Structure and function of chloroplast proteins: II. Effect of p-chloromercuribenzoate treatment on the ribulose 1,5-diphosphate carboxylase activity of spinach leaf fraction I protein

Abstract

Fraction I protein was purified from spinach leaves by Sephadex gel filtration and DEAE-cellulose column chromatography. To study the role of SH-groups in the enzyme molecule, p-chloromercuribenzoate (PCMB) titration of SH-groups was carried out with a parallel determination of enzyme activity. It was found that the rate and extent of reaction with PCMB was identical in the presence or absence of urea (4.5 m), sodium dodecyl sulfate (2 × 10 −2 m), and ribulose 1,5-diphosphate (RuDP) (1.1 × 10 −4 m). The total number of SH-groups per mole protein was 96, in agreement with chemical data. Approximately 10 SH-groups were blocked before an appreciable loss of the RuDP-carboxylase activity occurred, and complete inhibition of enzyme activity was associated with the blocking of about 30 SH-groups. The possible role of SH-groups in the structural rigidity of the protein molecule was suggested by the finding that proteolytic digestibility (chymotrypsin and Nagarse) of the protein was greatly enhanced by PCMB pretreatment as measured by decrements of RuDP-carboxylase activity. Full restoration of the enzyme activity by the addition of cysteine to the PCMB-inactivated enzyme protein was accompanied by restoration of resistance to proteolytic attack. That the molecule is restored to a conformation possibly identical with that of the native protein in tertiary structure was supported by electron microscopic observations of the reconstituted protein.