David O. Lambeth

Genetic Evidence for the Expression of ATP- and GTP-specific Succinyl-CoA Synthetases in Multicellular Eucaryotes

Highly ATP- and GTP-specific isoforms of succinyl-CoA synthetase in pigeon incorporate the same α-subunit, but different β-subunits (Johnson, J. D., Muhonen, W. W., and Lambeth, D. O. (1998) J. Biol. Chem. 273, 27573–27579). The sequences of the mature subunits were determined by methods based on reverse transcription-polymerase chain reaction. The 306-residue mature α-subunit in pigeon shows >88% identity to its homologues in pig and rat. The sequences of the mature ATP- and GTP-specific β-subunits (A-β and G-β, respectively) in pigeon are 54% identical. These sequences were used to identify expressed sequence tags for human and mouse that were highly homologous to G-β and A-β, respectively. The sequences for mature A-β and G-β in mouse and human were completed and verified by polymerase chain reaction. The sequence of A-β in pig was also obtained. The mammalian A-β sequences show >89% identity to each other; the G-β sequences are similarly related. However, pairwise comparisons of the A-β and G-β sequences revealed <53% identity. Alignment with two sequences of the β-subunit in Caenorhabditis elegans suggests that the A-β and G-β genes arose by duplication early in the evolution of multicellular eucaryotes. The expression of A-β is strong in numerous mouse and human tissues, which suggests that ATP-specific succinyl-CoA synthetase also plays an important role in species throughout the animal kingdom.

Implications for in vitro studies of the autoxidation of ferrous ion and the iron-catalyzed autoxidation of dithiothreitol

The influences of buffers and iron chelators on the rate of autoxidation of Fe2+ were examined in the pH range 6.0-7.4. The catalysis by Fe2+ and Fe3+ of the autoxidation of dithiothreitol was also investigated. In buffers which are non- or poor chelators of iron, 0.25 mM Fe2+, and 0.3 mM dithiothreitol when present with iron, oxidize within minutes at pH 7.4 and 30 degrees C. The stability of each increases as the pH is decreased and more than 90% of each remains after 1 h at pH 6.0. In the presence of buffers or oxy-ligands which preferentially and strongly chelate Fe3+ over Fe2+, Fe2+ autoxidizes rapidly in the pH range 6.0-7.4 while dithiothreitol is protected. Ligands which preferentially bind strongly to Fe2+ stabilize both Fe2+ and dithiothreitol at pH 7.4. Dithiothreitol readily reduces Fe3+ in non-chelating buffers or in the presence of strong chelators of Fe2+, however, the ferrous ions produced are prone to reoxidation at higher pH values. These results show that Fe2+ and dithiothreitol are very susceptible to autoxidation in the neutral pH range, and that the rates are strongly influenced by the presence of chelators of Fe2+ and Fe3+. The rapid autoxidations of these species need to be taken into account when designing and interpreting experiments involving Fe2+ or both dithiothreitol and iron.

The intracellular distribution and activities of phosphoenolpyruvate carboxykinase isozymes in various tissues of several mammals and birds

1. The intracellular distribution and/or activities of phosphoenolpyruvate carboxykinase isozymes were determined in liver, kidney, gastrointestinal mucosa, adipose, skeletal muscle, brain, spleen, lung and heart of fed and fasted rabbits, guinea pigs, rats, chickens and pigeons. 2. Liver and kidney of all species contained the highest enzyme activity/g. 3. Carboxykinase activity/g gastrointestinal mucosa of rabbits was quite high compared to the low activity in guinea pig and rat mucosa and essentially undetectable activity in chicken and pigeon mucosa. 4. Activity/g was high in rat brown adipose. 5. Low carboxykinase activity/g was found in skeletal muscle of all species and in white adipose of guinea pig, rabbit and rat although activity was undetectable in white adipose of chicken and pigeon. 6. Carboxykinase activity was essentially undetectable in brain, spleen, lung and heart of all species.

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

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 thekiller 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.

NME6: a new member of the nm23/nucleoside diphosphate kinase gene family located on human chromosome 3p21.3

Abstract The NME (nm23/nucleoside diphosphate kinase) gene family in human is involved in the phosphorylation of nucleoside diphosphates and a variety of regulatory phenomena associated with development, oncogenic transformation, and metastasis. Here we report the cDNA sequence for a sixth member of this family, NME6. The cDNA sequence predicts a 186-residue protein that includes the characteristic active site motif of a nucleoside diphosphate (NDP) kinase, as well as the other residues previously identified as crucial for nucleotide binding and catalysis. The NME6 protein sequence is only 34–41% identical to the five previously reported human NME proteins, and is similarly related to prokaryotic and primitive eukaryotic NDP kinases. Compared to typical proteins of this family such as NME1 and NME2, NME6 has three additional residues located in the Kpn loop, and a 22-residue extension at the COOH-terminal. Using radiation hybrid mapping, the NME6 gene was localized to chromosome 3p21.3. The 1.3-kb transcript of NME6 is expressed at a moderately low level in many human tissues, and is most abundant in kidney, prostate, ovary, intestine, and spleen. Homologous cDNAs were also cloned and sequenced for rat and mouse. The sequence of the first 171 residues of the mouse homologue (Nm23-M6) is 94% identical to the deduced human NME6 protein.

Characterization of the ATP- and GTP-specific succinyl-CoA synthetases in pigeon. The enzymes incorporate the same alpha-subunit.

