The long term objectives of the applicant's laboratory are to: (1) understand how GTP is involved in the regulation and integration of metabolism and energy transduction within the mitochondrial matrix; (2) understand how mitochondrial isoforms of nucleoside diphosphate kinase (NDPK) regulate the flow of phosphoryl groups between the mitochondrial pools of adenine and guanine nucleotides; and (3) elucidate the genetic relationships between the mitochondrial and """"""""cytosolic"""""""" isoforms of NDPK. Although cytosolic isoforms of NDPK are receiving intense scrutiny by a number of laboratories interested in regulatory biology, the roles, properties, and compartmentation of mitochondrial isoforms of NDPK have long been neglected. The matrix space of pigeon liver mitochondria contains an extraordinary high level of NDPK activity. GTP/GDP-specific succinate thiokinase and phosphoenolpyruvate carboxykinase are also present in these mitochondria. The matrix isoform of NDPK has been purified to homogeneity and has been partially characterized.
The specific aims of this proposal are to: (1) purify a second isoform of mitochondrial NDPK that is located outside the inner membrane of pigeon heart mitochondria and determine the physical and enzymatic characteristics of the two mitochondrial isoforms; and (2) determine the cDNA and amino acid sequences of each of the mitochondrial isoforms of NDPK. An initial attempt to clone and sequence a cDNA for a mitochondrial isoform of NDPK resulted in obtaining the sequence for an apparent cytosolic isoform. To obtain the sequence to a mitochondrial isoform, oligonucleotide primers will be used in PCR to amplify segments of the desired cDNA from a mRNA fraction of pigeon liver. The N-terminal sequence of the mitochondrial isoform of NDPK has been determined for the purified NDPK from pigeon liver mitochondria. Oligonucleotide primers will be made to several parts of this sequence. Other primers are being made to highly conserved regions within known NDPKs. Once a segment has been amplified, primers will be prepared to obtain the 3' and 5' ends by the RACE (rapid amplification of cDNA ends) procedure. If this approach does not work, one or more peptides obtained by proteolytic digestion of purified NDPK will be sequenced. Additional, degenerate oligonucleotide primers will be prepared based on the actual sequences. After obtaining the sequence to the matrix isoform of NDPK, the sequence of a mitochondrial isoform located outside the inner membrane will be sought by using the approaches described above. Sufficient amounts of each mitochondrial isoform of NDPK identified will be obtained to allow characterization of native molecular weight, number of subunits, substrate specificity, and kinetic constants. Both mitochondrial forms of NDPK may be obtained by over expressing the cDNAs in E. coli. Alternatively, the isoform located outside the inner membrane will be purified by conventional means. Antibodies against the matrix form are in hand and these may provide a quick way to purify the other isoform if there is sufficient cross-reactivity. Accomplishing the proposed specific aims will add important knowledge to the growing body of information about the properties, roles and functions of NDPK isoforms in various parts of the cell. The relatedness of any knowledge to health is uncertain at this time. However, work on NDPK within the past five years indicate that this enzyme is more than a house-keeping enzyme, it also plays regulatory functions in various cellular functions.
