Brain hexokinase and intramitochondrial compartments of ATP: fact and artifact.
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Adenosine Triphosphate↗
Biomedical subjects
K Nicolay
Publications and source records attributed to K Nicolay.
Effect of doxorubicin on the order of the acyl chains of anionic and zwitterionic phospholipids in liquid-crystalline mixed model membranes: absence of drug-induced segregation of lipids into extended domains.
We investigated the effect of the antineoplastic drug doxorubicin on the order of the acyl chains in liquid-crystalline mixed bilayers consisting of dioleoylphosphatidylserine (DOPS) or -phosphatidic acid (DOPA), and dioleoylphosphatidylcholine (DOPC) or -phosphatidylethanolamine (DOPE). Previous 2H-NMR studies on bilayers consisting of a single species of di[11,11-2H2]oleoyl-labeled phospholipid showed that doxorubicin does not affect the acyl chain order of pure zwitterionic phospholipid but dramatically decreases the order of anionic phospholipid [de Wolf, F. A., et al. (1991) Biochim. Biophys. Acta 1096, 67-80]. In the present work, we studied mixed bilayers in which alternatively the anionic or the zwitterionic phospholipid component was 2H-labeled so as to monitor its individual acyl chain order. Doxorubicin decreased the order parameter of the mixed anionic and zwitterionic lipids by approximately the same amount and did not induce a clear segregation of the lipid components into extended, separate domains. The drug had a comparable disordering effect on mixed bilayers of unlabeled cardiolipin and 2H-labeled zwitterionic phospholipid, indicating the absence of extensive segregation also in that case. Upon addition of doxorubicin to bilayers consisting of 67 mol% DOPE and 33 mol% anionic phospholipid, a significant part of the lipid adopted the inverted hexagonal (HII) phase at 25 degrees C. This bilayer destabilization, which occurred only in mixtures of anionic phospholipid and sufficient amounts of DOPE, might be of physiological importance. Even upon formation of extended HII-phase domains, lipid segregation was not clearly detectable, since the relative distribution of 2H-labeled anionic phospholipid and [2H]DOPE between the bilayer phase and HII phase was very similar. Our findings argue against a role of extensive anionic/zwitterionic lipid segregation in the mechanism of action and toxicity of doxorubicin.
Doxorubicin↗
On the mechanism of the mitochondrial decarboxylation of phosphatidylserine.
To study intramitochondrial phospholipid flow, radiolabeled phosphatidylserine was introduced into isolated rat liver mitochondria from donor vesicles through the action of a nonspecific lipid transfer protein. Imported phosphatidylserine was rapidly decarboxylated to phosphatidylethanolamine. Both the imported phosphatidylserine and the formed phosphatidylethanolamine were confined to the outer membrane. The enzyme phosphatidylserine decarboxylase was shown to be located exclusively in the inner membrane. It was not enriched in isolated contact site fractions. 1,4-Dinitrophenol caused an inhibition of the decarboxylation of phosphatidylserine. This inhibition was not due to the uncoupling of the oxidative phosphorylation itself, but possibly due to a decrease in the number of contact sites. This suggests that phosphatidylserine flows from the outer membrane to the inner membrane through contact sites between inner and outer membrane to become decarboxylated and that the formed phosphatidylethanolamine flows directly back to the outer membrane, without mixing with inner membrane phosphatidylethanolamine.
Animals↗
Intracellular compartmentation, structure and function of creatine kinase isoenzymes in tissues with high and fluctuating energy demands: the 'phosphocreatine circuit' for cellular energy homeostasis.
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Adenosine Triphosphate↗
1H-NMR imaging of fimbria fornix lesions in the rat brain.
Mechanical lesions of the fimbria fornix (FF) have been widely used as a model to investigate the recovery of damaged brain tissue. 1H-NMR imaging was employed to non-invasively measure changes in the brain after unilateral FF transection. Rats were subjected to NMR imaging at various times after the lesions were made. The experimental protocol included (multislice) T2-weighted and diffusion-weighted imaging thereby allowing the construction of two-dimensional maps of the relaxation time T2 (transverse or spine-spin relaxation time) and the apparent diffusion coefficient (ADC) of water. FF transection induced considerable changes in the status of the brain tissue at a number of different locations which were exclusively present in the affected hemisphere. At 1 day post-lesion the region of the lateral ventricle and hippocampus started to display pronounced changes in that T2- and diffusion-weighted images showed a hyperintensity and a hypointensity, respectively. These effects were maximal around day 2 to 4 whereafter a slow recovery towards the control situation was observed. Immediately after transection the FF lesion itself could be visualized. These early images pointed to an aspecific disruption of the tissue due to the mechanical intervention. Interestingly, however, from day 2 post-lesion a number of changes became evident in this region which seemed to be localized to specific structures, including the ventricle and hippocampus. After one month the presumably ventricle effect dominated and was predominantly localized to the anterior side of the FF lesion.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Temporal evolution of focal cerebral ischemia in the rat assessed by T2-weighted and diffusion-weighted magnetic resonance imaging.
