Relationship between 5'-nucleotidase, adenosine deaminase, AMP deaminase, ATP-(Mg2+)-ase activities and dTMP kinase activity in rat liver mitochondria.

The activities of dTMP kinase (ATP-deoxythymidine monophosphate phosphotransferase, EC 2.7.4.9), 5'-nucleotidase (5'-ribonucleoside phosphohydrolase, EC 3.1.3.5), adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), AMP deaminase (AMP aminohydrolase, EC 3.5.3.6) and ATP-(Mg2+)-ase (ATP phosphohydrolase, EC 3.6.1.3) were assayed in mitochondria of normal and regenerating rat liver. In regenerating mitochondria, the dTMP kinase activity increased 20 times, 5'-nucleotidase (5'Nase) activity for dTMP diminished by 65% and its activity for other nucleoside monophosphates did not change; adenosine deaminase activity for adenosine (AR) increased by 40%, but for deoxyadenosine (AdR) decreased by 70%. AMP deaminase and ATP-(Mg2+)-ase activities behaved similarly in mitochondria from regenerating liver, decreasing by 70 and 64% respectively. The changes of the amount of dTMP in mitochondria depend on enzyme activities which regulate the AdR concentration.     

A distinctive activity of 5'-nucleotidase for dTMP in rat liver mitochondria

The activities of 5'-nucleotidase (5'-ribonucleoside phosphohydrolase, EC 3.1.3.5); adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4); AMP deaminase (AMP aminohydrolase, EC 3.5.3.6), and ATP-(Mg2+)-ase (ATP phosphohydrolase, EC 3.6.1.3) were assayed in mitochondria of normal and regenerating rat liver 5'-Nucleotidase (5'Nase) and ATP-(Mg2+)-ase activities were compared with similar enzyme activities in the plasma membrane (PM) fraction, obtained from the same biological material. In the regenerating liver, 5'Nase for dTMP diminished its activity by 56% (24 h after partial hepatectomy) and 35 +/- 4% for all substrates in the PM fraction (48 h after operation). In mitochondria, 5'Nase for dTMP manifests sigmoidal substrate activity curve (in contrast with all substrates in the PM fraction and remaining substrates in mitochondria). In vivo 5-azacytidine (a) administered 1 h after partial hepatectomy, prevented changes of 5'Nase activity: (b) administered 24 or 48 h after partial hepatectomy, stabilized low 5'Nase activity (in mitochondria for dTMP, in the PM fraction for all substrates) and decreased ATP-(Mg2+)-ase activity by 51 and 31% in mitochondria and the PM fraction respectively.     

A mathematical model of human thymidine kinase 2 activity

The mitochondrial enzyme thymidine kinase 2 (TK2) phosphorylates deoxythymidine (dT) and deoxycytidine (dC) to form dTMP and dCMP, which in cells rapidly become the negative-feedback end-products dTTP and dCTP. TK2 kinetic activity exhibits Hill coefficients of ～0.5 (apparent negative cooperativity) for dT and ～1 for dC. We present a mathematical model of TK2 activity that is applicable if TK2 exists as two monomer forms in equilibrium.     

Intracellular localization of Neurospora crassa endo-exonuclease and its putative precursor.

Endo-exonuclease of rapidly growing mycelia of Neurospora crassa was found to be distributed in a ratio of about 1.6:1 in vacuoles and in mitochondria where it is associated with the inner membrane. Although the activity in vacuoles was readily released by osmotic shock, very little of that in mitochondria was released by this method. The mitochondrial activity was partially (60 to 70%) released by sonication, and the remaining activity was solubilized in the presence of Triton X-100. An inactive form of endo-exonuclease, activated in vitro by treatment with trypsin, is present in mycelia at a level over four times that of active enzyme. It was found to be distributed in a ratio of about 2.5:1 in the cytosol and in the inner membrane of mitochondria. The mitochondrial protein was more tightly bound than the active enzyme. Very little of the inactive enzyme was released by sonication, but it was solubilized in the presence of Triton X-100. The intracellular distribution of active and inactive forms of endo-exonuclease differs in a mutagen-sensitive mutant of Neurospora crassa (uvs-3) which shows many pleiotropic effects. The most striking difference in distribution is in the mitochondria where endo-exonuclease is present almost entirely in the inactive form at a level 30% higher than in wild-type mitochondria.     

