Effect of pH and inorganic phosphate on creatine kinase inactivation: an in vitro 31P NMR saturation-transfer study.

The pseudo-first-order rate constant of rabbit muscle creatine kinase (CK), in the direction of ATP synthesis (kf), was determined by saturation-transfer 31P NMR. When pH was varied between 6.0 and 7.4, kf increased linearly at both 20 degrees C and 37 degrees c. The corresponding flux is very small between pH 6.0 and 6.5, in contrast to previous studies. Up to 50 h exposure of the CK enzyme to high concentrations of inorganic phosphate (Pi), a known inhibitor in certain situations, had negligible effect on enzymatic flux in the physiological pH range. Thus under in vivo conditions, such as in stroke, where pH falls as low as 6.2 and Pi rises to high levels, the rate of the CK reaction may be severely reduced due to pH but not due to high Pi concentrations.     

31P NMR visibility and characterization of rat liver mitochondrial matrix adenine nucleotides

Compartmentation and NMR visibility of mitochondrial adenine nucleotides were quantitated in isolated rat liver mitochondria respiring on succinate and glutamate in vitro at 8 and 25 degrees C. Intra- and extramitochondrial nucleotides were discriminated by adding the chelator trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA). T1 values of about 0.2-0.3 s for magnesium-bound matrix nucleotides were determined. Adenine nucleotide T1 values were influenced by the ionic environment; only magnesium-free ATP T1's were affected by temperature. Intra- and extramitochondrial adenine nucleotide ratios were varied in ATP-loaded mitochondria with added ATP and phosphate using the mitochondrial inhibitors oligomycin and carboxyatractyloside, and adenine nucleotides were quantitated by using NMR and enzymatic analysis. There was good agreement between matrix ATP concentrations (magnesium-bound ATP) calculated by using NMR and standard biochemical techniques. Although matrix ADP could be detected by NMR, it was difficult to quantitate accurately by NMR. The data indicate that mitochondrial ATP is NMR-visible in isolated mitochondria in vitro.     

A 31P NMR study of the internal pH of yeast peroxisomes

The internal pH of peroxisomes in the yeasts Hansenula polymorpha, Candida utilis and Trichosporon cutaneum X4 was estimated by ³¹P nuclear magnetic resonance (NMR) spectroscopy. ³¹P NMR spectra of suspensions of intact cells of these yeasts, grown under conditions of extensive peroxisomal proliferation, displayed two prominent P_i-peaks at different chemical shift positions. In control cells grown on glucose, which contain very few peroxisomes, only a single peak was observed. This latter peak, which was detected under all growth conditions, was assigned to cytosolic P_i at pH 7.1. The additional peak present in spectra of peroxisome-containing cells, reflected P_i at a considerably lower pH of approximately 5.8–6.0. Experiments with the protonophore carbonyl cyanide m-chlorophenylhydrazon (CCCP) and the ionophores valinomycin and nigericin revealed that separation of the two P_i-peaks was caused by a pH-gradient across a membrane separating the two pools. Experiments with chloroquine confirmed the acidic nature of one of these pools. In a number of transfer experiments with the yeast H. polymorpha it was shown that the relative intensity of the P_i-signal at the low pH-position was correlated to the peroxisomal volume fraction. These results strongly suggest that this peak has to be assigned to P_i in peroxisomes, which therefore are acidic in nature. The presence of peroxisome-associated P_i was confirmed cytochemically.Abbreviations CCCP Carbonyl cyanide m-chlorophenylhydrazon - DCCD N,N-dicyclohexylcarbodiimide     

31P nuclear magnetic resonance study of alkaline phosphatase: the role of inorganic phosphate in limiting the enzyme turnover rate at alkaline pH.

