Origin of guanine nucleotides in isolated heart mitochondria.

Presence of guanine nucleotide within the matrix of mitochondria is uncontested; the mechanism by which GTP takes up residence in the matrix is unknown. In this report, we demonstrate for the first time that direct transport of guanine nucleotide across the inner membrane of heart mitochondria is possible. Transport of guanine nucleotides from the medium to the matrix was suggested by inhibition of translation in isolated rat heart mitochondria when GTP-gamma-S was added to the medium. This result suggested that GTP was one source of matrix GTP. Other sources were investigated by measuring matrix uptake and conversion to GTP of several purines, purine nucleosides, and purine nucleotides. Results demonstrated that [14C]-guanine and [3H]-guanosine were not taken up by isolated mitochondria and were not converted to any other compound. While [14C]-ATP and [3H]-AMP were taken up readily into the matrix, radioactivity was never associated with a guanine compound. [3H]-IMP was not taken up into the matrix and was never converted to another compound. Our data showed that label added as [3H]-GTP, [3H]-GDP, or [3H]-GMP was readily taken up and concentrated in the matrix of isolated mitochondria.

Guanine nucleotide transport by atractyloside-sensitive and -insensitive carriers in isolated heart mitochondria.

In previous work (McKee EE, Bentley AT, Smith RM Jr, and Ciaccio CE, Biochem Biophys Res Commun 257: 466-472, 1999), the transport of guanine nucleotides into the matrix of intact isolated heart mitochondria was demonstrated. In this study, the time course and mechanisms of guanine nucleotide transport are characterized. Two distinct mechanisms of transport were found to be capable of moving guanine nucleotides across the inner membrane. The first carrier was saturable, displayed temperature dependence, preferred GDP to GTP, and did not transport GMP or IMP. When incubated in the absence of exogenous ATP, this carrier had a V(max) of 946 +/- 53 pmol. mg(-1). min(-1) with a K(m) of 2.9 +/- 0.3 mM for GDP. However, transport of GTP and GDP on this carrier was completely inhibited by physiological concentrations of ATP, suggesting that this carrier was not involved with guanine nucleotide transport in vivo. Because transport on this carrier was also inhibited by atractyloside, this carrier was consistent with the well-characterized ATP/ADP translocase. The second mechanism of guanine nucleotide uptake was insensitive to atractyloside, displayed temperature dependence, and was capable of transporting GMP, GDP, and GTP at approximately equal rates but did not transport IMP, guanine, or guanosine. GTP transport via this mechanism was slow, with a V(max) of 48.7 +/- 1.4 pmol. mg(-1). min(-1) and a K(m) = 4.4 +/- 0.4 mM. However, because the requirement for guanine nucleotide transport is low in nondividing tissues such as the heart, this transport process is nevertheless sufficient to account for the matrix uptake of guanine nucleotides and may represent the physiological mechanism of transport.