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PubMed2001

A nonspecific nucleoside hydrolase from Leishmania donovani: implications for purine salvage by the parasite.

In contrast to their mammalian hosts, protozoan parasites do not synthesize purines de novo, but depend on preformed nucleotides that they purportedly obtain by salvage pathways. Nucleoside hydrolases may play a crucial role in that salvage process. By screening Leishmania donovani libraries with polyclonal antibodies against promastigote soluble exo-antigens, we have identified a cDNA encoding a protein with significant homology to nonspecific and uridine-inosine-preferring nucleoside hydrolases. Sequence comparison demonstrated that all the residues involved in Ca(2+)-binding and substrate recognition in the active site are conserved among the characterized protozoan nucleoside hydrolases. Genomic analysis suggests that it is a single copy gene in L. donovani, and its homologues are present in members representing other Leishmania species complexes. Both Northern blot and immunoblot analyses indicate that it is constitutively expressed in L. donovani promastigotes. The recombinant enzyme overexpressed in and purified from bacteria showed significant activity with all naturally occurring purine and pyrimidine nucleosides, and efficient utilization of p-nitrophenyl-beta-D-ribofuranoside as a substrate. Altogether, the sequence comparison and substrate specificity data identify this L. donovani nucleoside hydrolase as a nonspecific nucleoside hydrolase. Further, the nucleoside hydrolase was localized to specific foci in L. donovani promastigotes by immunofluorescent assays. Although the conservation of the nucleoside hydrolases among protozoan parasites offers promise for the design of broad-spectrum anti-parasitic drugs, the existence of multiple and distinct nucleoside hydrolases in a single species demands special consideration.

Amino Acid Sequence↗
PubMed2000

Molecular cloning of a Na+-dependent nucleoside transporter from rabbit intestine.

PURPOSE: Substantial species differences in the transport kinetics of nucleosides and therapeutic nucleoside analogs have been observed in various experimental systems. To explain these differences at a molecular level, it is necessary to clone the relevant transporters and examine their functional characteristics in heterologous expression systems. The goal of the present study was to clone the nucleoside transporters present in rabbit, an important preclinical animal model, and to functionally characterize the clone(s). METHODS: A Polymerase Chain Reaction (PCR)-based homology cloning approach in conjunction with Rapid Amplification of cDNA Ends (RACE) was used to isolate a full-length cDNA. Characterization of this transporter was accomplished through heterologous expression in Xenopus laevis oocytes. RESULTS: A full-length cDNA encoding a purine-selective, Na+-dependent nucleoside transporter, rbSPNT1, was isolated from rabbit small intestine. The encoded protein is 658 amino acid residues in length. Hydropathy analysis suggests that rbSPNT1 has 11 to 14 transmembrane domains. In Xenopus laevis oocytes expressing rbSPNT1, the uptake of uridine and inosine was enhanced significantly; uridine transport was inhibited by purine, but not pyrimidine nucleosides. mRNA transcripts for rbSPNT1 were detected primarily in intestine, lung, and kidney and at lower levels in liver, brain, and heart. CONCLUSIONS: A full-length functional nucleoside transporter was cloned. Sequence analysis and functional characterization suggest that rbSPNT1 is the rabbit homolog of the purine-selective nucleoside transporter, N1. The cloned rbSPNT1 can be used to understand the molecular mechanisms responsible for the observed species differences in the transport of nucleosides and therapeutic nucleoside analogs.

Amino Acid Sequence↗
PubMed2006

Structural basis for activation of the therapeutic L-nucleoside analogs 3TC and troxacitabine by human deoxycytidine kinase.

