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.     

核苷二磷酸激酶A的异构及其分子机制

对核苷二磷酸激酶A(NDPKA)的异构及其分子机制进行研究.还原和非还原SDSPAGE观察重组人核苷二磷酸激酶A(rhNDPKA)的异构;RPHPLC分析rhNDPKA异构体的反相色谱行为,并测定rhNDPKA异构体的酶活性;多角度激光散射法测定rhNDPKA异构体在溶液中的表观分子量;飞行质谱分析异构体的质量肽谱.结果发现,rhNDPKA在非还原SDSPAGE上表现为4条带,对应于NDPKA的氧化型、还原型、氧化型二聚体和还原型二聚体,其分子量分别为18.1kD、21.3kD、35.2kD和38.3kD.RPHPLC发现,还原型rhNDPKA和氧化型rhNDPKA疏水性有差异.新鲜制备的rhNDPKA在纯水溶液中,经空气氧化后,逐渐由还原型向氧化型过渡,而还原剂或生理盐水可使rhNDPKA稳定于还原型或氧化型.酶活测定结果表明,还原型rhNDPKA比活性为1965±166Umg,氧化型rhNDPKA比活性为974±53Umg.多角度激光散射检测发现,还原型rhNDPKA在溶液中仍可形成六聚体.质量肽谱结果证明,在氧化型rhNDPKA中,C4和C145位巯基形成二硫键,而C109位巯基游离存在.根据本文所确定的NDPKA单体中的二硫键位置,推导出rhNDPKA单体异构体和二聚体异构体的变构原理,这为进一步研究NDPKA的多能性调节机制打下了良好基础.     

Structural and Functional Characterization of Acinetobacter baumannii Nucleoside Diphosphate Kinase

近年来,鲍曼不动杆菌(Acinetobacter baumannii)在医院里越来越受到人们的关注,尤其是在重症监护病房(ICUs)．它以强大的多重耐药性(multiresistance)而闻名．核苷二磷酸激酶(nucleoside diphosphate kinase,NDK)是一种进化上非常保守的酶,它能催化核苷之间磷酸基团的转移．我们解析了鲍曼不动杆菌NDK野生型和C端氨基酸残基Arg141-Thr142-Arg143(RTR)截短突变体的结构．通过和黄色黏菌(Myxococcus xanthus)NDK的三维结构进行比较,推断鲍曼不动杆菌NDK的催化机制和黄色黏菌类似．通过激酶活性实验和圆二色谱实验,发现鲍曼不动杆菌NDK E28A突变体二级结构发生了改变,从而导致蛋白催化活性降低,说明Glu28是鲍曼不动杆菌NDK结构中非常关键的氨基酸残基．鲍曼不动杆菌NDK C端RTR截短突变体显示出催化活性极大的降低,这可能与C端RTR残基介导的二体间相互作用有关．虽然RTR截短突变体中的Lys33伸向了和野生型中不同的方向,和Val15产生相互作用弥补了一部分因为RTR截短丢失的相互作用,维持了RTR截短突变体和野生型类似的结构．但是,Lys33产生的相互作用依然太弱,不足以维持蛋白在催化的动态过程中整体结构的高效转换．我们解析的鲍曼不动杆菌NDK晶体高分辨率结构将有助于科学家设计针对鲍曼不动杆菌的药物．     

Nucleoside Diphosphate Kinase Is a Possible Component of the Ethylene Signal Transduction Pathway

In etiolated seedlings of Pisum sativum and leaves of Arabidopsis thaliana, in vivo ethylene treatment resulted in an increase in in vitro phosphorylation of 17 kD (P. sativum) or 16 and 17 kD (A. thaliana) polypeptides. These polypeptides were identified as nucleoside diphosphate kinase (NDPK) based on both biochemical properties and interaction with antibodies against NDPK from P. sativum. Using the receptor-directed antagonist of ethylene action 2,5-norbornadiene and the ethylene-insensitive mutants of A. thaliana etr1-1 and eti5, ethylene specificity and receptor dependence of NDPK phosphorylation have been demonstrated. In pea epicotyls, ethylene treatment also led to increase in nucleoside transferase activity unlike in A. thaliana leaves. The increases in nucleoside transferase activity and NDPK phosphorylation were very rapid and transient. The results suggest a role for NDPK as a possible component of the ethylene signal transduction chain.     

