Mitochondrial arginine kinase in the midgut of the tobacco hornworm (Manduca sexta)

Abstract

Mitochondria isolated from the posterior midgut of the tobacco hornworm (Manduca sexta) contain arginine kinase. The distribution of mitochondrial and cytoplasmic marker enzymes indicates that the presence of mitochondrial arginine kinase is not due to cytoplasmic contamination. Arginine is not oxidized by the midgut mitochondria but, when metabolic substrates and ATP are present, respiration can be initiated by the addition of arginine. Under these conditions, there is no return to State 4 respiration, indicating regeneration of ADP by the arginine kinase reaction. Respiration can be blocked, however, by atractyloside, an inhibitor of the adenine nucleotide translocator. These results indicate that arginine kinase resides outside the matrix. Mitochondrial arginine kinase is specific to l-arginine since analogs of l-arginine are ineffective in stimulating respiration in the presence of ATP. Coupling between the adenine nucleotide translocator and arginine kinase was investigated using kinetic and thermodynamic approaches. There were no differences in the activities of arginine kinase in respiring and non-respiring mitochondria when they were measured at different ATP or arginine concentrations. This result indicates that arginine kinase does not have preferential access to the ATP exported out of the matix. A comparison of the apparent equilibrium constant and the mass action ratio of the arginine kinase reaction also confirms that there is no microcompartmentation of the reaction.