Structural properties of phospholipids in the rat liver inner mitochondrial membrane. A P-NMR study

1. 1. The ³¹P-NMR characteristics of intact rat liver mitochondria, mitoplasts and isolated inner mitochondrial membranes, as well as mitochondrial phosphatidylethanolamine and phosphatidylcholine, have been examined.

2. 2. Rat liver mitochondrial phosphatidylethanolamine hydrated in excess aqueous buffer undergoes a bilayer-to-hexagonal (H_II) polymorphic phase transition as the temperature is increased through 10°C, and thus prefers the H_II) arrangement at 37°C. Rat liver mitochondrial phosphatidylcholine, on the other hand, adopts the bilayer phase at 37°C.

3. 3. Total inner mitochondrial membrane lipids, dispersed in an excess of aqueous buffer, exhibit ³¹P-NMR spectra consistent with a bilayer arrangement for the majority of the endogeneous phospholipids; the remainder exhibit spectra consistent with structure allowing isotropic motional averaging. Addition of Ca²⁺ results in hexagonal (H_II) phase formation for a portion of the phospholipids, as well as formation of ‘lipidic particles’ as detected by freeze-fracture techniques.

4. 4. Preparations of inner mitochondrial membrane at 4 and 37°C exhibit ³¹P-NMR spectra consistent with a bilayer arrangement of the large majority of the endogenous phospholipids which are detected. Approx. 10% of the signal intensity has characteristics indicating isotropic motional averaging processes. Addition of Ca²⁺ results in an increase in the size of this component, which can become the dominant spectral feature.

5. 5. Intact mitochondria, at 4°C, exhibit ³¹P-NMR spectra arising from both phospholipid and small water-soluble molecules (ADP, P_i, etc.). The phospholipid spectrum is characteristic of a bilayer arrangement. At 37°C the phospholipids again give spectra consistent with a bilayer; however, the labile nature of these systems is reflected by increased isotropic motion at longer (at least 30 min) incubation times.

6. 6. It is suggested that the uncoupling action of high Ca²⁺ concentrations on intact mitochondria may be related to a Ca²⁺-induced disruption of the integrity of the inner mitochondrial phospholipid bilayer. Further, the possibility that non-bilayer lipid structures such as inverted micelles occur in the inner mitochondrial membrane cannot be excluded.

A 31P-NMR study of the intact liver fluke Fasciola hepatica

³¹P-NMR techniques offer a useful method of studying changes in the metabolism of intact parasitic worms. The liver flukes, Fasciola hepatica, provide good quality ³¹P high resolution NMR spectra for at least 6 h under anaerobic conditions. The levels of ATP remain constant throughout this period. There is no signal for phosphocreatine or phosphoarginine. In contrast to the findings in mammalian tissues, there is a distinct peak for the terminal phosphate of ADP. A number of signals are observed in the phosphodiester region of the spectrum the largest of which is identified as l-α-glycerophosphoryl choline. Serotonin (5-hydroxytryptamine) causes an appreciable increase in the levels of sugar phosphates when the flukes are incubated in the absence of glucose. The addition of glucose also causes a marked increase in the signals for the hexose phosphate.     

31P-NMR studies on phospholipid structure in membranes of intact,functionally-active,rat liver mitochondria

³¹P-NMR studies of intact functional rat liver mitochondria at 37°C demonstrate that the large majority (?95%) of endogenous phospholipids exhibit motional properties consistent with bilayer structure. This property is unaffected by oxidative phosphorylation processes or the presence of Ca²⁺.     

A 31P-NMR study of the cross-membrane pH gradient induced by ATP hydrolysis in mitochondria

³¹P-NMR has been used to study the increase of ΔpH in mitochondria by externally added ATP. Freshly prepared mitochondria was treated with N-ethylmaleimide to inhibit the exchange between internal and external P_i. Upon addition of ATP, phosphocreatine (30 mM) and creatine kinase to a NMR sample of mitochondria suspension (approx. 120 mg protein/ml) at 0°C, an increase of ΔpH by approx. 0.5 pH unit was observed. However the increased ΔpH could not be maintained, but slowly decayed along with the increase of external ADP/ATP ratio. Further addition of valinomycin to the suspension induced a larger ΔpH (approx. 1) which was maintained by the increased rate of internal ATP hydrolysis as seen in the growth of the internal P_i peak intensity in NMR spectra and the concomitant decrease of the external phosphocreatine peak. The external P_i and ATP peaks stayed virtually constant. When carboxyatractyloside was added to inhibit the ATP/ADP translocase, the internal P_i increase was stopped and the ΔpH decayed. These observations in conjunction with those made earlier in respiring mitochondria clearly show the reversible nature of the ATPase function in which the internal ATP hydrolysis is associated with outward pumping of protons.     

