Functional and structural properties of immobilized subunits of Escherichia coli alkaline phosphatase.

Alkaline phosphatase from Escherichia coli has been immobilized on Sepharose CL-4B using low levels of cyanogen bromide activation in order to favor the attachment of the dimer to the support through a single covalent linkage.The matrix-bound subunits obtained after dissociation of the immobilized dimer were shown to be catalytically inactive by three different criteria:1) The level of phosphatase activity in the matrix-bound subunit population ,was dependent on the extent of cyanogen bromide activation of the support.2) After cross-linking reversibly immobilized subunit preparations, sodium dodecyl sulfate gel electrophoresis of protein released from the Sepharose demonstrated the presence of sufficient amounts of dimeric structures to account for the phosphatase activity in these populations.3) The protein in the matrix-bound subunits was more susceptible to proteolytic digestion than that in the matrix-bound dimer, while the catalytic activity in both immobilized derivatives remained constant.These results indicate that the subunits of alkaline phosphatase are catalytically inactive, and that the low levels of phosphatase activity observed are due to the presence of trace amounts of contaminating dimers.Titration of matrix-bound '261-labeled subunits with soluble nascent '311-labeled subunits restored 60% of the original phosphatase activity.The isotope ratio of I3'Ito "%protein reached a final value of 1.06 and provided a very sensitive indication of the extent of dimerization throughout the titration.When the immobilized apodimer and apomonomer were titrated with ssZnz+, the matrix-bound monomer bound 0.9 eq of Zn2+ while the matrix-bound dimer bound 4.1 eq of Zn2+; the Zn2+ binding sites in both immobilized derivatives had dissociation constants of 2 X M or lower.The preparation and characterization of immobilized subunits of alkaline phosphatase under solvent conditions optimal for renaturation should provide a valuable approach for studying the role of subunit interactions in this enzyme.Moreover, the techniques used in this investigation are generally applicable for the characterization of other immobilized enzyme derivatives.Although many proteins form oligomeric structures, the importance of subunit interactions for enzymatic function, regulation, or conformational stability has not yet been established.The major reason for this difficulty is that the contributions of secondary and tertiary structures to the properties