Subcellular Localization and Characterization of Nucleoside Diphosphate Kinase in Rat Retina: Effect of Diabetes

Nucleoside diphosphate kinase (NDP kinase) catalyzes the transfer of terminal phosphate from nucleotide triphosphates (e.g. ATP) to nucleotide diphosphates (e.g. GDP) to yield nucleotide triphosphates (e.g. GTP). Since guanine nucleotides play critical role(s) in GTP-binding protein (G-protein)-mediated signal transduction mechanisms in retina, we quantitated NDP kinase activity in subcellular fraction-derived from normal rat retina. A greater than 85% of the total specific activity was present in the soluble fraction, which was stimulated (up to 7 fold) by 2 mM magnesium. NDP kinase exhibited saturation kinetics towards di- and tri-phosphate substrates, and was inhibited by known inhibitors of NDP kinase, uridine diphosphate (UDP) or cromoglycate (CRG). We have previously reported significant abnormalities in the activation of G-proteins in streptozotocin (STZ)-diabetic rat retina (Kowluru et al. Diabetologia 35:624–631, 1992). Since NDP kinase hasbeen implicated in direct interaction with and/or activation of various G-proteins, we quantitated both basal and magnesium-stimulated NDP kinase activity in soluble and particulate fractions of retina derived from STZ-diabetic rats to examine whether abnormalities in G-protein function in diabetes are attributable to alterations in retinal NDP kinase. There was no effect of diabetes either on the basal or the magnesium-activated retinal NDP kinase activity. This study represents the first characterization of NDP kinase activity in rat retina, and suggests that in diabetes, this enzyme may not be rate-limiting and/or causal for the observed alterations in retinal G-protein functions.     

A Kinetic Study of the Rat Liver Adenosine Kinase Reverse Reaction

Adenosine kinase is an enzyme catalyzing the reaction: adenosine + ATP → AMP + ADP. We studied some biochemical properties not hitherto investigated and demonstrated that the reaction can be easily reversed when coupled with adenosine deaminase, which transforms adenosine into inosine and ammonia. The overall reaction is: AMP + ADP → ATP + inosine + NH₃. The exoergonic ADA reaction shifts the equilibrium and fills the energy gap necessary for synthesis of ATP. This reaction could be used by cells under particular conditions of energy deficiency and, together with myokinase activity, may help to restore physiological ATP levels.     

nHAP对大鼠肝线粒体的生物活性作用

为了探讨羟基磷灰石纳米粒子(nHAP)对大鼠肝线粒体生物活性的影响,将nHAP直接作用于线粒体,在不同浓度和时间下测定线粒体标志酶琥珀酸脱氢酶(SDH)比活性,并与对照组进行比较。结果显示,当nHAP中水含量在10%以下时,线粒体生物活性未发现改变;当nHAP浓度递增时,在等时间段内,对线粒体SDH比活性呈逐步抑制作用;在不等时间段内,nHAP对线粒体SDH比活性的抑制作用与对照组相比较差异有显著性(p<0.05)。因此,nHAP对线粒体SDH比活性的抑制有浓度和时间的依赖性。     

Reversibility of nucleoside diphosphate kinase solubilization from the surface of the outer mitochondrial membrane

It was found that in medium with low ionic strength nucleoside diphosphate kinase (NDPK) solubilization from the outer membrane of liver mitochondria could be partially reversed by the addition of 3.3 mM MgCl₂. Complete rebinding of the enzyme after the addition of MgCl₂ was observed when the mitochondrial washing and storage medium contained leupeptin, an inhibitor of cathepsins. It was demonstrated that leupeptin and another inhibitor of cysteine proteinases, E-64, do not influence the rate of NDPK solubilization as well as its solubilized and membrane-associated activity. We conclude that NDPK becomes sensitive to proteolysis only after its solubilization; proteolysis does not affect the part of the enzyme molecule that is responsible for catalysis. After solubilization of NDPK in the absence of leupeptin, cathepsins damage sites of its binding on the membranes. The rate of the enzyme solubilization is dependent on the pH of the storage medium (pH 6.0–8.0); it decreases with increase in pH. It was shown that in the medium with high ionic strength, MgCl₂ does not reverse pH-dependent NDPK solubilization, but solubilization could be reversed by increase in medium pH in the presence of E-64 and BSA. The physiological importance of these results is discussed. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 5, pp. 710–720.     

