Involvement of metals in enzymatic and nonenzymatic decomposition of C-terminal alpha-hydroxyglycine to amide: an implication for the catalytic role of enzyme-bound zinc in the peptidylamidoglycolate lyase reaction.

The peptide C-terminal amide group essential for the full biological activity of many peptide hormones is produced by consecutive actions of peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidylamidoglycolate lyase (PAL); PHM catalyzes the hydroxylation of C-terminal glycine, and PAL decomposes the peptidyl-alpha-hydroxyglycine to an amidated peptide and glyoxylate. PAL contains 1 mol of zinc, but its role, catalytic or structural, has not yet been clarified. In this study, we found that a series of transition metals, Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), and Cd(2+), catalyze the nonenzymatic decomposition of the hydroxyglycine intermediate in a concentration-dependent manner. The second-order rate constant of the metal catalysis increased with elevation of pH, indicating that the hydrated metal acts as a general base. Extensive removal of the enzyme-bound metals remarkably diminished the PAL activity; k(cat) of the metal-depleted enzyme retaining 0.1 mol of zinc decreased to 3.2 s(-1) from 25.7 s(-1) of the wild-type enzyme. Among a series of divalent metals tested, Zn(2+), Co(2+), and Cd(2+) could fully restore the PAL activity of the metal-depleted enzyme. Especially, Zn substitution reproduced the steady-state parameters of the wild-type enzyme. On the other hand, Co and Cd substitution largely altered the kinetic parameters; the k(cat) increased 3- and 5-fold and the K(m) for the substrate increased 2.5- and 4-fold, respectively. These observations support that the enzyme-bound zinc plays a catalytic role, rather than a structural role, in the PAL reaction through the action of zinc-bound water as a general base.

Heavy-metal distribution in river waters and sediments around a "firefly village", Shikoku, Japan: application of multivariate analysis.

River water and sediment samples were collected at the same site in a vicinity of an abandoned mine, and the concentrations of major elements and heavy metals were determined. The chemical correlations were observed by principal component analysis (PCA), and the samples were classified by cluster analysis (CA) based on the PCA scores. The PCA results presented a macroscopic viewpoint of covariance structure, i.e., the chemical elements could be classified into three groups: 1) major elements and heavy metals in the river water, 2) Cd, Fe and Mn in the sediments, and 3) Cu and Zn in the sediments. The CA results implied a similarity of chemical compositions in most parts of the study area, except the ranges close to the abandoned copper mine. At the mixing location of mining water with natural river water, major elements and cadmium showed simple physical mixing (conservative mixing). Other heavy metals (Cu, Fe, Mn and Zn) showed the massive precipitation at the mixing event. The PCA structure was mainly interpreted in terms of the mixing process between mining water and diluted natural river water.

Blood lactoferrin release induced by running exercise in normal volunteers: antibacterial activity.

BACKGROUND: The aim of this study was to determine serum lactoferrin concentrations and serum antibacterial activity before and after running exercise. METHODS: Twenty-four healthy young men were randomly assigned to high, middle, or low intensity of exercise groups (5000 steps running at 180, 130, and 80 steps/min, respectively). Blood samples were collected at baseline and immediately, 1 and 4 h after exercise. Concentrations of circulating neutrophils, serum lactoferrin, iron in whole blood, and serum iron were measured. Antibacterial activity of serum was evaluated using live Micrococcus luteus. RESULTS: The numbers of circulating neutrophils were increased by 20.0% and 15.5% 1 h after exercise in high and middle groups (both P<0.01), respectively. Serum lactoferrin concentrations were significantly increased immediately after exercise by 48.3% and 33.0% in the high and middle groups (both P<0.01), respectively. No significant changes in total iron or serum iron concentrations were observed during the study. Antibacterial activities of serum collected immediately after exercise in the high and middle groups were significantly stronger than those before exercise, by 31.2% and 25.4% (both P<0.05), respectively. CONCLUSIONS: Serum lactoferrin concentrations are increased immediately after running exercise and may play an antibacterial role in host defenses before mobilization of neutrophils into the circulating pool.

Expression and characterization of human bifunctional peptidylglycine alpha-amidating monooxygenase.

We report the purification and characterization of human bifunctional peptidylglycine alpha-amidating monooxygenase (the bifunctional PAM) expressed in Chinese hamster ovary cells. PAM is in charge of the formation of the C-terminal amides of biologically active peptides. The bifunctional PAM possesses two catalytic domains in a single polypeptide, peptidylglycine alpha-hydroxylating monooxygenase (PHM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PAL, EC 4.3.2.5). By introducing a stop codon at 835 Glu, we were able to eliminate the membrane-spanning domain in the C-terminal region and succeeded in purifying a soluble form of bifunctional PAM that was secreted into the medium. Through a three-step purification procedure, we obtained 0.3mg of the purified PAM, which showed a single band at 91 kDa on SDS-PAGE, from 1L of monolayer culture medium. Metals contained in the purified PAM were analyzed and chemical modifications were performed to gain insight into the mechanism of the PAL reaction. Inductively coupled plasma detected 0.62 mol of Zn(2+) and 1.25 mol of Cu(2+) per mol of bifunctional PAM. Further, the addition of 1mM EDTA reduced the PAL activity by about 50%, but the decreased activity was recovered by the addition of an excess amount of Zn(2+). In a series of chemical modifications, phenylglyoxal almost completely eliminated the PAL activity and diethyl pyrocarbonate suppressed activity by more than 70%. These findings implied that Arg and His residues might play crucial roles during catalysis.