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 alpha-subunit, but different beta-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 alpha-subunit in pigeon shows >88% identity to its homologues in pig and rat. The sequences of the mature ATP- and GTP-specific beta-subunits (A-beta and G-beta, 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-beta and A-beta, respectively. The sequences for mature A-beta and G-beta in mouse and human were completed and verified by polymerase chain reaction. The sequence of A-beta in pig was also obtained. The mammalian A-beta sequences show >89% identity to each other; the G-beta sequences are similarly related. However, pairwise comparisons of the A-beta and G-beta sequences revealed <53% identity. Alignment with two sequences of the beta-subunit in Caenorhabditis elegans suggests that the A-beta and G-beta genes arose by duplication early in the evolution of multicellular eucaryotes. The expression of A-beta 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.

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 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.

Synthesis and stability of nuclear matrix proteins in resting and serum-stimulated Swiss 3T3 cells.

The major [35S]methionine-radiolabeled nuclear matrix proteins of mouse 3T3 cells were isolated, and most of these were found to be similar in molecular weight, charge, and protease fingerprint to the nuclear matrix proteins of African green monkey kidney cells, which are found tightly bound to simian virus 40 chromosomes. These nuclear matrix proteins were found to be synthesized in quiescent and serum-stimulated cells and to be turned over slowly during pulse-chase experiments. In contrast, a 70-Kd (kilodalton) neutral protein identified as lamin a was found to be turned over rapidly, producing a 68-Kd protein with a similar isoelectric point. In addition, we observed a decrease in the amounts of two chromatin-bound matrix proteins and a relative increase in lamin a following labeling in the presence of 2 micrograms/ml actinomycin D. However, these effects do not appear to be a result of inhibition of transcription, since they were not observed with other inhibitors (alpha-amanitin and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole).

Identification of nuclear matrix proteins tightly bound to soluble simian virus 40 chromosomes.

Nuclear matrix proteins (defined as the nuclear proteins which were highly enriched in an insoluble fraction following extraction of lipids, loosely bound proteins, and nucleic acids) from mock- and SV40-infected cells were identified by two-dimensional polyacrylamide gel electrophoresis, consisting of nonequilibrium pH gradients in the first dimension and sodium dodecyl sulfate gel electrophoresis in the second dimension. The proteins identified in the mock-infected nuclear matrix included M1 (molecular weight, 71K), M2 (69K) M3 (58K), M4 (50K), M5 (49K), M6 (36K), M7 (36K), and M8 (31K), while the nuclear matrix from SV40-infected cells included, in addition to all these proteins, VP-1 (45K), VP-1' (44K), VP-3 (25K), V1 (36K), V2 (35K), and V3 (35K). Except for M7 all of these proteins sedimented with SV40 chromosomes isolated and partially purified by glycerol gradient sedimentation at low ionic strength, and only M6 and M8 were removed from the SV40 chromosomes during more extensive purification of the SV40 chromosomes by subsequent sedimentation at high ionic strength (0.5 M NaCl). When the structures of the SV40 chromosomes were destroyed by digestion with DNAase I, these tightly bound proteins no longer sedimented to the position of SV40 chromosomes. Further subfractionation of SV40 chromosomes indicated that the proteins M1 to M4 were preferentially associated with the nonreplicating SV40 chromosomes, whereas M5 was associated with encapsidating SV40 chromosomes and virions.

Two-dimensional analysis of proteins sedimenting with simian virus 40 chromosomes.

The nonhistone proteins sedimenting in low-salt glycerol gradients with simian virus 40 chromosomes were analyzed by two-dimensional gel electrophoresis, utilizing nonequilibrium pH gradients as the first dimension and sodium dodecyl sulfate-gel electrophoresis as the second dimension. By densitometric quantitation of the radiolabeled proteins present in each fraction of the gradients, it was possible to identify sedimenting with all or a fraction of the simian virus 40 chromosomes. VP-1 sedimented with simian virus 40 chromosomes; additional evidence for its binding to chromosomes was obtained by immunochemical techniques. Four proteins (Mr 25,000, pI 6.0; Mr 32,000, pI 7.2; Mr 35,000, pI 8.5; and Mr 80,000, pI 7.2) sedimented with specific subsets of chromosomes.

Study of ansamycin inhibition of a ribonucleic acid-directed deoxyribonucleic acid polymerase by an immobilized template assay.

A series of structurally related ansamycins have been analyzed, in a new immobilized template assay, to determine the mechanism by which they inhibit a ribonucleic acid-directed deoxyribonucleic acid (DNA) polymerase from Moloney murine leukemia virus. By this assay, we can better correlate specific structures of these drugs with inhibitory mechanisms. Using an immobilized template, we were also able to observe drug effects on the stability of complexes formed between the polymerase, a template (polyadenylic acid-agarose), and a primer, as well as to monitor the synthesis of DNA in the presence of drug. For each drug, we determined the complex (intermediate in DNA synthesis) which was primarily affected and whether the effect was due to a destabilization process. Although the activity and specificity of the unsubstituted ansamycins (streptovaricins and rifamycin SV) were modulated by conformation of the molecule and electron density of the aromatic ring, the principal mode of inhibition is, apparently, drug binding to a polymerase-template complex; the drug binds in a manner which prevents subsequent formation of a polymerase-template-primer complex. However, some derivatives of rifamycin SV, when substituted at carbon-3 with bulky or hydrophobic side chains, displayed markedly different modes of action. For example, demethyl dimethyl rifampin prevented the formation of polymerase-template complexes, whereas rifazacyclo 16 acted by promoting the dissociation of polymerase-template-primer complexes.