L-nucleoside analogs represent an important class of small molecules for treating both viral infections and cancers. These pro-drugs achieve pharmacological activity only after enzyme-catalyzed conversion to their tri-phosphorylated forms. Herein, we report the crystal structures of human deoxycytidine kinase (dCK) in complex with the L-nucleosides (-)-beta-2',3'-dideoxy-3'-thiacytidine (3TC)--an approved anti-human immunodeficiency virus (HIV) agent--and troxacitabine (TRO)--an experimental anti-neoplastic agent. The first step in activating these agents is catalyzed by dCK. Our studies reveal how dCK, which normally catalyzes phosphorylation of the natural D-nucleosides, can efficiently phosphorylate substrates with non-physiologic chirality. The capability of dCK to phosphorylate both D- and L-nucleosides and nucleoside analogs derives from structural properties of both the enzyme and the substrates themselves. First, the nucleoside-binding site tolerates substrates with different chiral configurations by maintaining virtually all of the protein-ligand interactions responsible for productive substrate positioning. Second, the pseudo-symmetry of nucleosides and nucleoside analogs in combination with their conformational flexibility allows the L- and D-enantiomeric forms to adopt similar shapes when bound to the enzyme. This is the first analysis of the structural basis for activation of L-nucleoside analogs, providing further impetus for discovery and clinical development of new agents in this molecular class.

Anti-HIV Agents↗
PubMed2001

[The contents and their change of nucleosides from natural Cordyceps sinensis and cultured cordyceps mycelia].

AIM: To compare the content of nucleosides from natural Cordyceps sinensis and cultured Cordyceps mycelia, and the effect of humidity and heat on the contents of nucleosides. METHODS: The contents of nucleosides were determined using high performance capillary electrophoresis (HPCE). Beckman P/ACE System 5010 apparatus equipped with a UV detector and a Beckman untreated fused-silica capillary (57 cm x 75 microns, 50 cm effective length) were used. Before sample injection, the capillary was rinsed with 1 mol.L-1 sodium hydroxide and running buffer for 5 minutes, respectively. 20 kV was applied during separation. Pressure injection was 586 kPa for 6 seconds, and the wavelength of detector was 254 nm. The running time was 20 minutes at 20 degrees C. The effect of humidity and heat on the content of nucleosides from natural Cordyceps sinensis and cultured Cordyceps mycelia was observed for 1, 3, 5 and 10 days at temperature 40 degrees C, and relative humidity 75%. RESULTS: The contents of nucleosides from cultured Cordyceps mycelia were higher than that of those from natural Cordyceps sinensis. The contents of nucleosides from freshly collected natural Cordyceps sinensis, were under the detection limit. The contents of nucleosides from natural Cordyceps sinensis were significantly increased by effect of humidity and heat, the phenomenon was not observed in cultured Cordyceps mycelia. CONCLUSION: There are difference in nucleosides content between natural Cordyceps sinensis and cultured Cordyceps mycelia. The nucleosides in natural Cordyceps sinensis may be derived from the degradation of macro-molecular nucleic acids. That means it is probably controversial that adenosine was used for quality control of natural Cordyceps sinensis.

Adenosine↗
PubMed1979

Nucleoside transport in cells and membrane vesicles from Escherichia coli K12.

Osmotic shock treatment of cells of Escherichia coli K12 caused a reduction in the transport of nucleosides into the cells. The strains used carried mutations in the nucleoside catabolizing enzymes. This indicated that the decrease in transport capacity was not due to loss of these enzymes during the shock treatment. Membrane vesicles, prepared from the same strains, showed a limited transport of cytidine, deoxycytidine, and uridine. Transport of purine nucleosides and of thymidine was very low in vesicles lacking the appropriate nucleoside phosphorylases and no significant stimulation was observed if the nucleoside phosphorylases were present in the membrane vesicles. These results all indicate that components outside the cytoplasmic membrane are important for nucleoside transport. Selection for resistance to fluorodeoxycytidine yielded mutants which were unable to transport any nucleoside, even when the nucleoside phosphorylases were present in high amounts. This finding is consistent with a requirement for a specific transport process prior to the initial enzymatic attack on the incoming nucleoside.