Interaction of AMP deaminase with RNA

tRNA, 18 S and 28 S ribosomal RNAs were found to activate muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) but inhibit liver and heart AMP deaminases. The macromolecular structures are essential for modulation of enzyme activity, since the effects of RNA disappeared after RNAase treatment. Sucrose density centrifugation experiments clearly demonstrated the binding of purified muscle AMP deaminase to tRNA, 18 S and 28 S RNAs. The binding is reversible and responsive to alterations of pH and KCl concentration. The binding was stable at pH 5.1-7.0 in 0.1 M KCl, but most of the enzyme dissociated at pH 7.5. KCl below 0.1 M concentration had no effect on dissociation of enzyme-RNA complex, but in 0.15 M KCl the complex was partially dissociated and in 0.2 M KCl most of the enzyme was released. Various nucleotides were also effective in dissociation of the enzyme from complex. The binding is saturable and the maximum number of muscle AMP deaminase molecules bound per mol 28 S RNA was calculated to be approx. 30. Liver and heart AMP deaminases were also found to interact with RNA.     

Properties and intramitochondrial localization of serine hydroxymethyltransferase in leaves of higher plants

The activity of serine hydroxymethyltransferase in mitochondria isolated from spinach leaves was absolutely dependent on tetrahydrofolate; pyridoxal phosphate has no effect on the activity. The stability of this activity in the isolated mitochondria was dependent on the presence of sulfhydryl compounds. It was apparently more stable at pH 7.0 to 7.5 than at higher pH even though the pH optimum of serine hydroxymethyltransferase was 8.5 for both the mitochondrial and cytoplasmic fractions. Distribution studies have indicated that serine hydroxymethyltransferase was predominantly located in the mitochondria. The activity of serine hydroxymethyltransferase was observed to be co-compartmented with glycine decarboxylation and malate dehydrogenase behind the mitochondrial inner membrane. This activity could be solubilized by KCl from osmotically ruptured mitochondrial membrane fractions but substantial activity (35 to 40%) was still retained with the membrane fractions at 0.3 m KCl. This suggests that the glycine decarboxylation-serine hydroxymethyltransferase complex may be closely bound to the internal surface of the mitochondrial inner membrane.The relationship of this integrated enzyme complex to CO(2) evolution and serine synthesis during photorespiration and the physiological role of the dicarboxylate shuttle were discussed.     

In Rat Liver Mitochondria All Nucleoside Diphosphate Kinase of the Outer Compartment Is Associated with the Outer Surface of the Outer Membrane

Data on localization of nucleoside diphosphate kinase (NDPK) in the outer mitochondrial compartment are contradictory. We have demonstrated that repeated quintuple wash of a mitochondrial pellet (protein concentration is about 2 mg/ml) solubilized only 60% of total NDPK activity. Since no release of adenylate kinase, the marker enzyme of the intermembrane space, was observed, it was concluded that the solubilized NDPK activity was associated with the outer surface of the outer mitochondrial membrane. Treatment of mitochondria with digitonin solutions in low (sucrose, mannitol) or high (KCl) ionic strength media revealed that solubilization of remaining NDPK activity basically coincided with the solubilization curve of monoamine oxidase, the marker enzyme of the outer mitochondrial membrane, but differed from solubilization behavior of adenylate kinase and malate dehydrogenase. We concluded that the remaining NDPK activity was also associated with the outer mitochondrial membrane and electrostatic interactions were not essential for NDPK binding to mitochondrial membranes. Results of polarographic determination of remaining adenylate kinase and NDPK activities of mitochondria incubated in ice for different time intervals and subjected to subsequent centrifugation suggest that all NDPK activity of the outer compartment of rat liver mitochondria is associated with the outer surface of the outer mitochondrial membrane. We suggest the existence of at least three NDPK fractions. They represent 70, 15, and 15% of total NDPK activity of the outer compartment and differ by tightness of membrane binding.     

Interactions between T4 phage-coded deoxycytidylate hydroxymethylase and thymidylate synthase as revealed with an anti-idiotypic antibody.