The present study was undertaken to characterize the formation of ischemic brain edema using diffusion-weighted and T2-weighted magnetic resonance imaging in a rat model of focal ischemia. The extent of edema formation was measured from multislice diffusion-weighted and T2-weighted spin-echo images acquired at various times after ischemia. The spin-spin relaxation time (T2) and the apparent diffusion coefficient in normal and ischemic tissue were also determined. The results show that on the diffusion-weighted images the lesion was clearly visible at 30 minutes after ischemia, while on the T2-weighted images it became increasingly evident after 2-3 hours. On both types of images the hyperintense area increased in size over the first 48 hours. After 1 week the hyperintensity on the diffusion-weighted images rapidly disappeared and evolved as a hypointense lesion in the chronic phase. These results confirm the high sensitivity of diffusion-weighted MRI for the detection of early ischemia. The temporal course of the edema observed on T2W-images is in agreement with the reported increase of total water content occurring in this model. The increase of the lesion observed on the diffusion-weighted images during the first 2 days points to an aggravation of cytotoxic edema that parallels the changes in free water shown by the T2-weighted images. It is shown that the highly elevated T2's of the infarcted area several days after ischemia can substantially contaminate the diffusion-weighted images.
Animals↗
Mitochondrial creatine kinase mediates contact formation between mitochondrial membranes.
Purified mitochondrial creatine kinase (Mi-CK) (EC 2.7.3.2) from chicken heart was shown to interact simultaneously with purified inner and outer mitochondrial membranes, thereby creating an intermembrane chondrial membranes, thereby creating an intermembrane were purified from rat liver and thus were fully devoid of Mi-CK. Intermembrane contact formation was demonstrated by measuring the binding of inner membrane vesicles to outer membranes spread at the air-water interface. Mi-CK also mediated intermembrane adhesion when membranes formed with total lipid extracts of both membranes were used, pointing to the role of lipids as potential membrane anchors of Mi-CK in the mitochondrial intermembrane space. Other enzymes of the intermembrane space that (like Mi-CK) are also cationic, as well as cytosolic isoenzymes of creatine kinase, failed to induce contact formation. Thus, of the proteins tested, membrane contact formation was specific for Mi-CK. The two oligomeric forms of Mi-CK (octamer and dimer) differed in their ability to mediate intermembrane adhesion, the octamer being more potent. Highly basic peptides, i.e. poly-L-lysines, were shown to strongly interact with membranes formed with lipid extracts of mitochondrial membranes: they both induced intermembrane binding and fusion. Interestingly, the extent of contact formation mediated by poly-L-lysines was lower than that of octameric Mi-CK. The implications of these findings on the function and localization of Mi-CK and on the structure of the mitochondrial intermembrane compartment are discussed.
Animals↗
Interaction of mitochondrial creatine kinase with model membranes. A monolayer study.
The interaction of mitochondrial creatine kinase (Mi-CK; EC 2.7.3.2) with phospholipid monolayers and spread mitochondrial membranes at the air/water interface has been investigated. It appeared that Mi-CK penetrated into these monolayers as evidenced by an increase in surface pressure upon incorporation of Mi-CK. The increase in surface pressure was dependent on (1) the amount and (2) the oligomeric form of Mi-CK in the subphase, as well as on (3) the initial surface pressure and (4) the phospholipid composition of the monolayer. In this experimental system Mi-CK was able to interact equally well with both inner and outer mitochondrial membranes.
Animals↗
Comparable interaction of doxorubicin with various acidic phospholipids results in changes of lipid order and dynamics.