Biological Transport↗
PubMed1986

Potential use of purine nucleosides and enzyme inhibitors for selective depletion of Thy-lymphoblasts from human bone marrow.

The toxicity of the purine nucleoside, deoxyadenosine in the presence of the adenosine deaminase inhibitor, deoxycoformycin and of deoxyguanosine in the presence of the purine nucleoside phosphorylase inhibitor, 8-aminoguanosine was measured against two Thy-leukemic cell lines. Toxicity was assessed by the survival of clonogenic cells in a colony assay. The kill of clonogenic Thy-leukemic cells was 99.99% with both nucleoside enzyme inhibitor combinations following 4-h incubations when 50 microM concentration of nucleoside were used. With these nucleoside concentrations some reduction in toxicity was apparent when drug treated cells were cultured in the presence of deoxycytidine (50 microM), however, this reduction in toxicity was not apparent when higher nucleoside concentrations were used (100 microM). Survival of bone marrow myeloid progenitor cells (CFU.GM) was only slightly reduced by these nucleoside concentrations following 4 hour incubations. The presence of a twenty-fold excess of normal bone marrow cells reduced the cytotoxic effect but clonogenic cell incubation still ranged from 99.98 to 99.99% for deoxyguanosine and deoxyadenosine respectively. These combinations of nucleosides and enzyme inhibitors may have a therapeutic role in the elimination of malignant Thy cells from human bone marrow.

Adenosine Deaminase Inhibitors↗
PubMed2003

Functional expression and characterization of a sodium-dependent nucleoside transporter hCNT2 cloned from human duodenum.

We have cloned and functionally expressed a sodium-dependent human nucleoside transporter, hCNT2, from a CNS cancer cell line U251. Our cDNA clone of hCNT2 had the same predicted amino acid sequence as the previously cloned hCNT2 transporter. Of the several cell lines studied, the best hCNT2 transport function was obtained when transiently expressed in U251 cells. Na(+)-dependent uptake of [3H]inosine in U251 cells transiently expressing hCNT2 was 50-fold greater than that in non-transfected cells, and uptake in Na(+)-containing medium was approximately 30-fold higher than that at Na(+)-free condition. The hCNT2 displayed saturable uptake of [3H]inosine with K(m) of 12.8 microM and V(max) of 6.66 pmol/mg protein/5 min. Uptake of [3H]inosine was significantly inhibited by the purine nucleoside drugs dideoxyinosine and cladribine, but not by acyclic nucleosides including acyclovir, ganciclovir, and their prodrugs valacyclovir and valganciclovir. This indicates that the closed ribose ring is important for binding of nucleoside drugs to hCNT2. Among several pyrimidine nucleosides, hCNT2 favorably interacted with the uridine analogue floxuridine. Interestingly, we found that benzimidazole analogues, including maribavir, 5,6-dichloro-2-bromo-1-beta-D-ribofuranosylbenzimidazole (BDCRB), and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), were strong inhibitors of inosine transport, even though they have a significantly different heterocycle structure compared to a typical purine ring. As measured by GeneChip arrays, mRNA expression of hCNT2 in human duodenum was 15-fold greater than that of hCNT1 or hENT2. Further, the rCNT2 expression in rat duodenum was 20-fold higher than rCNT1, rENT1 or rENT2. This suggests that hCNT2 (and rCNT2) may have a significant role in uptake of nucleoside drugs from the intestine and is a potential transporter target for the development of nucleoside and nucleoside-mimetic drugs.

Animals↗
PubMed1991

Multiple sodium-dependent nucleoside transport systems in bovine renal brush-border membrane vesicles.

