The Journal of Experimental Biology 200, 2789–2796 (1997) 2789 Printed in Great Britain © The Company of Biologists Limited 1997 JEB1062 MITOCHONDRIAL ARGININE KINASE IN THE MIDGUT OF THE TOBACCO HORNWORM (MANDUCA SEXTA) M. E. CHAMBERLIN* Department of Biological Sciences, Ohio University, Athens, OH 45701, USA Accepted 21 August 1997 Summary Mitochondria isolated from the posterior midgut of the arginine are ineffective in stimulating respiration in the tobacco hornworm (Manduca sexta) contain arginine presence of ATP. Coupling between the adenine nucleotide kinase. The distribution of mitochondrial and cytoplasmic translocator and arginine kinase was investigated using marker enzymes indicates that the presence of kinetic and thermodynamic approaches. There were no mitochondrial arginine kinase is not due to cytoplasmic differences in the activities of arginine kinase in respiring contamination. Arginine is not oxidized by the midgut and non-respiring mitochondria when they were measured mitochondria but, when metabolic substrates and ATP are at different ATP or arginine concentrations. This result present, respiration can be initiated by the addition of indicates that arginine kinase does not have preferential arginine. Under these conditions, there is no return to State access to the ATP exported out of the matix. A comparison 4 respiration, indicating regeneration of ADP by the of the apparent equilibrium constant and the mass action arginine kinase reaction. Respiration can be blocked, ratio of the arginine kinase reaction also confirms that however, by atractyloside, an inhibitor of the adenine there is no microcompartmentation of the reaction. nucleotide translocator. These results indicate that arginine kinase resides outside the matrix. Mitochondrial Key words: Manduca sexta, midgut, tobacco hornworm, arginine arginine kinase is specific to L-arginine since analogs of L- kinase, mitochondria, epithelia. Introduction Phosphagen kinases catalyze the reversible phosphorylation and ADP, are shuttled between the site of ATP synthesis and of guanidine compounds, and several different phosphagen its site of use. It has also been suggested that the presence of kinases have been identified in animals (Morrison, 1973). Of a mitochondrial creatine kinase may increase the efficiency of these phosphagen kinases, creatine kinase (CK; creatine + ATP oxidative phosphorylation by maintaining a low concentration ↔ creatine phosphate + ADP) from mammals is the best of ATP in the vicinity of ATP synthesis. understood. Several roles in cellular energetics have been Arginine kinase (AK) is the phosphagen kinase found in attributed to the CK system, three of which are (1) as a insects. Schneider et al. (1989) demonstrated that AK and temporal energy buffer, (2) as a spatial energy buffer, and (3) arginine phosphate served as a temporal energy buffer system to increase the efficiency of oxidative phosphorylation in locust femoral muscle. At rest, the arginine phosphate (reviewed by Wallimann et al. 1992). As a temporal energy concentration in femoral muscle is four times higher than that buffer, the CK system stabilizes ATP levels at the expense of of ATP. When the locust jumps, ATP levels are stabilized at creatine phosphate under conditions when ATP use exceeds the the expense of arginine phosphate. In contrast, the resting ability of the mitochondria to produce ATP. Spatial energy levels of ATP and arginine phosphate are nearly identical in buffering has been proposed for cells such as cardiac myocytes the highly aerobic flight muscle. Upon initiation of flight, ATP or spermatazoa, where there are cytosolic and mitochondrial levels fall and there is no change in arginine phosphate forms of the enzyme. Through the action of mitochondrial concentration, indicating that flight muscle AK plays little or creatine kinase, ATP produced at the mitochondria is no role in temporal energy buffering. Flight muscle, however, converted to creatine phosphate, which diffuses to a cytosolic is not the only aerobic insect tissue that contains AK. High site of ATP utilization (e.g. myofibrillar ATPase). There, the activities of this enzyme are found in ion-transporting epithelia cytosolic creatine kinase converts the creatine phosphate to such as locust hindgut (Chamberlin and Phillips, 1983) and ATP, and the creatine diffuses back to the mitochondria. lepidopteran midgut (Chamberlin, 1987; Gindling et al. 1995). Therefore, creatine and creatine phosphate, rather than ATP The present study explores the possibility that the AK system *e-mail: [email protected] 2790 M. E. CHAMBERLIN in the midgut of the tobacco hornworm (Manduca sexta) might Metabolite analysis serve as a temporal energy buffer. This function is most Metabolite levels were measured in the midgut after it had effective if the arginine phosphate levels exceed ATP levels at been quickly dissected from the animal, rinsed and weighed in rest. To determine whether the AK system could be involved a cold saline identical to that described by Chamberlin (1994) in an ATP buffering system in the midgut, ATP, arginine