cultural broth of Stm. globisporus 1829, were remarkably different in amino acid composition, immunological properties and modes of lytic action from each other. The M-1 enzyme was composed of 186 amino acid residues of which two moles were of half cystine, while the M-2 enzyme was composed of 99 amino acid residues with no cysteine. The hydrolyzing action of the M-2 enzyme was suppressed by the presence of an O-acetyl group on muramic acid residues in the peptidoglycan moiety, while that of the M-l enzyme was independent of the presence of O-acetyl groups. However, the hydrolyzing activity of both enzymes was enhanced when some muramic acid residues were substituted with stem peptides containing alanine, isoglutamine and lysine.     

Method for Enrichment of Rhodopseudomonas sphaeroides from Fresh Water

An inulinase was highly purified from the culture broth of Penicillium purpurogenum by chromatographies on DEAE-Sepharose CL-6B, Toyopearl HW-65, and Bio-Gel P-100. The enzyme was homogeneous by disc electrophoretic analysis. The molecular weight was 6.4 × 10⁴ by SDS-disc electrophoresis and gel filtration on Bio-Gel P-150. The isoelectric point was pH 3.6 by isoelectric focusing. The enzyme hydrolyzed inulin rapidly, but did not affect sucrose. By paper chromatography analysis, the major products from inulin were tri-, tetra-, penta-, and hexa-saccharides. The substrate specificity of the enzyme on hydrolyses of fructo-oligosaccharides[1^F(1-β-d-fructofuranosyl)n sucrose (n = 1 to 6 and n (average of polymerization degree) = 8)] were examined. The Km values and relative maximum velocities for the hydrolyses of inulin and fructo-oligosaccharides (GF_n, n = 2 to 7 and n = 9) were as follows: inulin, (DP = 35) 0.21 mM and 100; GF₉, 0.24 mM and 86.5; GF₇, 0.33 mM and 132; GF₆, 0.85 mM and 71.2; GF₅, 3.8 mM and 25.4; GF₄, 2.8 mM and 28.8; GF₃, (nystose) 16 mM and 0.8; GF₂ (1-kestose), 8.4 mM and 0.2. The molecular activities for the hydrolyses of fructo-oligosaccharides (GF_n, n = 2 to 6) were increased depending on the degree of polymerization of fructosyl residues, and were nearly constant if the polymerization degree was over seven. These results strongly suggested that the endo-type inulinase from Penicillium purpurogenum had a subsite structure consisting of at least seven subsites.     

Lipids and Related Substances Inducing the Lipase Production by Candida paralipolytica

Properties of autolytic breakdown of rat skeletal muscle proteins in the alkaline pH range have been reported. The activity is almost exclusively localized in the myofibrillar fraction, but is not solubilized with Triton X-100. The activity is affected by the KCI concentration in the reaction mixture. In 0.6 M and the more concentrated KCI solutions, the maximum activity is attained. The optimum pH of the activity is in the range of pH 7.5～9.5, and the optimum temperature is between 47～57°C.

This autolytic activity seems to be different from catheptic activity which shows its optimum pH in the acid pH range. Moreover, though more than half of the catheptic activity of rat skeletal muscle is recovered in the myofibrillar fraction, the catheptic activity in the myofibrillar fraction can be removed from the fraction by the extraction with dilute saline solution containing Triton X-100.     

Effect of Various Inhibitors on Lipase Action of Thermophilic Fungus Humicola lanuginosa S-38

The hydrolysis of olive oil by the Humicola lipase was inhibited by the addition of n-alcohols, fatty acids and surface active agents. The inhibition of n-alcohols was overcomed by the addition of more substrate but not by the addition of more enzyme. The inhibition of fatty acids and bile salts was eliminated by adding calcium ion. It was concluded that the inhibition of the Humicola lipase by n-alcohols, fatty acids and bile salts was not due to inactivation of the enzyme directly but due to the displacing of the substrate from the oil/water interface, thus blocking the enzyme from the substrate.     

In Vitro Slow Fluctuation in Horseradish Peroxidase Activity

In vitro slow fluctuations in the level of horseradish peroxidase activity were observed in long-range experiments (72–144 h). Besides random fluctuations, regular slow oscillatory patterns with period lengths ranging from 10.0 to 39.0 h were detected by statistical analysis. The possibility that these oscillations in enzyme activity could have reflected changes in the physical environment of the experimental setup has been thoroughly examined and ruled out. Periodic exposition of the enzyme solution, otherwise kept in darkness, to blue light illumination was shown to influence the period of the oscillations. The changes in enzyme activity were correlated with a modification of the Michaelis constant estimated using guaiacol as substrate. This result was confirmed by the action of chemical modifiers of the enzyme, such as ferulic acid and rutin. It is thought that the observed oscillations in horseradish peroxidase activity are due to spontaneous and specific changes in the tridimensional structure of the enzyme in the thermic reservoir.     

Chitosan-Induced Stomatal Closure Accompanied by Peroxidase-Mediated Reactive Oxygen Species Production in Arabidopsis

Chitosan induced stomatal closure in wild type-plants and NADPH oxidase knock-out mutants (atrbohD atrbohF), and reactive oxygen species (ROS) production in wild-type guard cells. Closure and production were completely abolished by catalase and a peroxidase inhibitor. These results indicate that chitosan induces ROS production mediated by peroxidase, resulting in stomatal closure.