Na(+)-dependent nucleoside transport was examined in bovine renal brush-border membrane vesicles. Two separate Na+/nucleoside cotransporters were shown to be present: (1) a system specific for purine nucleosides and uridine, designated as the N1 carrier, and (2) an Na(+)-dependent nucleoside transporter that accepts pyrimidine nucleosides, adenosine and analogues of adenosine, designated as the N2 system. Both systems exhibit a high affinity for nucleosides (apparent Km values approximately 10 microM), are insensitive to inhibition by facilitated-diffusion nucleoside transport inhibitors, are rheogenic and exhibit a high specificity for Na+. Na+ increases the affinity of the influx of guanosine and thymidine, nucleosides that serve as model permeants for the N1 and N2 nucleoside transporters respectively. The Na+/nucleoside coupling stoichiometry is consistent with 1:1 for both carriers.

Animals↗
PubMed1987

Residual nitrobenzylthioinosine-resistant nucleoside transport in a transport mutant (AE1) of S49 murine T-lymphoma cells.

The uptake of various nucleosides by S49 mouse T-lymphoma cells and that by a single-step nucleoside transport-defective mutant thereof (AE1) were compared. Residual nucleoside entry into AE1 cells occurred via two routes, nonmediated permeation and saturable, non-concentrative transport with broad substrate specificity and a Michaelis-Menten constant approximating that for thymidine transport in wild-type cells. However, in contrast to nucleoside transport in wild-type cells, residual nucleoside transport in AE1 cells was resistant to inhibition by nitrobenzylthioinosine. In its properties the latter resembled nitrobenzylthioinosine-resistant nucleoside transport observed in other types of mammalian cells. It amounted to less than 1% of the total nucleoside transport activity of wild-type S49 cells. The results indicate that nitrobenzylthioinosine-resistant and -sensitive nucleoside transports are genetically distinguishable. In wild-type cells, the salvage of thymidine, when present at concentrations higher than 1 to 10 microM, was limited by phosphorylation, because of the saturation of thymidine kinase. In AE1 cells, entry into the cells mainly limited thymidine salvage, but at high thymidine concentrations the combined entry via residual transport and nonmediated permeation was sufficiently rapid to support intracellular thymidine phosphorylation at rates comparable to those observed in wild-type cells.

Animals↗
PubMed1998

Es nucleoside transporter content of acute leukemia cells: role in cell sensitivity to cytarabine (araC).

Nucleoside analogs are important components of treatment regimens for acute leukemia in adults. Plasma membrane permeation of the nucleoside analog molecules, the initial event in the cellular conversion of nucleosides to active agents, is mediated by nucleoside-specific membrane transporters. The widely-expressed es nucleoside transporter accepts as substrates diverse nucleoside analogs, including cytarabine (araC), 2-chlorodeoxyadenosine, and fludarabine. The cellular content of es transporter sites has been measured in blasts from patients with acute lymphoblastic leukemia and acute myelogenous leukemia, by a sensitive, quantitative flow cytometry assay that employs the tightly-bound es ligand, SAENTA fluorescein. Values for es transporter expression varied ten-fold among samples from patients with acute myelogenous leukemia. In this article, we review current findings that document, in confocal fluorescence microscopy images and in flow cytometry assays of SAENTA fluorescein-stained cells, the patient-to-patient variance of es transporter expression in leukemic blasts from patients. Our data show a correlation between the expression of es transporters and the in vitro sensitivity to nucleoside drugs of blasts from acute leukemia patients. These findings show that the flow cytometry assay of es expression provides a facile means of predicting resistance of leukemia cells to the cytotoxicity of araC and other nucleosides.

Acute Disease↗
PubMed1992

A nucleoside transporter is functionally linked to ectonucleotidases in rat liver canalicular membrane.

