Assimilatory sulfate reduction in chloroplasts: Evidence for the participation of both stromal and membrane-bound enzymes

Abstract

The reduction of sulfate in osmotically shocked chloroplasts was investigated. Like intact (i.e., Class a) chloroplasts, this type carries out the complete sulfate reducing process. The conditions for formation of the intermediate bound sulfite and bound sulfide were examined. The amount of bound sulfide and bound sulfide formed from sulfate was proportional to the amount of chlorophyll. The Km of the overall reaction from sulfate to bound sulfite was 1.53 mmoles (at 500 μg chlorophyll). ATP was saturated at 5 mM over a concentration of 0.25 to 0.5 mg chlorophyll. At higher concentrations of chlorophyll, no saturation by ATP was observed. Cysteine formation showed an optimum concentration for O-acetyl serine at 4 mM. After separation of the osmotically ruptured chloroplasts into a soluble “chloroplast extract” and a paniculate “chloroplast thylakoid fraction,” bound sulfite was formed from sulfate by the thylakoid fraction but not by the chloroplast extract. From the chloroplast extract, a protein of low molecular weight was purified. It increased the amount of bound sulfite formed when a NADPH₂-regenerating system was present.