Submitochondrial location and electron transport characteristics of enzymes involved in proline oxidation

Isolated corn mitochondria (Zea mays cv. B73 x Mo17) were fractionated and the fragments were separated on a 20-45% (weight/weight) continuous sucrose gradient. Soluble enzymes remained at the top of the gradient overlapping with the outer membranes, while inner membrane vesicles and intact inner membranes were distributed farther down the gradient. Proline oxidase and Delta(1)-pyrroline-5-carboxylic acid dehydrogenase activities were associated only with the inner mitochondrial membrane. Glutamate dehydrogenase was confirmed as a matrix enzyme.Both proline and Delta(1)-pyrroline-5-carboxylic acid supported oxygen uptake in isolated mitochondria. Proline dependent oxygen uptake was relatively independent of pH with a maximum rate at pH 7.2. In contrast, Delta(1)-pyrroline-5-carboxylic acid-dependent oxygen uptake was sensitive to pH with an optimum at pH 6.1. The oxidation of proline and Delta(1)-pyrroline-5-carboxylic acid was inhibited by 10 micromolar rotenone. This indicates that electrons from these substrates enter the respiratory chain prior to at least one of the rotenone sensitive iron-sulfur proteins. Both substrates yielded ADP:O ratios of around 1.9 as compared to malate plus pyruvate (2.1), succinate (1.3), and exogenous NADH (1.2).