Mechanism of the Increase in Cytochrome c Oxidase Activity in Pea Cotyledons during Seed Hydration

Abstract

The increase in cytochrome c oxidase activity in pea cotyledons during seed hydration was investigated with 2 kinds of antibodies against the enzymes purified from mitochondria of pea shoot and sweet potato root. The antibody against the pea enzyme recognized only the largest of the enzyme subunits (subunit I), while that against the sweet potato one recognized 3 other pea enzyme subunits: the 2nd largest (subunit II) and the 2 smallest (subunits IV and V). There was no increase in the amount of protein immunoprecipitated with the anti-(pea enzyme) antibody in a crude fraction of cotyledon membranes during seed hydration, despite the marked increase in cytochrome c oxidase activity. Neither cycloheximide nor chloramphenicol had any effect on the activity or the amount. The crude membrane fraction from dry cotyledons contained an enzymatically inert protein precipitated with the anti-(pea enzyme)antibody. The inert protein could be separated from the active enzyme protein through solubilzation of the protein from the membranes with various concentrations of Triton X-100. It consisted of only 1 kind of polypeptide with the same MW as subunit I and decreased with the concomitant increase in the active enzyme protein during seed hydration. Dry pea cotyledons also contained a soluble protein that was immunoprecipitated by antibody to the sweet potato enzyme. The soluble form of the immunoreactive protein decreased with the concomitant increase in active cytochrome c oxidase protein in the crude membrane fraction from the cotyledons during seed hydration. None of these changes during seed hydration were inhibited by cycloheximide or chloramphenicol. The soluble, immunoreactive protein contained only 1 kind of polypeptide, of which the MW was identical to that of subunit IV. The mitochondrial inner membranes of dry cotyledons were separated into 3 fractions by sucrose density gradient centrifugation. The lightest was the richest of the three in the enzymatically inert protein immunoprecipitated with the anti-(pea enzyme)antibody; cytochrome c oxidase activity in this fraction increased immediately after the onset of seed hydration. A membrane-bound, free form of subunit I and a soluble, free form of subunit IV of cytochrome c oxidase apparently exist in dry pea cotyledons. These free forms of subunits are probably assembled with the other subunits to form the active cytochrome c oxidase during seed hydration.