Interactions of glycerol monooleate and dimethylsulphoxide with phospholipids A differential scanning calorimetry and 31P-NMR study

1. A comparative study has been made of the effects of the fusogens glycerol monooleate and dimethylsulphoxide on the polymorphic phase behaviour of dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidylethanolamine by differential scanning calorimetry and ³¹P-NMR techniques. 2. Glycerol monooleate induces a reduction in the temperature, cooperativity and enthalpy of the gel to liquid-crystal transitions of dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidylethanolamine, whereas dimethylsulphoxide induces an increase in the temperature and enthalpy and a reduction in the cooperativity of the gel to liquid-crystal transitions for those same phospholipids. 3. Glycerol monooleate induces the formation of isotropic and hexagonal (H_II) phases when mixed with either dipalmitoyl phosphatidylcholine or dipalmitoyl phosphatidylethanolamine. By contrast, in the presence of dimethylsulphoxide, those same phospholipids retain the lamellar configuration observed in the absence of fusogen. 4. These results are discussed in terms of the mechanisms of chemically induced cell fusion.     

A 31P-NMR saturation transfer study of the regulation of creatine kinase in the rat heart

(1) ³¹P nuclear magnetic resonance was used to measure the creatine kinase-catalysed fluxes in Langendorff-perfused rat hearts consuming oxygen at different rates and using either of two exogenous substrates (11 mM glucose or 5 mM acetate). (2) Fluxes in the direction of ATP synthesis were between 3.5–12-times the steady-state rates of ATP utilization (estimated from rates of O₂-consumption), demonstrating that the reaction is sufficiently rapid to maintain the cytosolic reactants near their equilibrium concentrations. (3) Under all conditions studied, the cytosolic free [ADP] was primarily responsible for regulating the creatine kinase fluxes. The enzyme displayed a K_m for cytosolic ADP of 35 μM and an apparent V_max of 5.5 mM/s in the intact tissue. (4) Although the reaction is maintained in an overall steady-state, the measured ratio of the forward flux (ATP synthesis) to the reverse flux (phosphocreatine synthesis) was significantly greater than unity under some conditions. It is proposed that this discrepancy may be a consequence of participation of ATP in reactions other than the PCr /ag ATP or ATP /ag ADP + P_i interconversions specifically considered in the analysis. (5) The results support the view that creatine kinase functions primarily to maintain low cytosolic concentrations of ADP during transient periods in which energy utilization exceeds production.     

Voltage activation of heart inner mitochondrial membrane channels

The patch clamp records obtained from mitoplast membranes prepared in the presence of a calcium chelator generally lack channel activity. However, multiconductance channel (MCC) activity can be induced by membrane potentials above ±60mV [Kinnallyet al., Biochem. Biophys. Res. Commun. 176, 1183–1188 (1991)]. Once activated, the MCC activity persists at all voltages. The present report characterizes the activation by voltage of multiconductance channels of rat heart inner mitochondrial membranes using patch-clamping. In some membrane patches, the size of single current transitions progressively increases with time upon application of voltage. The inhibitor cyclosporin has also been found to decrease channel conductance in steps. The results suggest that voltage-induced effects which are inhibited by cyclosporin Aare likely to involve either an increase in effective pore diameter or the assembly of low-conductance units. In activated patches, we have found at high membrane potentials (e.g., 130 mV) changes in conductance as high as 5 nS occurring in large steps (up to 2.7 nS). These were generally preceded by a smaller transition. Similar results were obtained less frequently at lower voltages. These results can be explained on the assumption that once assembled the channels may act in unison.     