Prevention of nucleoside loss in bile is physiologically desirable because hepatocytes are the main source of nucleosides for animal cells which lack de novo nucleoside biosynthesis. We have demonstrated a Na+ gradient-energized, concentrative nucleoside transport system in canalicular membrane vesicles (CMV) from rat liver by studying [3H]adenosine uptake using a rapid filtration technique. The Na(+)-dependent nucleoside transporter accepts purine, analogues of purine nucleosides and uridine; exhibits high affinity for adenosine (apparent Km, 14 microM); is not inhibited by nitrobenzylthioinosine or dipyridamole, and is present in CMV but not in rat liver sinusoidal membrane vesicles. Adenosine transport in right side-out CMV was substantially greater than with inside-out CMV. CMV also contain abundant ecto-ATPase and ecto-AMPase (5'-nucleotidase). These ectoenzymes were shown to degrade nucleotides into nucleosides which were conserved by the Na(+)-dependent nucleoside transport system.

Adenosine↗
PubMed2005

Recent advances in studies on biochemical and structural properties of equilibrative and concentrative nucleoside transporters.

Nucleoside transporters (NT) facilitate the movement of nucleosides and nucleobases across cell membranes. NT-mediated transport is vital for the synthesis of nucleic acids in cells that lack de novo purine synthesis. Some nucleosides display biological activity and act as signalling molecules. For example, adenosine exerts a potent action on many physiological processes including vasodilatation, hormone and neurotransmitter release, platelet aggregation, and lipolysis. Therefore, carrier-mediated transport of this nucleoside plays an important role in modulating cell function, because the efficiency of the transport processes determines adenosine availability to its receptors or to metabolizing enzymes. Nucleoside transporters are also key elements in anticancer and antiviral therapy with the use of nucleoside analogues. Mammalian cells possess two major nucleoside transporter families: equilibrative (ENT) and concentrative (CNT) Na(+)-dependent ones. This review characterizes gene loci, substrate specificity, tissue distribution, membrane topology and structure of ENT and CNT proteins. Regulation of nucleoside transporters by various factors is also presented.

Animals↗
PubMed2001

Nucleoside transporter subtype expression: effects on potency of adenosine kinase inhibitors.

1. Adenosine kinase (AK) inhibitors can enhance adenosine levels and potentiate adenosine receptor activation. As the AK inhibitors 5' iodotubercidin (ITU) and 5-amino-5'-deoxyadenosine (NH(2)dAdo) are nucleoside analogues, we hypothesized that nucleoside transporter subtype expression can affect the potency of these inhibitors in intact cells. 3. Three nucleoside transporter subtypes that mediate adenosine permeation of rat cells have been characterized and cloned: equilibrative transporters rENT1 and rENT2 and concentrative transporter rCNT2. We stably transfected rat C6 glioma cells, which express rENT2 nucleoside transporters, with rENT1 (rENT1-C6 cells) or rCNT2 (rCNT2-C6 cells) nucleoside transporters. 3. We tested the effects of ITU and NH(2)dAdo on [(3)H]-adenosine uptake and conversion to [(3)H]-adenine nucleotides in the three cell types. NH(2)dAdo did not show any cell type selectivity. In contrast, ITU showed significant inhibition of [(3)H]-adenosine uptake and [(3)H]-adenine nucleotide formation at concentrations < or =100 nM in rENT1-C6 cells, while concentrations > or =3 microM were required for C6 or rCNT2-C6 cells. 4. Nitrobenzylthioinosine (NBMPR; 100 nM), a selective inhibitor of rENT1, abolished the effects of nanomolar concentrations of ITU in rENT1-C6 cells. 5. This study demonstrates that the effects of ITU, but not NH(2)dAdo, in whole cell assays are dependent upon nucleoside transporter subtype expression. Thus, cellular and tissue differences in expression of nucleoside transporter subtypes may affect the pharmacological actions of some AK inhibitors.

Adenine Nucleotides↗
PubMed2006

Direct determination of nucleosides in the urine of patients with breast cancer using column-switching liquid chromatography-tandem mass spectrometry.