31P nuclear magnetic resonance (NMR) was used to directly observe the binding of inorganic phosphate to alkaline phosphatase. Evidencq for the tight binding of 1.5-2.0 mol of inorganic phosphate per dimer of alkaline phosphatase is presented. Two distinct forms of bound phosphate are observed, one predominating above pH 7 and representing the non-covalent E-P1 complex and the other predominating below pH 5 and representing the covalent E-P1 complex. The 31P NMR line width of the E-P1 complex indicates that the dissociation of noncovalent phosphate is the rate-limiting step in the turnover of the enzyme at high pH.     

The regulation of ATPase-ATPase interactions in sarcoplasmic reticulum membrane. I. The effects of Ca2+, ATP, and inorganic phosphate

Two-dimensional crystalline arrays of Ca2+-ATPase molecules develop after treatment of sarcoplasmic reticulum vesicles with Na3VO4 in calcium-free medium (Dux, L., and Martonosi, A. (1983) J. Biol. Chem. 258, 2599-2603). The formation of Ca2+-ATPase crystals is inhibited by Ca2+ (2 microM), or ATP (5 mM), but not by ADP, 5'-adenylylimidodiphosphate, or adenylylmethylenediphosphonate. ATPase crystals did not form at 37 degrees C and exposure of preformed crystals to 37 degrees C for 1 h caused the disappearance of crystal lattice. Inorganic orthophosphate (1 mM at pH 6.0) promoted the formation of a distinct crystal form of Ca2+-ATPase, which was different from that produced by Na3VO4. These observations indicate that Ca2+, ATP, inorganic phosphate, pH, and temperature influence the interactions between ATPase molecules in the sarcoplasmic reticulum membrane.     

31P NMR of phosphate and phosphonate complexes of metalloalkaline phosphatases.

31P NMR spectra of phosphate and phosphonate complexes of Escherichia coli alkaline phosphatase have been obtained by Fourier transform NMR methods. One equivalent of P1i, bound to Zn(II) alkaline phosphatase, pH 8, gives rise to a single 31P resonance 2 ppm downfield from that for Pi, and assignable to the noncovalent complex, E-P. Inorganic phosphate in excess of 1 eq per enzyme dimer gives rise to a resonance at the position expected for free Pi. At pH 5.1, a second resonance appears 8.5 ppm downfield from that for free Pi, and is assignable to the covalent complex, E-P. The large downfield shift suggests that the enzyme phosphoryl group is highly strained with an O-P-O bond angle of under 100 degrees.     

31P NMR study of intracellular pH during the respiratory burst of macrophages

The metabolic events occurring during the respiratory burst of macrophages previously primed in vivo with lipopolysaccharide were studied by 31P nuclear magnetic resonance, using the P388D1 cell line as a model of the mature macrophages. Using perchloric acid extracts, the presence of phosphocreatine was shown in the primed cells, indicating that in control P388 D1 macrophages, in which no phosphocreatine was seen, in vivo maturation was incomplete. The cells primed in vivo exhibited greater maturation than the control cells, as well as greater creatine kinase activity. Perfusion of gel-embedded macrophages allowed the monitoring of phosphorylated metabolite peak intensities and of the intracellular pH. After the respiratory burst of the primed macrophages had been triggered by concanavalin A, these intensities did not alter significantly, but the intracellular pH decreased. 31P NMR spectra reflected transient acidification in the primed cells, possibly due to the formation of endocytic vesicles and their fusion with lysosomes.     

An intracellular pH gradient in the anammox bacterium Kuenenia stuttgartiensis as evaluated by 31P NMR

The cytoplasm of anaerobic ammonium oxidizing (anammox) bacteria consists of three compartments separated by membranes. It has been suggested that a proton motive force may be generated over the membrane of the innermost compartment, the “anammoxosome”. ³¹P nuclear magnetic resonance (NMR) spectroscopy was employed to investigate intracellular pH differences in the anammox bacterium Kuenenia stuttgartiensis. With in vivo NMR, spectra were recorded of active, highly concentrated suspensions of K. stuttgartiensis in a wide-bore NMR tube. At different external pH values, two stable and distinct phosphate peaks were apparent in the recorded spectra. These peaks were equivalent with pH values of 7.3 and 6.3 and suggested the presence of a proton motive force over an intracytoplasmic membrane in K. stuttgartiensis. This study provides for the second time—after discovery of acidocalcisome-like compartments in Agrobacterium tumefaciens—evidence for an intracytoplasmic pH gradient in a chemotrophic prokaryotic cell.     

