Structure and Function of Chloroplast Proteins<subtitle>XIX. Dissociation of Spinach Leaf Ribulose-1, 5-diphosphate Carboxylase by Mercuribenzoate<xref ref-type="fn" rid="fn1"><sup>*</sup></xref></subtitle>

Abstract

The treatment of spinach ribulose-1,5-diphosphate carboxylase [EC 4.1.1.39] (AmBnn) with p-mercuribenzoate at pH 7.5 followed by continued incubation at pH 9.0 causes dissociation of the enzyme into eight oligomeric forms of the large subunit designated as Am, A−1, …, A−7 and two forms of the small subunit (B1 and B2), as discernible from band patterns on the polyacrylamide gel electrophoresis at pH 8.9. The separation of two types of subunits upon the -mercuribenzoate treatment was achieved with a Sephadex G-200 gel column equilibrated at pH 9.0. The molecular weights of the constituent monomeric subunits (A and B) were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 5.4 × 104 with A and 1.3 × 104 with B, respectively. It was found that upon removal of mercuribenzoate by the addition of β-mercaptoethanol the molecular species Am exhibits the carboxylase activity without the presence of the smaller subunit. The molecular weight of the catalytic oligomer Am estimated from polyacrylamide gel electrophoresis and Sephadex G-200 gel filtration yields value of 4.3 x 105 and 4.7×105, respectively. Parallel analysis of the smaller subunits gives the values of 1.1 ×l04 and 2.1 ×l04 with B1 and B2, respectively. There is a fairly good agreement within experimental error between these values and those obtained by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. From these results we conclude that the catalytic large subunit Am of spinach leaf ribulose-1,5-diphosphate carboxylase is probably an octamer A8.