Structure and Function of Chloroplast Proteins*

Abstract

Fraction-I protein, isolated from wheat leaves, was purified by Sephadex gel filtration and DEAE cellulose column chromatography. Its component subunits were studied after cleavage with SDS.^** Parallel to the disruption of the protein into subunits, as demonstrated by polyacrylamide gel electrophoresis, a proportionate decline in ribulosedi-phosphate carboxylase [EC 4.1.1.39] activities was observed. The structural and functional role of SH-groups in the protein molecule was studied by the reaction with PGMB. Mercaptide formation was paralleled by loss of carboxylase activity. Preincubation of the enzyme with RuDP prevented the inhibitory action of PGMB, indicating possible participation of the SH-group in the active site. Extent of mercaptide formation as determined by spectrophotometry was the same for enzyme with and without RuDP preincubation. Total SH-groups per protein molecule (M.W. 5.15×1O⁵) were 96. Thus only a minor fraction of the SH-groups in the protein may take part in the enzymatic reaction. Polyacrylamide gel electrophoresis provides evidence for conformational changes of the protein subunit structure by PCMB treatment. The protein molecule appears to cleave into large and small fragments, accompanied by a concomitant loss of the enzyme activity. Addition of cysteine caused the restoration of enzyme activity and reformation of a protein band indistinguishable from that of original fraction-I protein.