We developed an analytical method for a simple, sensitive and simultaneous determination of oxidized nucleosides in urine using column-switching liquid chromatography-electrospray/tandem mass spectrometry (LC-ESI/MS/MS). We connected two columns through a six-way switching valve and effectively separated nucleosides in the urine from the interference by column-switching liquid chromatography. We monitored separated nucleosides using positive ionization tandem mass spectrometry in selective reaction monitoring (SRM) mode. The calibration ranges of nucleosides were 0.2-100 nmol/mL. The linearity of the method was 0.994-0.999, and the limits-of-detection (LOD) at a signal-to-noise (S/N) ratio of 3 were 0.1-0.2 nmol/mL. The coefficients of variation were in the range 2.28-11.74% for within-day variation and 4.36-11.15% for day-to-day variation, respectively. To explore the relationship between breast cancer and the nucleosides level in human urine, we measured the concentrations of nucleosides in female patients with breast cancer (n = 30) and in normal female subjects (n = 30). The concentration of nucleosides was significantly increased in patients with breast cancer when compared with the normal controls (1-methyladenosine; p < 0.005, N(2),N(2)-dimethylguanosine; p < 0.01, 5-hydroxymethyl-2'-deoxyuridine; p < 0.001, 8-hydroxy-2-deoxyguanosine; p < 0.001). Therefore, the elevated levels of nucleosides could be used as an important biomarker for breast-cancer research.

Adult↗
PubMed1983

Comparison of urinary modified nucleosides and bases in rats with hepatomas and nephroblastomas.

Hepatomas were induced in rats with aflatoxin B1, and nephroblastomas with dimethylnitrosamine. Microscopic examination of livers of aflatoxin-treated rats revealed multinodular hepatocyte hyperplasia at 8 months, and by 13 months all rats had hepatomas. Nephroblastomas were observed by 4 months and by 8 months all rats had developed them. The urinary excretion of several modified nucleosides and bases by normal rats is dependent on body weight and reflects, to a certain extent, their concentrations in tissue tRNA. Increased levels of several modified nucleosides and bases were found in all rats that had cancer. Rats with hepatomas excreted essentially the same modified nucleosides and bases as did those with nephroblastomas; the quantitative patterns of excretion were different, however, suggesting that the urinary modified nucleosides and bases may be used to differentiate between neoplasms. Although the increase in urinary modified nucleosides and bases by tumor-bearing animals results primarily from more rapid turnover of neoplastic tRNAs, the data indicate that increased turnover of mRNA and possibly rRNA may occur in neoplastic tissue. Preliminary data suggest that increases in urinary modified nucleosides and bases may occur during a precancerous stage. The urinary pattern of modified nucleosides and bases by rats with hepatomas is altered if another primary tumor is present. The results obtained from these studies support the use of modified nucleosides and bases in urine as biochemical markers of cancer.

Aflatoxin B1↗
PubMed1987

Membrane transport and the antineoplastic action of nucleoside analogues.

This article summarizes recent studies characterizing nucleoside transport in mammalian cells and discusses evidence for a role of membrane transport in the pharmacologic action of nucleoside analogues. Some of these studies have also addressed the controversy concerning the multiplicity in transport routes. It seems clear that erythrocytes and, perhaps, some other mammalian cells possess a single, broadly specific system for transporting nucleosides. However, substantial evidence from valid studies discriminating between transport and intracellular metabolism suggests that at least some mammalian cells, including some tumor cells, possess more than a single system. Evidence now exists for a determining role of membrane transport of nucleoside analogues in their cytotoxicity and, in the case of one pyrimidine nucleoside (AraC), in therapeutic responsiveness in leukemic patients. There are also numerous examples of transport-related resistance to nucleoside analogues. Included in this article are the results of studies from the authors' laboratory pertaining to the therapeutic activity of the purine nucleoside, FAraA, in murine tumor models. These studies provide evidence for a determining role of both membrane transport and intracellular phosphorylation in the selective antitumor action of this agent against murine leukemia. Substantially increased transport inward of FAraA occurs at pharmacologically achievable concentrations of this agent in tumor cells as compared to drug-limiting, normal proliferative epithelium of the small intestine. The basis for this differential appears to be the kinetic duality of FAraA and adenosine transport inward found in tumor cells, but not in proliferative intestinal epithelial cells. Tumor cells have highly saturable (low influx Km) and poorly saturable (high influx Km) systems for adenosine transport, both of which are shared by FAraA. In contrast, proliferative epithelial cells have only a poorly saturable system for these substrates. If a similar kinetic duality of nucleoside transport is found in other tumor cells certain implications arise concerning the significance of the duality to neoplastic transformation.