We have characterized the interaction of the antitumor drug doxorubicin with model membranes of the anionic phospholipids dioleoylphosphatidic acid (DOPA), dioleoylphosphatidylserine (DOPS), cardiolipin and dioleoylphosphatidylglycerol (DOPG) as compared to the zwitterionic dioleoylphosphatidylcholine (DOPC) or dioleoylphosphatidylethanolamine (DOPE). The saturating binding levels were: 2.4 (DOPA), 1.3 (cardiolipin), 1.5 (DOPS, DOPG) and 0.02 (DOPC) doxorubicin per lipid phosphorus (mol/mol). The half-saturating free drug concentrations were comparable for DOPA, cardiolipin, DOPS and DOPG: 20, 16, 35 and 18 microM, respectively. Doxorubicin fluorescence revealed the simultaneous existence of at least two populations of bound drug in the various anionic phospholipids: (1) fluorescent molecules with chromophores that reside between the lipid molecules and (2) above 0.01-0.02 doxorubicin bound per lipid phosphorus: non-fluorescent drug-stacks that are closer to the aqueous phase than the fluorescent molecules. Small-angle X-ray scattering indicated that doxorubicin can reorganize anionic phospholipid dispersions into closely-packed multilamellar structures. Addition of the drug caused leakage of entrapped 6-carboxyfluorescein. Neither 2H-NMR on [2-2H]serine-labelled DOPS nor 31P-NMR revealed any significant effect of doxorubicin on headgroup conformation, but 2H-NMR on di[11,11-2H2]oleoyl-labelled phospholipids showed that the drug had a strong acyl chain-disordering effect on anionic phospholipids. 2H-NMR relaxation measurements indicated that the drug immobilized the headgroups and acyl chains of anionic phospholipids. The implications of these observations for the cellular activity of the drug are indicated.
Animals↗
The role of contact sites between inner and outer mitochondrial membrane in energy transfer.
Three functions have been suggested to be localized in contact sites between the inner and the outer membrane of mitochondria from mammalian cells: (i) transfer of energy from matrix to cytosol through the action of peripheral kinases; (ii) import of mitochondrial precursor proteins; and (iii) transfer of lipids between outer and inner membrane. In the contact site-related energy transfer a number of kinases localized in the periphery of the mitochondrion play a crucial role. Two examples of such kinases are relevant here: (i) hexokinase isoenzyme I which is capable of binding to the outer aspect of the outer membrane; and (ii) the mitochondrial isoenzyme of creatine kinase which is localized in the intermembrane space. Recently, evidence was presented that both hexokinase and creatine kinase are preferentially localized in contact sites (Adams, V. et al. (1989) Biochim. Biophys. Acta 981, 213-225). The aim of the present experiments was two-fold. First, to establish methods which enable the bioenergetic aspects of energy transfer mediated by kinases in contact sites to be measured. In these experiments emphasis was on hexokinase, while 31P-NMR was the major experimental technique. Second, we wanted to develop methods which can give insight into factors playing a role in the formation of contact sites involved in energy transfer. In the latter approach, mitochondrial creatine kinase was studied using monolayer techniques.
Animals↗
The phosphatidylcholine-transfer protein catalyzed import of phosphatidylcholine into isolated rat liver mitochondria.
In order to study the individual steps involved in the import of phosphatidylcholine (PC) into rat liver mitochondria, a number of PC analogues were introduced into the outer membrane of isolated mitochondria. Two fluorescent PC species, i.e. 1-palmitoyl-2-(16-bimanylthio)hexadecanoyl-PC (bimane-PC) and 1-palmitoyl-2-(10-pyrene)decanoyl-PC (pyrene-PC), and one radiolabeled PC species, i.e. 1-palmitoyl-2-[1-14C]oleoyl-PC (14C-POPC), were studied. The PC analogues were introduced from small unilamellar vesicles with the use of PC-specific transfer protein. The amount of PC imported was quantified by reisolation of the mitochondria. Import of the fluorescent PC species was monitored by on-line fluorescence spectroscopy. The distribution of the newly inserted PC between the outer and the inner membrane was assessed by separation of the two membranes using digitonin treatment. All analogues tested remained exclusively localized in the outer membrane thereby suggesting that additional (extramitochondrial) factors are required to initiate transfer of PC to the inner membrane.
Androgen-Binding Protein↗
Improved methods to isolate and subfractionate rat liver mitochondria. Lipid composition of the inner and outer membrane.
Rat liver mitochondria were isolated by a combination of differential and Percoll gradient centrifugation, resulting in a highly pure and intact preparation, as assessed by marker enzyme analysis, latency of cytochrome-c oxidase, respiratory control index and electron microscopy. Two different methods were compared for the separation of inner and outer membranes. In the swell-shrink-sonicate procedure glycerol was included resulting in the isolation of one outer membrane and two inner membrane fractions of high purity. Using digitonin a highly selective and gradual solubilization of the outer membrane could be accomplished. Analysis of the phospholipid composition of the intact mitochondria and all subfractions showed that the inner membrane was virtually devoid of phosphatidylinositol and -serine, while the outer membrane contained 23% of the total mitochondrial cardiolipin, which did not originate from inner membrane contamination and therefore is a true component of the outer membrane.