Two succinyl-CoA synthetases, one highly specific for GTP/GDP and the other for ATP/ADP, have been purified to homogeneity from pigeon liver and breast muscle. The two enzymes are differentially distributed in pigeon, with only the GTP-specific enzyme detected in liver and the ATP-specific enzyme in breast muscle. Based on assays in the direction of CoA formation, the ratios of GTP-specific to ATP-specific activities in kidney, brain, and heart are ∼7, 1, and 0.1, respectively. Both enzymes have the characteristic α- and β-subunits found in other succinyl-CoA synthetases. Studies of the α-subunit by electrophoresis, mass spectrometry, reversed-phase high performance liquid chromatography, and peptide mapping showed that it was the same in the two enzymes. Characterization of the β-subunits by the same methods indicated that they were different, with the tryptic peptide maps providing evidence that the β-subunits likely differ along their entire sequences. Because the two succinyl-CoA synthetases incorporate the same α-subunit, the determinants of nucleotide specificity must reside within the β-subunit. Determination of the apparent Michaelis constants showed that the affinity of the GTP-specific enzyme for GDP is greater than that of the ATP-specific enzyme for ADP (7 versus 250 μm). Rather large differences in apparentK m values were also observed for succinate and phosphate.

Apparent ATP-linked succinate thiokinase activity and its relation to nucleoside diphosphate kinase in mitochondrial matrix preparations from rabbit

The relative abilities of ATP and GTP to support succinyl-CoA synthesis by mitochondrial matrix fractions prepared from rabbit heart and liver mitoplasts were investigated. The activity supported by ATP in rabbit heart preparations was less than 15% of that obtained with GTP, while no ATP-supported activity was observed in rabbit liver preparations. However, the addition of 30 micromolar GDP to matrix fractions from either heart or liver stimulated the ATP-supported activity to 40% of that observed with GTP, and the further addition of bovine liver nucleoside diphosphate kinase in the presence of 8 microM added GDP increased the activity to near that observed with GTP. The specific activity of nucleoside diphosphate kinase assayed directly in mitochondrial matrix from heart was about 15% of the specific activity of ATP-supported succinate thiokinase induced upon adding GDP. Evidence for a complex between nucleoside diphosphate kinase and succinate thiokinase in mitochondrial matrix from rabbit heart was obtained by glycerol density gradient centrifugation. It is proposed that binding of nucleoside diphosphate kinase to succinate thiokinase activates the former enzyme, accounts for the ATP-supported succinyl-CoA synthetase activity observed, and is involved in the channeling of high energy phosphate from GTP produced in the Krebs cycle to the ATP pool.

The compartmentation of nucleoside diphosphate kinase in mitochondria

The compartmentation of nucleoside diphosphate kinase (NDPK) was studied in mitochondria isolated from heart and liver of rat, rabbit, and pigeon. Compartmentation was assessed by determining latencies of enzyme activities, fractionating mitochondria with digitonin, and treating mitochondria with trypsin in the presence and absence of digitonin. NDPK activity in pigeon liver mitochondria was five- and seven-fold higher than in rat and rabbit liver mitochondria. The ratios of NDPK activities in liver vs. heart mitochondria were about 15 for rat, 2 for rabbit, and more than 40 for pigeon. Nearly all NDPK in pigeon liver mitochondria is in the matrix space, but outside the matrix in rat and rabbit liver mitochondria. Most NDPK in pigeon heart mitochondria was located outside the matrix while a significant fraction may be in the matrix of rat and rabbit heart mitochondria. These results are discussed relative to the assumed role that mitochondrial NDPK transfers the phosphoryl group of GTP produced in the Krebs cycle to the adenine nucleotide pool.

Purification and characterization of the isozymes of phosphoenolpyruvate carboxykinase from rabbit liver

Procedures are described for the purification of the mitochondrial and cytosolic isozymes of phosphoenolpyruvate carboxykinase from rabbit liver. Examination of the purified isozymes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated apparent homogeneity and identical molecular weights of approximately 65,000. Gel filtration chromatography of the native isozymes, however, yielded apparent molecular weights of 68,000 and 56,000 for the cytosolic and mitochondrial isozymes, respectively. The isoelectric points as determined by chromatofocusing were 5.8 for the mitochondrial isozyme and 5.0 for the cytosolic isozyme. The purified isozymes were readily separable on ion-exchange columns, with the cytosolic isozyme showing the greater affinity. A minor amount of cross-reactivity was apparent when each isozyme was immunotitrated with polyclonal antibodies raised in goat against the opposite isozyme. Peptide maps obtained by high pressure liquid chromatography of both tryptic digests and cyanogen bromide digests of the isozymes showed that many of the peaks were not coincident, suggesting that differences in the sequences are found throughout the primary structures of the isozymes.

Synthesis of malate from phosphoenolpyruvate by rabbit liver mitochondria: implications for lipogenesis

(1) Rabbit liver mitochondria can convert exogenous phosphoenolpyruvate to malate. (2) Malate production is dependent on phosphoenolpyruvate and HCO3- and is stimulated by CN- or malonate alone and especially in combination. (3) Malate production is inhibited 70% by 3-mercaptopicolinate, a specific inhibitor of phosphoenolpyruvate carboxykinase, and 50-60% by 1,2,3-benzenetricarboxylate, an inhibitor of the tricarboxylate transporter. (4) Rat liver mitochondria incubated with phosphoenolpyruvate under identical conditions do not produce malate. (5) Malate production from phosphoenolpyruvate is stimulated by exogenous GDP or IDP but not by ADP. (6) Data support the conclusion that malate is being produced from oxalacetate generated by reversal of mitochondrial phosphoenolpyruvate carboxykinase. A possible role for this enzyme in hepatic lipogenesis is suggested.