Development of a coliforms monitoring system using an enzymatic fluorescence method.

A coliforms monitoring system in treated effluent of a wastewater treatment plant has been developed. In order to achieve rapid monitoring within 1 hour, an enzymatic fluorescence method without a culturing process was introduced to this system. It converts the increase rate of fluorescence intensity as enzymatic activity into the number of coliforms instead of converting fluorescence intensity itself. A flow injection analysis is used in this system for automatic measurement. Moreover, it is equipped with the pre-filtering unit to remove the interfering substances in the suspended solids causing deterioration in measurement precision. The good relationship (correlation coefficient of 0.90) between the obtained values using this system and the analysed values using the conventional direct counting method was observed in a test at an existing wastewater treatment plant.

Characterization of rat monoamine oxidase A with noncovalently-bound FAD expressed in yeast cells.

The FAD-binding cysteine of rat liver monoamine oxidase A (MAO A), Cys406, was converted to an alanine by site-directed mutagenesis of the cDNA. The wild-type and mutated enzymes were expressed in yeast cells and catalytic activities were assayed, using as substrates serotonin, tyramine, and kynuramine. Specific activities of the Ala-mutant for these substrates, calculated as the activities per pargyline-sensitive molecule, were about half of those of the wild-type enzyme. The Km values of the mutant enzyme for the substrates were similar to those of the wild-type enzyme. An adduct between FAD and pargyline, a mechanism-based inhibitor, was attached to the apoprotein in the wild-type enzyme, while in the Ala-mutant it was detached from the apoprotein, thereby indicating the presence of noncovalently bound FAD in the mutant enzyme. The Ala-mutant rapidly lost activity during incubation, whereas the wild-type enzyme retained the initial activity. Partial protection from inactivation occurred in the presence of FAD, but not of FMN. Recovery of the enzyme activity was nil when FAD was added after the inactivation. Thus, while the covalent attachment of FAD in MAO A is not required for the catalytic activity, it may function as a structural core for the active conformation in the membrane.

Regions of the molecule responsible for substrate specificity of monoamine oxidase A and B: a chimeric enzyme analysis.

To examine regions of the monoamine oxidase (MAO, EC 1.4.3.4) molecule responsible for substrate recognition, a series of these enzymes, in which discrete regions in one molecule were substituted by corresponding sequences of the other, were constructed from the cDNAs of rat liver MAO A and MAO B and were expressed in yeast, Saccharomyces cerevisiae. Substrate specificities of the original and chimeric enzymes were examined in terms of the maximum activity (Vmax) and the affinity (Km) for serotonin, beta-phenylethylamine (PEA), and benzylamine. Chimeric enzymes with the amino-terminal portion (about 220 residues) and the amino-terminal and middle portions (about 400 residues) of MAO A and MAO B, respectively, exhibited substantially the same Km values as those of the parent enzymes. Extension of the substitution in the middle portion of a chimeric enzyme to the second half of the amino-terminal portion resulted in conversion of the Km values for serotonin to those of the counterpart. Data on relative Vmax values of the chimeric enzymes for the three substrates revealed that the relative catalytic activities were mainly determined by the presence of the middle portion. We conclude from these observations that the region between about residues 120-220 and about residues 50-400 is responsible for determination of the substrate specificity of MAO A and MAO B, respectively, while the middle portion, of about residues 220-400, may relate to the relative catalytic activity towards substrates.

Rat monoamine oxidase B expressed in Escherichia coli has a covalently-bound FAD.

Rat liver monoamine oxidase B (MAO B) was expressed in E. coli as catalytically active form, though inclusion bodies of the enzyme were also formed as a major protein in the cell. The active form of the recombinant MAO B exhibited similar properties as rat liver enzyme and localized in membrane of the bacteria. Covalent attachment of FAD to polypeptide chain of the recombinant enzyme was revealed by a labeling experiment with [3H]-pargyline, an irreversible mechanism-based inhibitor, indicating that the covalent linkage of FAD to the apoprotein was formed even in the prokaryotic cell. This observation suggests autocatalytic formation of the linkage in MAO B.

Purification and complex formation analysis of a cysteine proteinase inhibitor (cystatin) from seeds of Wisteria floribunda.

Seeds of Wisteria floribunda contain several kinds of cysteine proteinase inhibitor (cystatin). We purified and characterized one of these inhibitors, named WCPI-3. The molecular weight of WCPI-3 was estimated to be 17,500 and 15,700 by gel filtration and SDS-PAGE, respectively. The isoelectric point was 5.7. WCPI-3 formed an equimolar complex with native papain and the dissociation constant was estimated to be 6.1 nM. Complex formation between WCPI-3 and Cys25-modified papain, such as S-carboxy-methylated or S-carbamoylmethylated papain, could not be observed by gel filtration or native PAGE analysis. A peptide fragment derived from WCPI-3 digested by Achromobacter proteinase (lysyl endopeptidase) had the amino acid sequence of VVAGVNYRFVLK. The VVAG sequence in this fragment corresponds to the conserved sequence QVVAG which is considered to be one of binding regions to cysteine proteinases. The amino acid sequence of the amino-terminal portion (34 residues) of WCPI-3 was highly homologous to that of oryzacystatin from rice seeds.