Elucidation of the quaternary structure of reversibly immobilized alkaline phosphatase derivatives

Abstract

Escherichia coli alkaline phosphatase has been reversibly immobilized on Sepharose CL-4B through two different methods, both based on a disulfide linkage, under conditions selected to favour the coupling of the enzyme to the solid support through one covalent linkage. The quaternary structure of the reversibly immobilized subunit, produced by dissociation of the matrix-bound dimer, was examined by cross-linking with the bifunctional reagent dimethyl suberimidate. Following release from the solid support, the protein was analysed by sodium dodecyl sulfate gel electrophoresis demonstrating the presence of a sufficient amount of dimeric structures in the immobilized subunit preparation to account for all the enzyme activity observed in this sample. These results suggest that the subunit of alkaline phosphatase may be catalytically inactive. This approach to studying the quaternary structure of immobilized subunit derivatives offers the opportunity to directly determine the homogeneity and structure of matrix-bound 'monomer' preparations and is particularly useful in determining if low levels of catalytic activity observed in some immobilized subunit populations are due to the presence of contaminating oligomeric structures.

Characterization of a Monomeric Escherichia coli Alkaline Phosphatase Formed upon a Single Amino Acid Substitution

“…Monomers of the wild-type enzyme have been obtained through a variety of techniques such as thiol reduction in the presence of urea (11), acid treatment (12), metal chelation, followed by incubation in 5% formamide (9), or by isolation of matrix bound monomers (13). However, the characterization of these monomeric forms of E. coli AP have been conducted under very specialized conditions.…”

Alkaline Phosphatase, Solution Structure, and Mechanism

“…Many of the anomalies may arise from poorly defined systems as far as metal and endogenous phosphate content are concerned. A recent study on immobilized monomeric and dimeric forms of alkaline phosphatase reversibly bound to Sephadex (138) showed that whereas the former binds only one Zn(I1) ion per subunit, the latter binds two under the same conditions, that is, four per dimer. This clearly indicates an interaction between subunits at the most basic level; namely, the influence of a large surface area of contact between monomers on the conformation and consequent properties of the individual subunits.…”

Subunit interactions in tyrosinase from frog epidermis in immobilized enzyme systems

“…One approach to understanding the function of quaternary structures in enzymes is to study the properties of single subunits and to compare them with those of the native oligomer. Chan (1970) introduced the use of immobilization techniques in order to study possible activity of the isolated subunits under conditions in which reassociation was prevented, and subsequent reports demonstrated the existence of active subunits bound to a solid matrix (Chan & Mawer, 1972;Grazi et al, 1973;Green & Toms, 1973;Chan, , 1974Fell & White, 1975;Bickerstaff & Price, 1976;Bruch et al, 1976;Chan & Mosbach, 1976;Carvajal et al, 1977;Gurne et al, 1977;Batlle et al, 1978;Chantler & Gratzer, 1978;Kochetov & Solovieva, 1978;Andersson & Mosbach, 1979;Arrio-Dupont & Coulet, 1979;McCracken & Meighen, 1979). The use of immobilized enzyme as a tool for evaluating subunit interactions has been reviewed (Chan, 1976a,b;Bickerstaff, 1980).…”