Detection of a covalent intermediate in the mechanism of action of porcine pancreatic alpha-amylase by using 13C nuclear magnetic resonance

The catalytic mechanism of porcine pancreatic alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) has been examined by nuclear magnetic resonance (NMR) at subzero temperatures by using [1-13C]maltotetraose as substrate. Spectral summation and difference techniques revealed a broad resonance peak, whose chemical shift, relative signal intensity and time-course appearance corresponded to a beta-carboxyl-acetal ester covalent enzyme-glycosyl intermediate. This evidence supports a double-displacement covalent mechanism for porcine pancreatic alpha-amylase-catalyzed hydrolysis of glycosidic linkages, based on the presence of catalytic aspartic acid residues within the active site of this enzyme.     

Interference between dinitrophenyl and azobenzenearsonate determinants on in vitro binding to lymphoid cells. II. Type of binding cells and studies at the level of individual lymphoid cells.

The specific binding of DNP-T₄ on lymphoid cells occurs on the surface of B-cells. This was proved both by the absence of DNP-T₄ binding in cells pretreated with anti-total mouse Ig or anti-mouse IgM sera and by the absence of significant binding on thymocytes. Moreover, splenocytes of nu/nu mice bound similar amounts of DNP-T₄ as splenocytes of CBA/C₃H or BALB/c mice. Removal of adherent cells from normal spleen populations did not decrease the amounts of DNP-T₄ bound onto the non-adherent cell population. Azobenzenearsonate (ARS) conjugates partially inhibited the specific binding of DNP-T₄ to both splenocytes of nu/nu mice and spleen-cell suspensions depleted from adherent cells. The problem of whether the inhibition of specific DNP binding brought about by treatment of the cells with ARS derivatives was expressed by the reduction of the number of binding cells was investigated by two methods. In the first, the number of lytic plaques formed by DNP-T₄ around single lymphoid cells was counted with populations treated or not with ARS derivatives. In the second, anti-TNP producing MOPC-315 cells were used for rosette formation with TNP-conjugated sheep erythrocytes, in the absence or presence of ARS derivatives. Both these methods showed that this inhibition was due to partial reduction of the number of B-cells specifically binding either DNP or TNP determinant, thus indicating that only a certain percentage of the cells bearing the specific hapten receptors are affected by treatment with ARS derivatives.