Purification and Characterization of the Reconstitutively Active Adenine Nucleotide Carrier from Maize Mitochondria

Abstract

The adenine nucleotide carrier from maize (Zea mays L. cv B 73) shoot mitochondria was solubilized with Triton X-100 and purified by sequential chromatography on hydroxyapatite and Matrex Cel Blue B in the presence of cardiolipin and asolectin. Sodium dodecyl sulfate-gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular m a s of 32 kD. When reconstituted i n liposomes, the adenine nucleotide carrier catalyzed a pyridoxal 5'-phosphate-sensitive ATP/ATP exchange. It was purified 168-fold with a recovery of 60% and a protein yield of 0.25% with respect to the mitochondrial extract. Among the various substrates and inhibitors tested, the reconstituted protein transported only ADP, ATP, CDP, and CTP, and was inhibited by atractyloside, bongkrekate, phenylisothiocianate, pyridoxal 5'-phosphate, and mersalyl (but not N-ethylmaleimide). Maximum initial velocity of the reconstituted ATP/ATP exchange was determined to be 2.2 pmol min-' mg-' protein at 25°C. The half-saturation constants and the corresponding inhibition constants were 17 PM for ATP, 26 p~ for ADP, 59 p~ for CTP, and 125 p~ for CDP. The activation energy of the ATP/ATP exchange was 48 kilojoule/mol between O and 15"C, and 22 kilojoule/mol between 15 and 35°C. Partia1 amino acid sequences showed that the purified protein was the product of the ANT-C1 gene sequenced previously (8. Bathgate, A. Baker, C.J. Leaver [1989] Eur J Biochem 183: 303-310).The inner membrane of plant mitochondria contains at least 10 specific carrier systems for the transport of metabolites (Day

Plant mitochondrial carriers: an overview

“…All together, these studies have demonstrated that ATP/ADP transport in plant mitochondria involves an ANT similar to the mammalian one, being inhibited by BGK, CATR and ATR, although the inhibitory effect of the latter was less pronounced than in mammalian mitochondria. The ANT has been successfully purified from maize shoot mitochondria using hydroxylapatite chromatography followed by Matrex Gel Blue B chromatography [53] (table 1). This procedure led to the isolation of a single polypeptide with an apparent molecular mass of 32 kDa.…”

Characterization of the ATP-translocating properties of the predictedArabidopsis thalianamitochondrial adenine nucleotide translocator 2

“…For example, the ANT from bovine mitochondria binds (but does not transport) GDP and CTP (Brustovetsky and Klingenberg 1994). By contrast, the reconstituted ANT from maize mitochondria are capable of transporting GDP and GTP, although not as effectively as ATP (Genchi et al 1996). These nucleotides were identified as competitive inhibitors for the reconstituted maize ANT.…”

“…Specifically the value of 15 mmolÁL -1 is within the range of K m values previously documented for both native mitochondrial and plastidic ANT (3-30 mmolÁL -1 ), as well as other recombinant reconstituted ANT systems (10-70 mmolÁL -1 )( K r ämer and Klingenberg 1982;Bisaccia et al 1985;Knirsch et al 1989;Schünemann et al 1993;Genchi et al 1996;Tjaden et al 1998;Millar and Heazlewood 2003;Leroch et al 2008). However, it is important to note that the K m and V max values for ANT2 are reported here only as approximations of the actual kinetic parameter.…”

“…For example, the ANT from pea root plastids also displayed low affinity for UTP, UDP, and AMP (Schünemann et al 1993;Genchi et al 1996). In fact, in general, it has been shown that even though ANT substrate recognition is highly specific for ATP and ADP, some variations in transport selectivity do occur (Goto et al 2002).…”

“…To date, one endoplasmic reticulum (ER-ANT1), two plastidic (At1g80300 and At1g15500), and at least three mitochondrial [At5g13490 (ANT2), At3g8580 (ANT1), and At4g01100 (ADNT1)] ANTs have been identified (Neuhaus et al 1997;Klingenberg 2008;Palmieri et al 2008). While the plastidic ANTs from A. thaliana have been extensively studied, their structures are not homologous to, and their biochemical function differs from, the mitochondrial ANTs (Knirsch et al 1989;Schünemann et al 1993;Brustovetsky and Klingenberg 1994;Mozo et al 1995;Genchi et al 1996;Fiore et al 1998;Möhlmann et al 1998;Tjaden et al 1998;Trentmann et al 2000;Leroch et al 2008). ER-ANT1 is unique in its localization and has also been fairly well characterized (Leroch et al 2008).…”