Animals↗
In vivo inactivation of peroxisomal alcohol oxidase in Hansenula polymorpha by KCN is an irreversible process.
The fate of alcohol oxidase (AO) in chemostat-grown cells of Hansenula polymorpha, after its inactivation by KCN, was studied during subsequent cultivation of the cyanide-treated cells in fresh methanol media. Biochemical experiments showed that the cyanide-induced inactivation of AO was due to the release of flavin adenine dinucleotide (FAD) from the holo enzyme. However, dissociation of octameric AO into subunits was not observed. Subsequent growth of intact cyanide-treated cells in fresh methanol media was paralleled by proteolytic degradation of part of the peroxisomes present in the cells. The recovery of AO activity, concurrently observed in these cultures, was accounted for by synthesis of new enzyme protein. Reactivation of previously inactivated AO was not observed, even in the presence of FAD in such cultures. Newly synthesized AO protein was incorporated in only few of the peroxisomes present in the cells. 31P nuclear magnetic resonance (NMR) studies showed that cyanide-treatment of the cells led to a dissipation of the pH gradient across the peroxisomes membrane. However, restoration of this pH gradient was fast when cells were incubated in fresh methanol medium after removal of the cyanide.
Alcohol Oxidoreductases↗
Formation of trans-caffeoyl-CoA from trans-4-coumaroyl-CoA by Zn2+-dependent enzymes in cultured plant cells and its activation by an elicitor-induced pH shift.
A novel hydroxylase activity catalyzing the formation of trans-caffeoyl-CoA from trans-4-coumaroyl-CoA was identified in crude extracts from cultured parsley cells. The extracts were less active (Vmax/Km) in converting trans-4-coumaric to trans-caffeic acid. Optimal hydroxylase activity was found at pH 6.5 with a steep decline toward both pH 7.4 and pH 5.0. The enzyme activity requires ascorbate and Zn2+ at optimal concentrations of 50 and 0.5 mM, respectively. No other reductant could replace ascorbate, whereas high concentrations of Ca2+ partially substituted for Zn2+. The enzyme is soluble and appears to be located in the cytoplasm. The unusual pH optimum suggests that the hydroxylase is inactive at the normal cytoplasmic pH. Upon treatment of parsley cells with an elicitor derived from Phytophthora megasperma f. sp. glycinea, the cytoplasmic pH dropped by approximately 0.25 pH unit within 55 min as determined by 31P NMR spectroscopy. Our results suggest that this shift in the cytoplasmic pH is sufficient for the activation of the hydroxylase, eventually leading to the formation of caffeoyl and feruloyl esters. Such esters may be a part of a very rapid resistance response of the plant cells, which would leave no time for de novo enzyme synthesis.
Acyl Coenzyme A↗
A comparative model membrane study on structural effects of membrane-active positively charged anti-tumor drugs.
The interaction of a number of positively charged anti-tumor drugs with cardiolipin-containing model membranes has been investigated using 31P nuclear magnetic resonance (31P-NMR), differential scanning calorimetry (DSC) and monolayer techniques. It appeared that the ellipticines used (i.e., celiptium and 2-N-methylellipticinium), and also ethidium bromide, completely blocked Ca2+-induced HII phase formation in pure cardiolipin liposomes at molar ratios of drug-to-lipid of approx. 1:1. For the anthracyclines adriamycin and 4'-epi-adriamycin, a similar effect was observed, but now a 2:1 ratio was required. 31P-NMR experiments on dioleoylphosphatidylethanolamine/cardiolipin mixed liposomes indicated that the two anthracyclines, but not the other three drugs, were capable of inducing macroscopic phase separation into domains enriched in drug-cardiolipin complexes and domains enriched in the zwitterionic phospholipid species. DSC experiments on dipalmitoylphosphatidylcholine/cardiolipin mixtures led, with the exception of 2-N-methylellipticinium, to the same conclusion. Measurements of surface pressure and surface potential of cardiolipin monolayers at the air/water interface as well as conformational analysis of the various drug-cardiolipin recombinants showed that the ellipticines are deeply embedded in the acyl chain region of the bilayer, while the anthracyclines and ethidium bromide are preferentially localized in the interface. All drugs share an important electrostatic interaction with the negatively charged phosphates of cardiolipin.
1,2-Dipalmitoylphosphatidylcholine↗
Regulatory aspects of mitochondrial phospholipase A2: correlation of hydrolysis rates with substrate configuration as evidenced by 31P-NMR.