Mitochondrial nucleoside diphosphate kinase: Mode of interaction with the outer mitochondrial membrane and proportion of catalytic activity functionally coupled to oxidative phosphorylation

In the present study, we found that ionic interactions are not essential for the binding of nucleoside diphosphate kinase of liver mitochondria outer compartment to outer mitochondrial membrane and that the proportion of the enzyme activity involved in functional coupling with oxidative phosphorylation (we demonstrated the existence of functional coupling earlier) is only 17%. Additional evidence was obtained that functionally coupled activity of nucleoside diphosphate kinase is associated with the outer surface of mitochondria. Dextran (10%) did not increase functional coupling. The physological importance of these effects is discussed. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 3, pp. 395–407.     

Response of Isolated Rat Liver Mitochondria to Variation of External Osmolarity in KCl Medium: Regulation of Matrix Volume and Oxidative Phosphorylation

When isolated rat liver mitochondria are incubated in KCl medium, matrix volume, flux, and forces in both hypo- and hyperosmolarity are time-dependent. In hypoosmotic KCl medium, matrix volume is regulated via the K⁺/H⁺ exchanger. In hyperosmotic medium, the volume is regulated in such a manner that at steady state, which is reached within 4 min, it is maintained whatever the hyperosmolarity. This regulation is Pi- and -dependent, indicating Pi-K salt entry into the matrix. Under steady state, hyperosmolarity has no effect on isolated rat liver mitochondria energetic parameters such as respiratory rate, proton electrochemical potential difference, and oxidative phosphorylation yield. Hypoosmolarity decreases the NADH/NAD⁺ ratio, state 3 respiratory rate, and , while oxidative phosphorylation yield is not significantly modified. This indicates kinetic control upstream the respiratory chain. This study points out the key role of potassium on the regulation of matrix volume, flux, and forces. Indeed, while matrix volume is regulated in NaCl hyperosmotic medium, flux and force restoration in hyperosmotic medium occurs only in the presence of external potassium.     

THE SUBMITOCHONDRIAL LOCALIZATION OF MONOAMINE OXIDASE : An Enzymatic Marker for the Outer Membrane of Rat Liver Mitochondria

Controlled osmotic lysis (water-washing) of rat liver mitochondria results in a mixed population of small vesicles derived mainly from the outer mitochondrial membrane and of larger bodies containing a few cristae derived from the inner membrane. These elements have been separated on Ficoll and sucrose gradients. The small vesicles were rich in monoamine oxidase, and the large bodies were rich in cytochrome oxidase. Separation of the inner and outer membranes has also been accomplished by treating mitochondria with digitonin in an isotonic medium and fractionating the treated mitochondria by differential centrifugation. Treatment with low digitonin concentrations released monoamine oxidase activity from low speed mitochondrial pellets, and this release of enzymatic activity was correlated with the loss of the outer membrane as seen in the electron microscope. The low speed mitochondrial pellet contained most of the cytochrome oxidase and malate dehydrogenase activities of the intact mitochondria, while the monoamine oxidase activity could be recovered in the form of small vesicles by high speed centrifugation of the low speed supernatant. The results indicate that monoamine oxidase is found only in the outer mitochondrial membrane and that cytochrome oxidase is found only in the inner membrane. Digitonin treatment released more monoamine oxidase than cytochrome oxidase from sonic particles, thus indicating that digitonin preferentially degrades the outer mitochondrial membrane.     

In vivo cross-linking of nm23/nucleoside diphosphate kinase to the PDGF-A gene promoter

Human isoforms A and B of nm23/nucleoside diphosphate (NDP) kinase, functionally important in development and cancer, have been reported to bind to DNA, and in particular isoform A to the PDGF-A promoter and isoform B to the c-myc promoter and to telomeric repeats. However, no direct proof of the binding in vivo has yet been obtained. To demonstrate this interaction, human erythroleukemic K562 cells were incubated with two different cross-linking reagents, formaldehyde or cis-diammine dichloro platinum II. The DNA-protein covalent complexes were isolated and analyzed by Western blotting. The positive immunochemical staining showed that in both conditions NDP kinase isoforms A and B were efficiently cross-linked to DNA in vivo. NDP kinase-linked DNA fragments obtained by immunoprecipitation, subjected to hybridization with different probes, showed a definite enrichment in the nuclease-hypersensitive silencer element of the PDGF-A promoter. No conclusive evidence was found by this technique of preferential hybridization with a nuclease-hypersensitive element of the c-myc promoter and with the telomeric TTAGGG repeats. The immunoprecipitated NDP kinase-DNA complexes are a promising material for the detection of other specific DNA sequences interacting with NDP kinase.     