Differential solubilization of inner plasma membrane leaflet components by Lubrol WX and Triton X-100

A commonly-used method for analysing raft membrane domains is based on their resistance to extraction by non-ionic detergents at 4 °C. However, the selectivity of different detergents in defining raft membrane domains has been questioned. We have compared the lipid composition of detergent-resistant membranes (DRMs) obtained after Triton X-100 or Lubrol WX extraction in MDCK cells in order to understand the differential effect of these detergents on membranes and their selectivity in solubilizing or not proteins. Both Lubrol and Triton DRMs were enriched with cholesterol over the lysate, thus exhibiting characteristics consistent with the properties of membrane rafts. However, the two DRM fractions differed considerably in the ratio between lipids of the inner and outer membrane leaflets. Lubrol DRMs were especially enriched with phosphatidylethanolamine, including polyunsaturated species with long fatty acyl chains. Lubrol and Triton DRMs also differed in the amount of raft transmembrane proteins and raft proteins anchored to the cytoplasmic leaflet. Our results suggest that the inner side of rafts is enriched with phosphatidylethanolamine and cholesterol, and is more solubilized by Triton X-100 than by Lubrol WX.     

A protective effect of insulin on reperfusing the ischaemic rat heart shown using 31P-NMR

(1) The effect on the recovery of mechanical function, ATP, phosphocreatine, P_i and pH of various lengths of total global ischaemia in the insulin-treated, perfused rat heart has been studied using ³¹P-NMR. (2) Insulin-treated hearts recovered stable mechanical function after 18 min ischaemia when their intracellular pH was 6.0 and 70% of the pre-ischaemic ATP remained. Hearts perfused without insulin fail to recover after 18 min ischaemia, having an intracellular pH of 6.3 and 40% of ATP remaining (Bailey, I.A., Seymour, A.-M.L. and Radda, G.K. (1981) Biochim. Biophys. Acta 637, 1–7). Thus, ATP maintenance in ischaemia is more important to recovery on reperfusion than is maintaining intracellular pH. (3) The importance of this observation in devising biochemical strategies for the clinical protection of the myocardium is discussed.     

Modulation of inner mitochondrial membrane channel activity

Three classes of inner mitochondrial membrane (IMM) channel activities have been defined by direct measurement of conductance levels in membranes with patch clamp techniques in 150 mM K Cl. The 107 pS activity is slightly anion selective and voltage dependent (open with matrix positive potentials). Multiple conductance channel (MCC) activity includes several levels from about 40 to over 1000 pS and can be activated by voltage or Ca²⁺. MCC may be responsible for the Ca²⁺-induced permeability transition observed with mitochondrial suspensions. A low conductance channel (LCC) is activated by alkaline pH and inhibited by Mg²⁺. LCC has a unit conductance of about 15 pS and may correspond to the inner membrane anion channel, IMAC, which was proposed from results obtained from suspension studies. All of the IMM channels defined thus far appear to be highly regulated and have a low open probability under physiological conditions. A summary of what is known about IMM channel regulation and pharmacology is presented and possible physiological roles of these channels are discussed.     

Differential protein distributions define two sub-compartments of the mitochondrial inner membrane in yeast

The mitochondrial inner membrane exhibits a complex topology. Its infolds, the cristae membranes, are contiguous with the inner boundary membrane (IBM), which runs parallel to the outer membrane. Using live cells co-expressing functional fluorescent fusion proteins, we report on the distribution of inner membrane proteins in budding yeast. To this end we introduce the enlarged mitochondria of Deltamdm10, Deltamdm31, Deltamdm32, and Deltammm1 cells as a versatile model system to study sub-mitochondrial protein localizations. Proteins of the F(1)F(0) ATP synthase and of the respiratory chain complexes III and IV were visualized in the cristae-containing interior of the mitochondria. In contrast, proteins of the TIM23 complex and of the presequence translocase-associated motor were strongly enriched at the IBM. The different protein distributions shown here demonstrate that the cristae membranes and the IBM are functionally distinct sub-compartments.     

