|Effect of divalent cations on the porcine kidney cortex membrane-bound form of dipeptidyl peptidase IV. |
Isel Pascual,Hansel Gómez,Tirso Pons,Mae Chappé,Miguel Angel Vargas,Gilberto Valdés,Alí Lopéz,Angélika Saroyán,Jean-Louis Charli,María de los Angeles Chávez
The international journal of biochemistry & cell biology
Dipeptidyl peptidase IV is an ectopeptidase with multiple physiological roles including the degradation of incretins, and a target of therapies for type 2 diabetes mellitus. Divalent cations can inhibit its activity, but there has been little effort to understand how they act. The intact membrane-bound form of porcine kidney dipeptidyl peptidase IV was purified by a simple and fast procedure. The purified enzyme hydrolyzed Gly-Pro-p-nitroanilide with an average V(max) of 1.397±0.003 μmol min(-1) mL(-1), k(cat) of 145.0±1.2 s(-1), K(M) of 0.138±0.005 mM and k(cat)/K(M) of 1050 mM(-1) s(-1). The enzyme was inhibited by bacitracin, tosyl-L-lysine chloromethyl ketone, and by the dipeptidyl peptidase IV family inhibitor L-threo-Ile-thiazolidide (K(i) 70 nM). The enzyme was inhibited by the divalent ions Ca(2+), Co(2+), Cd(2+), Hg(2+) and Zn(2+), following kinetic mechanisms of mixed inhibition, with K(i) values of 2.04×10(-1), 2.28×10(-2), 4.21×10(-4), 8.00×10(-5) and 2.95×10(-5) M, respectively. According to bioinformatic tools, Ca(2+) ions preferentially bound to the β-propeller domain of the porcine enzyme, while Zn(2+) ions to the α-β hydrolase domain; the binding sites were strikingly conserved in the human enzyme and other homologues. The functional characterization indicates that porcine and human homologues have very similar functional properties. Knowledge about the mechanisms of action of divalent cations may facilitate the design of new inhibitors.
|Retention of specific yolk IgY in the human oral cavity. |
David Carlander, Hans Kollberg, Anders Larsson
BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy
INTRODUCTION: The increasing prevalence of antibiotic-resistant bacteria emphasises the need for new treatments that can replace traditional antibiotics. Oral immunotherapy with yolk antibodies from hyperimmunised hens is a new promising treatment strategy, primarily for infections in the mouth and gastrointestinal tract. Several studies show that bacterial and viral infections can be prevented with egg yolk immunoglobulin (IgY) in a dose-dependent manner. Oral treatment could potentially be used against many frequently encountered diseases (e.g. common cold, tonsillitis and caries). GROUP STUDIED: Healthy volunteers. STUDY DESIGN: We studied the presence of yolk anti-Pseudomonas aeruginosa antibodies in saliva from healthy volunteers over time after 1 or 2 minutes' mouth rinse, performed in the evening, with an aqueous IgY antibody preparation. The test persons rinsed the mouth with 8.0ml phosphate buffered saline before gargling with the antibody preparation 8 and 24 hours later. Statistical analysis was performed with the Mann-Whitney U test. METHODS: The antibody titres in the mouth rinses were tested for their specific activity against P. aeruginosa by ELISA. RESULTS AND CONCLUSION: The next morning there were still active antibodies detected in the saliva from 18 of 19 subjects. After 24 hours, active antibodies could be detected in saliva from only a few of the subjects. A 2-minute mouth rinse resulted in higher mean ELISA absorbance values than a 1-minute rinse.