The influence of variation of the phospholipid composition in model membranes composed of phosphatidylcholine and phosphatidylethanolamine on the hydrolysis of these phospholipids by rat liver mitochondrial phospholipase A2 was investigated. With the pure phospholipids, phosphatidylethanolamine was hydrolyzed over 30-times faster than phosphatidylcholine. Upon increasing the mole percentage of phosphatidylethanolamine in mixtures, a gradual, though non-linear, increase in the initial rate of hydrolysis of this phospholipid was observed. By contrast, phosphatidylcholine hydrolysis remained constant up to about 50 mol% phosphatidylethanolamine, whereafter a sudden fall-off of activity was observed. This drop in the hydrolysis rate coincided with a transition of the phospholipid structure from bilayer to an as yet unidentified organization characterized by an isotropic signal in the 31P-NMR spectra recorded in the presence of Ca2+. The occurrence of this phase was clearly dependent on Ca2+, since mixtures with identical composition in the absence of Ca2+ remained largely in bilayer configuration. That the structure adopted by phospholipids is of importance for their susceptibility to attack by this intracellular phospholipase A2 became evident also in studies with the single phospholipids in the absence or presence of Triton X-100 above the critical micellar concentration. While phosphatidylcholine hydrolysis was inhibited in mixed micelles as compared to its bilayer organization, the hydrolysis of phosphatidylethanolamine in mixed micelles was 3-fold that in the hexagonal HII phase.
Animals↗
Mechanism of stimulation of endogenous fermentation in yeast by carbonyl cyanide m-chlorophenylhydrazone.
Addition of the uncoupler and protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) to starved yeast cells starts endogenous alcoholic fermentation lasting about 20 min. Hexose 6-phosphates, fructose 2,6-bisphosphate, and pyruvate accumulate in less than 2 min after addition of CCCP from almost zero concentration to concentrations which correspond to 1/5-1/10 of the steady-state concentrations during fermentation of glucose. CCCP immediately causes a decrease of the intracellular cytosolic pH from 6.9 to 6.4. This change activates adenylate cyclase (Purwin, C., Nicolay, K., Scheffers, W.A., and Holzer, H. (1986) J. Biol. Chem. 261, 8744-8749) and leads to the previously observed transient increase of cyclic AMP. It is shown here that the following enzymes known from in vitro experiments to be activated by cyclic AMP-dependent phosphorylation are activated in the CCCP-treated starved yeast cells in vivo: glycogen phosphorylase, trehalase (pH 7), 6-phosphofructo-2-kinase. The activation of 6-phosphofructo-2-kinase leads to an accumulation of fructose 2,6-bisphosphate, which is known from in vitro experiments to activate 6-phosphofructo-1-kinase and to inhibit fructose-1,6-bisphosphatase. All effects observed in the intact yeast cells fit with the idea that the CCCP-initiated activation of adenylate cyclase leads to a sequence of events which by protein phosphorylation and allosteric effects initiates endogenous alcoholic fermentation.
Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
Effects of adriamycin on respiratory chain activities in mitochondria from rat liver, rat heart and bovine heart. Evidence for a preferential inhibition of complex III and IV.
The inhibition of respiratory chain activities in rat liver, rat heart and bovine heart mitochondria by the anthracycline antibiotic adriamycin was measured in order to determine the adriamycin-sensitive sites. It appeared that complex III and IV are efficiently affected such that their activities were reduced to 50% of control values at 175 +/- 25 microM adriamycin. Complex I displayed a minor sensitivity to the drug. Of the complex-I-related activities tested, only duroquinone oxidation appeared sensitive (50% inhibition at approx. 450 microM adriamycin). Electron-transfer activities catalyzed by complex II remained essentially unaltered up to high drug concentrations. Of the activities measured for this complex, only duroquinone oxidation was significantly affected. However, the adriamycin concentration required to reduce this activity to 50% exceeded 1 mM. Mitochondria isolated from rat liver, rat heart and bovine heart behaved essentially identical in their response to adriamycin. These data support the conclusion that, in these three mitochondrial systems, the major drug-sensitive sites lie in complex III and IV. Cytochrome c oxidase and succinate oxidase activity in whole mitochondria exhibited a similar sensitivity towards adriamycin, as inner membrane ghosts, suggesting that the drug has direct access to its inner membrane target sites irrespective of the presence of the outer membrane. By measuring NADH and succinate oxidase activities in the presence of exogenously added cytochrome c, it appeared that adriamycin was less inhibitory under these conditions. This suggests that adriamycin competes with cytochrome c for binding to the same site on the inner membrane, presumably cardiolipin.
Animals↗