A Single-Nucleotide Polymorphism in Serine-Threonine Kinase 11, the Gene Encoding Liver Kinase B1, Is a Risk Factor for Multiple Sclerosis

We identified a family in which five siblings were diagnosed with multiple sclerosis (MS) or clinically isolated syndrome. Several women in the maternal lineage have comorbidities typically associated with Peutz Jeghers Syndrome, a rare autosomal-dominant disease caused by mutations in the serine-threonine-kinase 11 (STK11) gene, which encodes liver kinase B1. Sequence analysis of DNA from one sibling identified a single-nucleotide polymorphism (SNP) within STK11 intron 5. This SNP (dbSNP ID: rs9282860) was identified by TaqMan polymerase chain reaction (PCR) assays in DNA samples available from two other siblings. Further screening was carried out in samples from 654 relapsing-remitting MS patients, 100 primary progressive MS patients, and 661 controls. The STK11-SNP has increased frequency in all female patients versus controls (odds ratio = 1.66, 95% CI = 1.05, 2.64, p = .032). The STK11-SNP was not associated with disease duration or onset; however, it was significantly associated with reduced severity (assessed by MS severity scores), with the lowest scores in patients who also harbored the HLA-DRB1*1501 allele. In vitro studies showed that peripheral blood mononuclear cells from members of the family were more sensitive to the mitochondrial inhibitor metformin than cells from MS patients with the major STK11 allele. The increased association of SNP rs9282860 in women with MS defines this variant as a genetic risk factor. The lower disease severity observed in the context of HLA-DRB1*1501 combined with limited in vitro studies raises the provocative possibility that cells harboring the STK11-SNP could be targeted by drugs which increase metabolic stress.     

Localization and characterization of the mitochondrial isoform of the nucleoside diphosphate kinase in the pancreatic beta cell: evidence for its complexation with mitochondrial succinyl-CoA synthetase

Nucleoside diphosphate kinase (NDPK) catalyzes the transfer of terminal phosphates from nucleoside triphosphates to nucleoside diphosphates to yield nucleotide triphosphates. The present study was undertaken to localize and characterize the mitochondrial isoform of NDPK (mNDPK) in the pancreatic beta cell since it could contribute to the generation of mitochondrial nucleotide triphosphates and, thereby, to the mitochondrial high-energy phosphate metabolism of the pancreatic beta cell. Mitochondrial fractions from the insulin-secreting beta cells were isolated by differential centrifugation. mNDPK activity was assayed as the amount of [(3)H]GTPgammaS formed from ATPgammaS and [(3)H]GDP. Incubation of isolated mitochondrial extracts with either [gamma-(32)P]ATP or GTP resulted in the formation [(32)P]NDPK, which could be immunoprecipitated by an anti-NDPK serum. mNDPK exhibited saturation kinetics with respect to its nucleoside diphosphate acceptors and nucleoside triphosphate donors and sensitivity to known inhibitors of NDPK (e.g., uridine diphosphate and cromoglycate). By Western blot analyses, at least three isoforms of NDPK were identified in various subcellular fractions of the beta cell. The nm23-H1 (NDPK-A) was predominantly soluble whereas nm23-H2 (NDPK-B) was associated with the soluble as well as membranous fractions. The mitochondrial isoform of NDPK, nm23-H4, was uniformly distributed in the beta cell mitochondrial subfractions. A significant amount of NDPK (as determined by the catalytic activity and immunological methods) was recovered in the immunoprecipitates of mitochondrial fraction precipitated with an antiserum directed against succinyl-CoA synthetase (SCS), suggesting that NDPK might remain complexed with SCS. We provide the first evidence for the localization of a mitochondrial isoform of the NDPK in the islet beta cell and thus offer a potential mechanism for the generation of intramitochondrial GTP which, unlike ATP, is not transported into mitochondria via the classical nucleotide translocase. Further work will be required to determine the importance of the NDPK/SCS complex to normal beta cell function in the secretion of insulin.     

