The influence of Al3+ ion on porcine pepsin activity in vitro.
The in vitro effect of Al(3+) ions in the concentration range 1.7 x 10(-6) M-8.7 x 10(-3) M on pepsin activity at pH 2, via kinetic parameters and its electrophoretic mobility was evaluated. Kinetic study demonstrated the existence of an activation effect of Al(3+) at pH 2 on pepsin molecule. Kinetic analysis with respect to concentrations of haemoglobin showed that Al(3+) ions increase the maximal velocity (V(max)) and k(cat) values rather than apparent affinity for substrate (K(S)) implying the non-competitive nature of activation which indicated that aluminium was a non-essential activator of partial non-competitive type. The values of the equilibrium constants K(S) and K(mA) for dissociation of corresponding complexes were evaluated as 0.904 +/- 0.083 mM and 8.56 +/- 0.51 microM, respectively. Dissociation constant K(A), of activator from enzyme-activator complex calculated via kinetic and direct measurement of Al(3+) binding data, as well as activation constant A(50), the activator concentration that gives a rate equal to half at a saturating concentration of activator, were found to be 8.82 +/- 0.90 microM, 8.39 +/- 0.76 microM, and 8.05 +/- 0.48 microM respectively. Native PAGE electrophoresis shows the decrease in electrophoretic mobility of pepsin and confirms modification of the electric charge and conformational changes of pepsin caused by bound Al(3+) on the pepsin molecule. Al(3+) induced conformational changes of pepsin were verified by UV-VIS and IR spectra. Moreover, the absence of conformational changes in the haemoglobin molecule in the presence of Al(3+) ions confirms that the obtained activation is a consequence of conformational changes caused only in the pepsin molecule.
Pavelkic
Vesna M
VM
Department of Physical Chemistry, Vinca-Institute of Nuclear Science, Belgrade, Serbia. vesnap@vin.bg.ac.yu
Gopcevic
Kristina R
KR
Krstic
Danijela Z
DZ
Ilic
Marija A
MA
D000535:Aluminum / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D002412:Cations / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D010434:Pepsin A / Q000737:chemistry / Q000378:metabolism*; D013053:Spectrophotometry; D013552:Swine
D002412:Cations; D006454:Hemoglobins; D000535:Aluminum; D010434:Pepsin A