Characterization and Cloning of a Nucleoside-diphosphate Kinase Targeted to Matrix of Mitochondria in Pigeon

David O Lambeth,James G Mehus,Mary A Ivey,Barry I Milavetz

doi:10.1074/jbc.272.39.24604

Abstract

Nucleoside-diphosphate kinase (NDP kinase) from the matrix space of mitochondria in pigeon liver was purified to homogeneity. Degenerate oligonucleotide primers to the N-terminal sequence of the purified protein and the region containing the active site histidine were used in reverse transcriptase-polymerase chain reaction to obtain a major portion of the coding sequence for the mature protein. The sequences of the C and N termini of the mature protein, and eight residues in the signal peptide, were obtained by rapid amplification of cDNA end procedures. The entire coding sequence of a cytosolic form of NDP kinase was also determined. Both isoforms, which share 53% sequence identity, possess the characteristically conserved regions of known NDP kinases. The mature mitochondrial NDP kinase protein migrates in molecular sieving columns with an apparent molecular mass of about 66 kDa. It shows very high thermal stability even though it lacks the proline residue in the killer of prune loop, and the Tyr/Glu C termini that are important in stabilizing other NDP kinases. The affinity of the mitochondrial isoform for adenine and guanine nucleotides is much higher than for pyrimidine nucleotides, but the enzyme is especially susceptible to substrate inhibition by GDP. Semi-quantitative reverse transcriptase-polymerase chain reaction showed that the relative levels of expression of the mitochondrial isoform are liver > kidney >> heart = brain > breast muscle. The cytosolic isoform is strongly and approximately equally expressed in these same five tissues. This work is the first characterization of a NDP kinase isoform that is found in the matrix space of mitochondria.

Highlights

Nucleoside diphosphate kinase (NDP1 kinase) catalyzes the transfer of a phosphoryl group from nucleoside triphosphates to nucleoside diphosphates
Purification of NDP Kinase Located in Mitochondrial Matrix—Most of the NDP kinase activity in pigeon liver mitochondria is located inside the inner membrane barrier [30]
We report the first characterization of an isoform of NDP kinase that is clearly targeted to the matrix space of mitochondria

Summary

EXPERIMENTAL PROCEDURES

Materials—Reagents used for enzyme assays and protein purification/characterization were of the highest purity available from Sigma. For each nucleoside diphosphate studied, a stock solution of NDP kinase was diluted with 65 mM Tris-HCl containing 1 mg/ml bovine serum albumin so that the maximum rate was less than 0.2 absorbance units/min. Assays contained 65 mM Tris-HCl, pH 7.5, 4.8 mM MgCl2, 10 mM KCl, 150 ␮M ATP, 0.15 mM NADH, 0.60 mM phosphoenolpyruvate, 0.2 IU of pyruvate kinase, and 0.4 IU of lactate dehydrogenase. Cloning cDNA for Cytosolic NDP Kinase—Since the cytosolic form of NDP kinase for pigeon was expected to be very homologous to nm23/ NDP kinase sequences of higher vertebrates, multiple sequence alignments were used to identify regions with strong identity These regions were used to design degenerate oligonucleotide primers for PCR. A 0.3-␮g aliquot of purified mRNA from pigeon liver was reverse transcribed with the mixture of active site primers to yield a cDNA pool

Purification ml Supernatant

RESULTS

DISCUSSION