Mitochondria as the target for mildronate's protective effects in azidothymidine (AZT)-induced toxicity of isolated rat liver mitochondria

Previously mildronate, an aza-butyrobetaine derivative, was shown to be a cytoprotective drug, through its mechanism of action of inhibition of carnitine palmitoyltransferase-1, thus protecting mitochondria from long-chain fatty acid accumulation and subsequent damage. Recently in an azidothymidine (AZT)-induced cardiotoxicity model in vivo (in mice), we have found mildronate's ability of protecting heart tissue from nuclear factor kappaB abnormal expression. Preliminary data also demonstrate cerebro- and hepatoprotecting properties of mildronate in AZT-toxicity models. We suggest that mildronate may target its action predominantly to mitochondria. The present study in isolated rat liver mitochondria was designed to clarify mitochondrial targets for mildronate by using AZT as a model compound. The aim of this study was to investigate: (1) whether mildronate may protect mitochondria from AZT-induced toxicity; and (2) which is the most critical target in mitochondrial processes that is responsible for mildronate's regulatory action. The results showed that mildronate protected mitochondria from AZT-induced damage predominantly at the level of complex I, mainly by reducing hydrogen peroxide generation. Significant protection of AZT-caused inhibition of uncoupled respiration, ADP to oxygen ratio, and transmembrane potential were also observed. Mildronate per se had no effect on the bioenergetics, oxidative stress, or permeability transition of rat liver mitochondria. Since mitochondrial complex I is the first enzyme of the respiratory electron transport chain and its damage is considered to be responsible for different mitochondrial diseases, we may account for mildronate's effectiveness in the prevention of pathologies associated with mitochondrial dysfunctions.     

Features of the Uncoupling Effect of Fatty Acids in Liver Mitochondria of Mammals with Different Body Weight

In the presence of oligomycin, EGTA, and magnesium ions, the protonophore uncoupling activity of palmitate (V(Pal)) is determined as the ratio of the acceleration of respiration with palmitate to its concentration. Under these conditions, V(Pal) in liver mitochondria of one-month-old rats with the body weight of 50 g is 1.46-fold higher than in liver mitochondria of adult rats with the body weight of 250 g, whereas the uncoupling activity of FCCP does not depend on the age of the animals. The difference in V(Pal) is mainly due to its component insensitive to carboxyatractylate and glutamate (V(Ins)). This value is 2.9-fold higher in mitochondria of one-month-old rats than in those of adult rats. The protonophore activity of palmitate is similar in liver mitochondria of four-day-old and adult rats. In liver mitochondria of adult mammals (mouse, rat, guinea pig, rabbit), V(Pal) decreases with increase in the body weight of the animals. In double logarithmic coordinates, the dependence of the V(Pal) value on the body weight is linear with slope angle tangent of -0.18. The V(Pal) value is mainly contributed by its component V(Ins). In the presence of calcium ions, palmitate induces the nonspecific permeability of the inner membrane of liver mitochondria (pore opening). This Ca2+-dependent uncoupling effect of palmitate is less pronounced in mitochondria of one-month-old rats than in those of adult rats. In mitochondria of adult animals (mice, rats, and guinea pigs), the Ca2+-dependent uncoupling activity of palmitate is virtually the same. It is concluded that the protonophore uncoupling effect of palmitate in liver mitochondria of mammals, unlike its Ca2+-dependent effect, is associated with thermogenesis at rest and also with production of additional heat on cooling of the animals.     

Reconstruction of Pathways Associated with Amino Acid Metabolism in Human Mitochondria

We have used a bioinformatics approach for the identification and reconstruction of metabolic pathways associated with amino acid metabolism in human mitochon- dria. Human mitochondrial proteins determined by experimental and computa- tional methods have been superposed on the reference pathways from the KEGG database to identify mitochondrial pathways. Enzymes at the entry and exit points for each reconstructed pathway were identified, and mitochondrial solute carrier proteins were determined where applicable. Intermediate enzymes in the mito- chondrial pathways were identified based on the annotations available from public databases, evidence in current literature, or our MITOPRED program, which pre- dicts the mitochondrial localization of proteins. Through integration of the data derived from experimental, bibliographical, and computational sources, we recon- structed the amino acid metabolic pathways in human mitochondria, which could help better understand the mitochondrial metabolism and its role in human health.