Anti-idiotypic antibodies were used to mimic the binding surface of the T4 bacteriophage deoxycytidylate hydroxymethylase enzyme, providing an immunological probe for protein-protein interactions involving this enzyme. Polyclonal dCMP hydroxymethylase antibodies were affinity-purified and used to generate anti-idiotypic antibodies. The anti-idiotypic serum immunoprecipitated two native viral proteins, deoxycytidylate hydroxymethylase (EC 2.1.2.8) and thymidylate synthase (EC 2.1.1.45), from a sonicated detergent-treated extract of T4-infected Escherichia coli. The anti-anti-dCMP hydroxymethylase antibody was found to be specific in binding to the T4 dTMP synthase, with no detectable affinity for the host dTMP synthase. Previous work in our laboratory has demonstrated the viral dCMP hydroxymethylase and dTMP synthase to be associated in a deoxyribonucleotide synthetase enzyme complex. Our current approach, using anti-idiotypic antibodies as probes for protein-protein interactions, and complementary studies involving dCMP hydroxymethylase enzyme affinity columns indicate a direct association between bacteriophage T4 dCMP hydroxymethylase and dTMP synthase.     

Analyses of deoxycytidylate deaminase molecular forms in human-mouse and monkey-mouse somatic cell hybrids

Disc polyacrylamide gel electrophoresis (disc PAGE) analyses have revealed that mouse, human, and monkey cytosol deoxycytidylate (dCMP) deaminases differ in electrophoretic mobility, so that mixtures of mouse and human, mouse and monkey, and human and monkey enzymes can be separated. To learn whether the genes for dCMP deaminase and thymidine (dT) kinase are genetically linked, disc PAGE analyses of cytosol fractions from human-mouse and monkey-mouse somatic cell hybrids were carried out. The interspecific somatic cell hybrids were derived from the fusion of cytosol dT kinase deficient mouse cells with cytosol dT kinase-positive human and monkey cells: they contained mostly mouse chromosomes and a few primate chromosomes, including the determinant for primate cytosol dT kinase. The disc PAGE analyses demonstrated that the human-mouse and monkey-mouse somatic cell hybrids contained a dCMP deaminase activity with an electrophoretic mobility characteristic of mouse dCMP deaminase. Enzymes with electrophoretic mobilities characteristic of human and monkey dCMP deaminases were not demonstrable. These findings suggest that primate cytosol dT kinase and dCMP deaminase are coded on different chromosomes, or that the formation in hybrid cells of an active primate dCMP deaminase is suppressed. Chick-mouse somatic cell hybrids containing chick but not mouse cytosol dT kinase were also analyzed. The chick-mouse hybrid cells contained cytosol dCMP deaminase activity, but it was not possible to establish whether the enzyme was of murine or avian origin because of the similarity in electrophoretic mobility between the chick and mouse enzymes. Human and mouse cells contained low levels of mitochondrial dCMP deaminase activity. In contrast to dT kinase isozymes, however, mitochondrial and cytosol dCMP deaminases were electrophoretically indistinguishable.This investigation was aided by Grant Q-163 from the Robert A. Welch Foundation and by USPHS Grants CA-06656-12 and 1-K6-AI 2352 from the National Cancer Institute and the National Institute of Allergy and Infectious Diseases.     

