Effect of calcium on rat intestinal alkaline phosphatase activity and molecular aggregation

Abstract

Two fractions of rat intestinal alkaline phosphatase (IAP) were detected by Western blot: 168 +/- 6 and 475 +/- 45 kDa. The low molecular weight fraction constitutes 43% of the isolated proteins exhibiting 82% of the enzymatic activity, and a heavier fraction constitutes 57% of the isolated proteins and has 18% of the enzymatic activity. Calcium produced an increase of the 475-kDa form to the detriment of the 168-kDa form. This work also describes the kinetic and structural changes of IAP as a function of calcium concentration. With [Ca2+] < 10 mmole/L, the Ca(2+)-IAP interaction fitted a binding model with 7.8 +/- 4.4 moles of Ca2+ /mole of protein, affinity constant = 19.1 +/- 8.4 L/mmole, and enzymatic activity increased as a linear function of [Ca2+] (r = 0.946 p < 0.01). On the other hand, with [Ca2+] > 10 mmole/L the data did not fit this model and, the enzymatic activity decreased as a function of [Ca2+] (r = - 0.703 p < 0.05).

Aggregation and inhibition of rat intestinal alkaline phosphatase by high concentrations of calcium. Reversibility of the processes

“…The purification process [4] involved: lipid extraction with butanol, gel filtration, and chromatography on DEAE-cellulose with a NaCl gradient. The enzyme was recovered by precipitation with acetone at 2 208C, and dissolved in Tris-HCl buffer.…”

“…There is no strong evidence about the importance of calcium in the formation of the dimer, but that results indicate that the putative calcium binding site is important for alkaline phosphatase activity. We have demonstrated that calcium (Ca 2 þ ) favors the formation of a 475 kDa-aggregated form with scarce phosphatase activity [4].…”

“…When calcium concentrations are lower that 10 mmole/L the activity of IAP increases following a linear function of calcium concentration, and IAP binds calcium following a reversible binding model with approximately 8 equivalent and independent binding sites with a dissociation constant of 5.2 £ 10 25 mole/L [4]. When calcium is higher than 20 mmole/L the activity decreases as a linear function of calcium concentration in the solution.…”

“…As the aggregated form persists after elution in a column of gel filtration and PAGE, where the concentration of calcium is not higher than 20 mmole/L, it is suspected that the binding of calcium is stable or the effect of calcium persist even in the absence of calcium in the solution. PAGE of IAP that was previously exposed to 45 Ca 2 þ , demonstrated that, after electrophoresis, calcium is still bound to the bands containing the aggregated form [4].…”

“…In this case IAP binds calcium without following the model of independent and equivalent sites described above. The process of inhibition is simultaneous to molecular aggregation [4].…”