13C- and 31P-NMR studies of the conformation of carcinogen-modified nucleic acid dimers

The effect of the carcinogen acetylaminofluorene (AAF) on nucleic acid structure was examined using ¹³C- and ³¹P-NMR spectroscopies. Conformational effects were compared in two AAF-modified dinucleoside monophosphates (ApG and GpA) and two AAF-modified deoxydinucleotides (dpApG and dpGpA). Changes in adenine ¹³C chemical shifts on formation of the AAF-adduct and as a function of temperature provided evidence of base stacking. Differences in fluorene ¹³C chemical shifts between the AAF-modified dimer and AAF-modified monomer provided evidence of fluorene stacking. The effect of forming the adduct on the phosphate backbone was examined using ³¹P-NMR. A correlation was demonstrated between the degree of adenine-fluorene stacking on one hand and the change in conformation of the backbone conformation on the other.     

Cytofluorometric quantitation of apoptosis-driven inner mitochondrial membrane permeabilization

The mitochondrial matrix can be specifically labeled by loading cells with calcein and simultaneous quenching of the non-mitochondrial calcein fluorescence with cobalt (Co²⁺). Positive staining of mitochondria thus requires that the inner mitochondrial membrane functions as a barrier separating calcein (within the matrix) from Co²⁺ (outside of the matrix). Upon induction of apoptosis, such calcein/Co²⁺-labeled cells, demonstrate a decrease in the overall calcein fluorescence resulting from inner mitochondrial membrane permeabilization. This decrease can be quantified by cytofluorometry and can be dissociated from other apoptosis-associated mitochondrial perturbations such as the loss of the mitochondrial transmembrane potential ( _m ), the local overproduction of reactive oxygen species, and the mitochondrial release of cytochrome c. In some paradigms of apoptosis the loss of calcein/Co²⁺ (CC) staining can be dissociated from the _m loss, both of which may occur in a caspase-dependent or caspase-independent fashion, depending on the apoptosis inducer. Importantly, inner membrane permeabilization to CC may occur without a permanent _m dissipation in apoptosis, suggesting that transient permeabilization events could participate at the apoptotic cascade. Altogether, our data demonstrate that inner mitochondrial membrane permeabilization constitutes an early event in the apoptotic cascade.     

31P-NMR spectrum of phosphocreatine: Deuterium-induced splitting of the signal

It has been found in experiments with high resolution ³¹P-NMR spectroscopy (200 MHz) that the phosphocreatine peak is splitted into two different peaks in the mixtures of H₂O and D₂O and is single but with different chemical shifts in pure H₂O and D₂O. This phenomenon is explained by substitution of protons of guanidino group in phosphocreatine by deuterium. The effect of splitting disappeared at extreme pH values (>8.5 or <4.0) and at temperatures higher than 45°C due to accelerated proton-deuterium exchange. Creatine kinase added to phosphocreatine solution also lowered its temperature of peaks' collapse by 5°–10°C. A saturation (spin) transfer method was used to show that the phosphoryl group transfer to ADP in creatine kinase active center is slower with deuterium-substituted phosphocreatine than with H-phosphocreatine. The data are taken to show the importance of the proton transfer step in the creatine kinase reaction mechanism and acceleration of phosphocreatine proton-deuterium exchange by creatine kinase.     

Identification of anion and cation pathways in the inner mitochondrial membrane by patch clamping of mouse liver mitoplasts

Summary Alkalinization of the matrix side of the mitochondrial inner membrane by pH shifts from 6.8 to 8.3 caused a reversible increase in current of 3.2±0.2 pA (mean±se,n=21) at±40 mV measured using patch-clamp techniques. The current increase was reversed in a graded fashion by the addition of Mg²⁺ in 0.15m KCl corresponds to approximately 15 pS. Reversal potentials derived from whole patch currents indicated that the inner mitochondrial membrane was primarily cation selective at pH 6.8 with aP _k/P _Cl=32 (n=6). Treatment with alkaline pH (8.3) increased the current and anion permeability (P _K/P _Cl=16,n=6). The membrane becomes completely cation selective when low concentrations (12 m) of the drug propranolol are added. The amphiphilic drugs amiodarone (4 m), propranolol (70 m) and quinine (0.6mm) blocked almost all of the current. The pH-dependent current was also inhibited by tributyltin. These results are consistent with the presence of two pathways in the inner mitochondrial membrane. One is cation selective and generally open and the other is anion selective and induced by alkaline pH. The alkaline pH-activated channel likely corresponds to the inner membrane anion channel postulated by others from suspension studies.