and except that it lacked arginine. The tissue was then frozen in arginine phosphate levels were measured in this tissue. liquid nitrogen and homogenized in 5 vols of 6 % perchloric If, like CK in vertebrates, AK is to play a role in increasing acid. The homogenate was centrifuged at 12 000 g for 1 min, the efficiency of oxidative phosphorylation, it must be present and the supernatant removed and neutralized with 3 mol l−1 in the mitochondria. Focusing on insect flight muscle, a tissue K2CO3. The neutralized extract was then frozen in liquid with a high mitochondrial density and high AK activity, nitrogen and stored at −80 °C until it was analyzed for arginine, several researchers (Ellington and Hines, 1991; Schneider et arginine phosphate and ATP levels as described below. al. 1989; Wyss et al. 1995) have failed to detect a Preliminary studies revealed that this extraction procedure did mitochondrial form of AK. Like the insect flight muscle, the not lead to acid hydrolysis of arginine phosphate. tobacco hornworm midgut also has a high mitochondrial Arginine, arginine phosphate, ATP and ADP levels were content, and the present study demonstrates the presence of AK measured spectrophotometrically in a Gilford Response in mitochondria isolated from this tissue. spectrophotometer. Arginine and arginine phosphate were If mitochondrial AK from the midgut acts in a manner measured according to a modified method of Gäde (1985). The analogous to that of CK in mammalian mitochondria, then assay medium contained the tissue or mitochondrial extract, there should be a functional coupling between AK and the 5 mmol l−1 ADP, 0.4 mmol l−1 NADH, 2.5 mmol l−1 pyruvate −1 −1 adenine nucleotide translocase (ANT), the transporter that and 5 mmol l MgCl2 in 100 mmol l imidazole, pH 7.2. The exchanges ATP and ADP across the inner mitochondrial arginine concentration was determined by measuring the membrane. A functional coupling between CK and ANT has change in NADH concentration upon the addition of 1 unit ml−1 been demonstrated in mammalian heart mitochondria using octopine dehydrogenase (1 unit=1 µmol product min−1). Once both kinetic and thermodynamic approaches. For example, the the octopine dehydrogenase reaction had reached equilibrium, Km for ATP of the creatine kinase reaction is over eight times AK (5 units ml−1) was added to determine the arginine lower in respiring mitochondria than in rotenone- and phosphate concentration in the extract. oligomycin-poisoned mitochondria because the CK reaction ATP was determined by monitoring the increase in NADPH has preferred access to the ATP transported out of the when hexokinase (2 units ml−1) was added to the assay medium mitochondria (Wyss et al. 1992). A comparison of the mass containing tissue or mitochondrial extract, 0.4 mmol l−1 NADP, −1 −1 action ratio ([ATP][creatine]/[ADP][creatine phosphate]) with 5 mmol l MgCl2, 50 mmol l glucose and excess glucose-6- ′ the apparent equilibrium constant (Keq) of the CK reaction in phosphate dehydrogenase (1 unit ml−1) in 100 mmol l−1 heart mitochondria (DeFuria et al. 1980) or mitoplasts (a imidazole, pH 7.2. preparation consisting of inner membrane and matrix only; ADP was determined by monitoring the change in NADH Saks et al. 1985) revealed that creatine phosphate was absorbance when pyruvate kinase (2 units ml−1) was added to ′ synthesized even when the mass action ratio was less than Keq. the assay medium containing mitochondrial extract, −1 −1 −1 This apparent violation of thermodynamics has been 100 mmol l KCl, 10 mmol l MgCl2, 2 mmol l interpreted to mean that the CK reaction is not occurring in the phosphoenolpyruvate, 0.3 mmol l−1 NADH and lactate bulk solution but in a microenvironment near ANT (Saks et al. dehydrogenase (2.5 units ml−1) in 100 mmol l−1 imidazole, 1985). In contrast to the CK system, Doumen and Ellington pH 7.2. (1990b) failed to show any coupling between AK and ANT in mitochondria isolated from the horseshoe crab (Limulus Isolation of the mitochondria polyphemus) heart. In the present study, kinetic and Mitochondria were isolated using the method described in thermodynamic studies were undertaken to determine whether Gibellato and Chamberlin (1994). Briefly, this entails gently there is compartmentation of the AK reaction in mitochondria homogenizing the posterior midguts in isolation medium and isolated from the tobacco hornworm midgut. separating the mitochondria by differential centrifugation. To assess the cytoplasmic contamination of the mitochondrial preparation, the activities of AK, citrate synthase Materials and methods (mitochondrial enzyme) and phosphoglucoisomerase Insects (cytoplasmic enzyme) were measured in the homogenates and Manduca sexta (L.) larvae were raised from eggs provided isolated mitochondria. Prior to the measurement of these by the United States Department of Agriculture (USDA) or a enzyme activities, the initial tissue homogenate used to prepare colony derived from the USDA stock and maintained in the mitochondria as well as the final mitochondrial suspension Biological Sciences Department at Ohio University.