Animals↗
PubMed1986

The inhibition of nucleic acid synthesis in encephalomyocarditis virus-infected L929 cells: effects on nucleoside transport.

Picornavirus infection induces a profound inhibition of labelling of newly synthesized RNA in some cell lines. EMC virus blocks transcription in L929 cells, particularly at early times during infection. This inhibition is not dependent on virus gene expression, since it occurs with UV-inactivated virus and also in the presence of translation inhibitors. The inhibition can be largely accounted for by the blockade of [3H]nucleoside transport, as suggested by the transport kinetics and incorporation of labelled nucleoside from preloaded cells. The inhibition of transport and incorporation into TCA-precipitable material was observed with pyrimidine (uridine, thymidine and cytosine) and purine nucleosides (adenosine and guanosine), but the blockade by EMC virus was higher with the latter nucleosides. Preloading of cells with any of these nucleosides resulted in a decreased effect on nucleoside incorporation into nucleic acid after virus infection. These results suggest that the inhibition of incorporation of labelled nucleosides into nucleic acid in EMC virus-infected cells can be explained, at least in part, by the decreased pool size of the phosphorylated nucleosides. These effects are not specific for L cells, because they are also observed in other cell lines.

Animals↗
PubMed2005

Functional coupling between nucleoside diphosphate kinase of the outer mitochondrial compartment and oxidative phosphorylation.

In rat liver mitochondria all nucleoside diphosphate kinase of the outer compartment is associated with the outer surface of the outer membrane (Lipskaya, T. Yu., and Plakida, K. N. (2003) Biochemistry (Moscow), 68, 1136-1144). In the present study, three systems operating as ADP donors for oxidative phosphorylation have been investigated. The outer membrane bound nucleoside diphosphate kinase was the first system tested. Two others employed yeast hexokinase and yeast nucleoside diphosphate kinase. The two enzymes exhibited the same activity but could not bind to mitochondrial membranes. In all three systems, muscle creatine phosphokinase was the external agent competing with the oxidative phosphorylation system for ADP. Determination of mitochondrial respiration rate in the presence of increasing quantities of creatine phosphokinase revealed that at large excess of creatine phosphokinase activity over other kinase activities (of the three systems tested) and oxidative phosphorylation the creatine phosphokinase reaction reached a quasi-equilibrium state. Under these conditions equilibrium concentrations of all creatine phosphokinase substrates were determined and K(eq)app of this reaction was calculated for the system with yeast hexokinase. In samples containing active mitochondrial nucleoside diphosphate kinase the concentrations of ATP, creatine, and phosphocreatine were determined and the quasi-equilibrium concentration of ADP was calculated using the K(eq)app value. At balance of quasi-equilibrium concentrations of ADP and ATP/ADP ratio the mitochondrial respiration rate in the system containing nucleoside diphosphate kinase was 21% of the respiration rate assayed in the absence of creatine phosphokinase; in the system containing yeast hexokinase this parameter was only 7% of the respiration rate assayed in the absence of creatine phosphokinase. Substitution of mitochondrial nucleoside diphosphate kinase with yeast nucleoside diphosphate kinase abolished this difference. It is concluded that oxidative phosphorylation is accompanied by appearance of functional coupling between mitochondrial nucleoside diphosphate kinase and the oxidative phosphorylation system. Possible mechanisms of this coupling are discussed.

Adenosine Diphosphate↗