Nonidentity of Some Simian Virus 40-induced Enzymes with Tumor Antigen

The complement-fixing tumor (T) antigen induced by simian virus 40 (SV40) has been prepared from SV40-infected cell cultures, from infected cell cultures treated at the time of infection with 1-beta-d-arabinofuranosylcytosine (ara-C), and from SV40-transformed cells. Upon partial purification, the T antigen exhibited the following properties: it was tightly adsorbed by calcium phosphate gel, it was precipitated by acetic acid at pH 5 or by ammonium sulfate at about 20 to 32% saturation, and it had a molecular weight greater than 250,000, as estimated by Sephadex G-200 gel chromatography. In contrast, deoxycytidylate (dCMP) deaminase, thymidylate (dTMP) kinase, and thymidine (dT) kinase were less strongly bound to calcium phosphate and were not precipitated at pH 5; these enzymes also had much lower molecular weights than the T antigen, as did dihydrofolic (FH(2)) reductase. Furthermore, higher ammonium sulfate concentrations were required to precipitate dCMP deaminase, dTMP kinase, and FH(2) reductase activities than to precipitate the T antigen. Another difference was that the T antigen was not stabilized, but dCMP deaminase, dTMP kinase, and dT kinase, were stabilized, respectively, by dCTP, dTMP, and dT or dTTP. Deoxyribonucleic acid (DNA) polymerase activity resembled the T antigen in adsorption to calcium phosphate, in precipitation by ammonium sulfate or at pH 5, and in the rate of inactivation when incubated at 38 C. However, the polymerase activity could be partly separated from the T antigen by Sephadex G-200 gel chromatography. The cell fraction containing partially purified T antigen also contained a soluble complement-fixing antigen (presumably a subunit of the viral capsid) which reacted with hyperimmune monkey sera. The latter antigen was present in very low titers or absent from cell extracts prepared from SV40-infected monkey kidney cell cultures which had been treated with ara-C at the time of infection, or from SV40-transformed mouse kidney (mKS) or hamster tumor (H-50) cells. The T antigen, however, was present in usual amounts in SV40-transformed cells or ara-C treated, infected cells.     

Heart mitochondria in physiological salt solution: not ionic strength but salt composition is important for association of creatine kinase with the inner membrane surface

In physiological salt solution (PSS) which mimicks the cardiac cells cytoplasm and contains 120 mM K-MES, 10 mM NaCl, 20 mM imidazole, pH 7.2, 20 mM taurine, 15 mM creatine, 15 mM Na2phosphocreatine, 5 mM Na2ATP, 8 mM MgCl2, 5 mM K2HPO4, 3 mM glutamate, 3 mM malate, 0.5 mM dithiothreitol and 10 mg/ml of bovine serum albumine both isolated mitochondria and intracellular structures in skinned fibers stay intact. In PSS mitochondrial creatine kinase remains firmly attached to the inner membrane surface. CKmi-mi is extracted from cardiac mitoplasts in 0.125 M KCl solution, but addition of 10 mM sodium borate to this KCl solution completely inhibits dissociation of CKmi-mi. Therefore, not ionic strength but ion composition is important for association of CKmi-mi with mitochondrial membrane. Functional and structural studies using antibodies against CKmi-mi showed that in PSS CKmi-mi is bound to the inner mitochondrial membrane in spatially close relationship to adenine nucleotide translocase (ANT). Thus, under physiological conditions CKmi-mi is structurally and functionally coupled to ANT in cardiac mitochondria and functions to catalyze almost complete utilization of mitochondrial ATP for aerobic phosphocreatine synthesis.     

Purification and properties of the major nuclease from mitochondria of Saccharomyces cerevisiae

The vast majority of nuclease activity in yeast mitochondria is due to a single polypeptide with an apparent molecular weight of 38,000. The enzyme is located in the mitochondrial inner membrane and requires non-ionic detergents for solubilization and activity. A combination of heparin-agarose and Cibacron blue-agarose chromatography was employed to purify the nuclease to approximately 90% homogeneity. The purified enzyme shows multiple activities: 1) RNase activity on single-stranded, but not double-stranded RNA, 2) endonuclease activity on single- and double-stranded DNA, and 3) a 5'-exonuclease activity on double-stranded DNA. Digestion products with DNA contain 5'-phosphorylated termini. Antibody raised against an analogous enzyme purified from Neurospora crassa (Chow, T. Y. K., and Fraser, M. (1983) J. Biol. Chem. 258, 12010-12018) inhibits and immunoprecipitates the yeast enzyme. This antibody inhibits 90-95% of all nuclease activity present in solubilized mitochondria, indicating that the purified nuclease accounts for the bulk of mitochondrial nucleolytic activity. Analysis of a mutant strain in which the gene for the nuclease has been disrupted supports this conclusion and shows that all detectable DNase activity and most nonspecific RNase activity in the mitochondria is due to this single enzyme.     

