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Published on: January 7, 2013
Characterization and cloning of a nucleoside-diphosphate kinase targeted to matrix of mitochondria in pigeon
D O Lambeth1, J G Mehus, M A Ivey
1Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58202, USA. dlambeth@mail.med.und.nodak.edu
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.
Insights
Mitochondrial nucleoside-diphosphate kinase (NDP kinase) was purified and characterized, revealing unique stability and nucleotide binding properties. This study provides the first characterization of this crucial mitochondrial enzyme.
Area of Science:
- Biochemistry
- Molecular Biology
- Mitochondrial Biology
Background:
- Nucleoside-diphosphate kinase (NDP kinase) enzymes play vital roles in cellular metabolism.
- Mitochondria, the powerhouses of the cell, have unique enzymatic requirements.
- Previous NDP kinase research has primarily focused on cytosolic isoforms.
Purpose of the Study:
- To purify and characterize the NDP kinase isoform located in the mitochondrial matrix space of pigeon liver.
- To determine the coding sequence and structural features of the mitochondrial NDP kinase.
- To investigate the kinetic properties, stability, and tissue expression patterns of this mitochondrial isoform.
Main Methods:
- Protein purification to homogeneity.
- Reverse transcriptase-polymerase chain reaction (RT-PCR) for gene sequencing.
- Rapid amplification of cDNA ends (RACE) for full-length sequence determination.
- Enzyme kinetics assays and thermal stability studies.
- Semi-quantitative RT-PCR for expression analysis.
Main Results:
- Purification of a 66 kDa mitochondrial NDP kinase.
- Determination of the complete coding sequence for both mitochondrial and cytosolic isoforms, sharing 53% identity.
- Mitochondrial NDP kinase exhibits high thermal stability and preferential affinity for adenine and guanine nucleotides.
- The enzyme is susceptible to substrate inhibition by GDP.
- Tissue expression is highest in liver and kidney for the mitochondrial isoform, while the cytosolic form is uniformly expressed.
Conclusions:
- This study presents the first comprehensive characterization of a mitochondrial matrix NDP kinase.
- The identified mitochondrial NDP kinase possesses distinct biochemical properties compared to known cytosolic isoforms.
- Understanding this isoform's function and regulation may offer new insights into mitochondrial energy metabolism and disease.
