Submitochondrial Location and Electron Transport Characteristics of Enzymes Involved in Proline Oxidation

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Abstract
Isolated corn mitochondria (Zea mays cv. B73 x Mo17) were fractionated and the fragments were separated on a 20-45% (wt/wt) 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 O2 uptake in isolated mitochondria. Proline dependent O2 uptake was relatively independent of pH with a maximum rate at pH 7.2. In contrast, .DELTA.1-pyrroline-5-carboxylic acid-dependent O2 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 .mu.M rotenone. Electrons from these substrates enter the respiratory chain prior to at least one of the rotenone sensitive Fe-S 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).