Specific inhibition of ATP-ADP translocase in cardiac mitoplasts by antibodies against mitochondrial creatine kinase

Mitochondrial creatine kinase was purified from rat hearts and used to produce antibodies in chicken and rabbits. Antibodies were purified to a high degree of homogeneity by an affinity chromatography method. Chicken antibodies against mitochondrial creatine kinase inhibited this enzyme in rat-heart mitochondrial inner membrane and matrix preparation, and simultaneously blocked oxidative phosphorylation. Under these conditions respiratory chain activities remained unchanged, but adenine nucleotide translocase was inhibited. Removal of mitochondrial creatine kinase from the membrane by pretreatment with 0.15 M KCl and 20 mM ADP completely abolished the effect of antibodies against mitochondrial creatine kinase on oxidative phosphorylation. Noninhibitory antibodies from rabbit with high affinity to rat mitochondrial creatine kinase inhibited neither creatine kinase activity nor oxidative phosphorylation. These data show close and specific spatial arrangement of mitochondrial creatine kinase and adenine nucleotide translocase in mitochondria. It is supposed that there is a fixed orientation of these proteins in the cardiolipin domain in the membrane and that their interaction may occur by a frequent collision due to their lateral movement.     

Identification and characterization of tyrosine kinase activity associated with mitochondrial outer membrane in sarcoma 180 cells

Tyrosine protein kinase activity has been detected in the mitochondrial fraction purified from sarcoma 180 tumor cells. Following hypotonic disruption of mitochondria, tyrosine kinase activity appeared to cosediment with monamine oxidase, marker enzyme of mitochondrial outer membrane; meanwhile, serine and threonine kinases were found to be associated with the inner membrane and matrix of mitochondria. Mitochondrial tyrosine kinase(s) showed thermosensitivity and Mn2+ dependence, useful properties for its characterization and separation from tyrosine kinases associated with other particulate fraction and from serine and threonine kinases associated with mitochondria. Following in vitro incubation of mitochondria with labelled ATP as substrate and analysis by PAGE, a complex pattern of phosphotyrosine containing proteins with a major band of 50-55 kilodaltons resulted.     

The localization of some coenzyme A-dependant enzymes in rat liver mitochondria

1. CoA, acetyl-CoA, l-carnitine and acetyl-l-carnitine when added to rat liver mitochondria equilibrate with approximately two-thirds of the total intramitochondrial water. The mitochondrial space calculated to be freely permeable to these solutes was identical with that obtained for sucrose. 2. Acetyl-CoA is rapidly deacylated by rat liver mitochondria at 0 degrees C, and special precautions are required to measure its mitochondrial permeation. 3. Rat liver mitochondria were separated into fractions that correspond to the inner membrane, the outer membrane, and the soluble proteins of the matrix and intermembrane compartment. Soluble enzymes considered to be located in the matrix were citrate synthase (EC 4.1.3.7), palmitoyl-CoA dehydrogenase (EC 1.3.2.2), electron-transferring flavoprotein, medium-chain-length ATP-specific fatty acyl-CoA synthetase (EC 6.2.1.2), l-3-hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.35) and 3-keto-acyl-CoA thiolase (EC 2.3.1.16). Carnitine palmitoyltransferase (EC 2.3.1.-) is largely associated with the inner-membrane fraction. A long-chain-length ATP-specific fatty acyl-CoA synthetase (EC 6.2.1.3) is associated with the outer-membrane fraction.     

The First Crystal Structure of a dTTP-bound Deoxycytidylate Deaminase Validates and Details the Allosteric-Inhibitor Binding Site

Deoxycytidylate deaminase is unique within the zinc-dependent cytidine deaminase family as being allosterically regulated, activated by dCTP, and inhibited by dTTP. Here we present the first crystal structure of a dTTP-bound deoxycytidylate deaminase from the bacteriophage S-TIM5, confirming that this inhibitor binds to the same site as the dCTP activator. The molecular details of this structure, complemented by structures apo- and dCMP-bound, provide insights into the allosteric mechanism. Although the positioning of the nucleoside moiety of dTTP is almost identical to that previously described for dCTP, protonation of N3 in deoxythymidine and not deoxycytidine would facilitate hydrogen bonding of dTTP but not dCTP and may result in a higher affinity of dTTP to the allosteric site conferring its inhibitory activity. Further the functional group on C4 (O in dTTP and NH₂ in dCTP) makes interactions with nonconserved protein residues preceding the allosteric motif, and the relative strength of binding to these residues appears to correspond to the potency of dTTP inhibition. The active sites of these structures are also uniquely occupied by dTMP and dCMP resolving aspects of substrate specificity. The methyl group of dTMP apparently clashes with a highly conserved tyrosine residue, preventing the formation of a correct base stacking shown to be imperative for deamination activity. The relevance of these findings to the wider zinc-dependent cytidine deaminase family is also discussed.     

Functional changes in mitochondrial properties as a result of their membrane cryodestruction. II. Influence of freezing and thawing on ATP complex activity of intact liver mitochondria

The influence of the freeze-thawing rates on ATP synthetase (ATPase) complex of intact liver mitochondria was investigated. It was shown that the increase in latent ATPase activity and decrease in ATP synthetase activity resulted from an influence on the inner mitochondrial membrane. An increase in freeze-thawing rates led to the preservation of ATP synthetase activity and ATP hydrolysis reduction. Kinetic parameter changes of the ATP synthetase reaction resulted from an insignificant nonspecific increase in the inner mitochondrial membrane permeability and changes in its electrochemical potential level.     

BIOCHEMICAL AND ULTRASTRUCTURAL PROPERTIES OF A MITOCHONDRIAL INNER MEMBRANE FRACTION DEFICIENT IN OUTER MEMBRANE AND MATRIX ACTIVITIES

Treatment of the inner membrane matrix fraction of rat liver mitochondria with the nonionic detergent Lubrol WX solubilized about 70% of the total protein and 90% or more of the following matrix activities: malate dehydrogenase, glutamate dehydrogenase, and isocitrate dehydrogenase (NADP). The Lubrol-insoluble fraction was enriched in cytochromes, phospholipids, and a Mg⁺⁺-stimulated ATPase activity. Less than 2% of the total mitochondrial activity of monoamine oxidase, an outer membrane marker, or adenylate kinase, an intracristal space marker could be detected in this inner membrane fraction. Electron micrographs of negatively stained preparations showed vesicles (≤0.4 µ diameter) literally saturated on the periphery with the 90 A ATPase particles. These inner membrane vesicles, which appeared for the most part to be inverted with respect to the normal inner membrane configuration in intact mitochondria, retained the succinicoxidase portion of the electron-transport chain, an intact phosphorylation site II with a high affinity for ADP, and the capacity to accumulate Ca⁺⁺. A number of biochemical properties characteristic of intact mitochondria and the inner membrane matrix fraction, however, were either absent or markedly deficient in the inner membrane vesicles. These included stimulation of respiration by either ADP or 2,4-dinitrophenol, oligomycin-sensitive ADP-ATP exchange activity, atractyloside sensitivity of adenine nucleotide requiring reactions, and a stimulation of the Mg⁺⁺-ATPase by 2,4-dinitrophenol.     

Isolation of a Saccharomyces cerevisiae mutant strain deficient in deoxycytidylate deaminase activity and partial characterization of the enzyme.

Deoxycytidylate deaminase activity in Saccharomyces cerevisiae has been partially characterized. The yeast enzyme was found to exhibit properties similar to those of dCMP deaminases isolated from higher eucaryotes. A mutant strain completely deficient in dCMP deaminase activity was isolated by selection for resistance to 5-fluoro-2'-deoxycytidylate followed by screening for cross sensitivity to 5-fluoro-2'-deoxyuridylate, a potent inhibitor of the yeast thymidylate synthetase. We have designated this new allele dcd1 . A strain exhibiting an auxotrophic requirement for dUMP was isolated after mutagenesis of a dcd1 tup7 haploid. Genetic analysis revealed that this auxotrophic phenotype resulted from a combination of the dcd1 allele and a second, unlinked, nuclear mutation that we designated dmp1 . This allele, which by itself conveys no readily discernible phenotype, presumably impairs efficient synthesis of dUMP from UDP. The auxotrophic requirement of dcd1 dmp1 tup7 strains also can be satisfied by exogenous dTMP but not deoxyuridine.