Elucidation of the cytoplasmic and vacuolar components in the inorganic phosphate region in the 31P NMR spectrum of yeast

Subcellular compartments, such as the vacuole in yeast, play important roles in cell metabolism and in cell response to external conditions. Concentrations of inorganic phosphate and pH values of the vacuole and cytoplasm were determined for anaerobic Saccharomyces cerevisiae cells based upon (31)P NMR spectroscopy. A new approach allows the determination of these values for the vacuole in cases when the resonance for inorganic phosphate in the cytoplasm overlaps with the resonance for inorganic phosphate in the vacuole. The intracellular inorganic phosphate resonance was first decomposed into two components by computer analysis. The assignments of the components were determined from in vivo correlations of P(i) chemical shift and the chemical shifts of the cytoplasmic sugar phosphates, and the pH dependency of the resonance of pyrophosphate and the terminal phosphate of poly-phosphate (PP(1)) which reside in the vacuole. An in vivo correlation relating PP(1) and P(i) (vac) chemical shifts was established from numerous evaluations of intracellular compositions for several strains of S. cerevisiae. This correlation will aid future analysis of (31)P NMR spectra of yeast and will extend NMR studies of compartmentation to cellular suspensions in phosphate-containing medium. Application of this method shows that both vacuolar and extracellular P(i) were phosphate reserves during glycolysis in anaerobic S. cerevisiae. Net transport of inorganic phosphate across the vacuolar membrane was not correlated with the pH gradient across the membrane.     

CO2 induced acute respiratory acidosis and brain tissue intracellular pH: a 31P NMR study in swine

High concentration carbon dioxide (CO(2)) is used to promote pre-slaughter anaesthesia in swine and poultry, as well as short-lasting surgical anaesthesia and euthanasia in laboratory animals. Questions related to animal welfare have been raised, as CO(2) anaesthesia does not set in momentarily. Carbon dioxide promotes anaesthesia by lowering the intracellular pH in the brain cells, but the dynamics of the changes in response to a high concentration of CO(2) is not known. Based on (31)P NMR spectroscopy, we describe CO(2)-induced changes in intracellular pH in the brains of five pigs inhaling 90% CO(2) in ambient air for a period of 60 s, and compare the results to changes in arterial blood pH, P(CO2), O(2) saturation and HCO(3)(-) concentration. The intracellular pH paralleled the arterial pH and P(CO2) during inhalation of CO(2); and it is suggested that the acute reaction to CO(2) inhalation mainly reflects respiratory acidosis, and not metabolic regulation as for example transmembrane fluxes of H(+)/HCO(3)(-). The intracellular pH decreased to approximately 6.7 within the 60 s inhalation period, and the situation was metabolically reversible after the end of CO(2) inhalation. The fast decrease in intracellular pH supports the conclusion that high concentration CO(2) leads to anaesthesia soon after the start of inhalation.     

High-pressure 31P NMR study of dipalmitoylphosphatidylcholine bilayers.

High-pressure 31P NMR was used for the first time to investigate the effects of pressure on the structure and dynamics of the phosphocholine headgroup in pure 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) multilamellar aqueous dispersions and in DPPC bilayers containing the positively charged form of the local anesthetic tetracaine (TTC). The 31P chemical shift anisotropies, delta sigma, and the 31P spin-lattice relaxation times, T1, were measured as a function of pressure from 1 bar to 5 kbar at 50 degrees C for both pure DPPC and DPPC/TTC bilayers. This pressure range permitted us to explore the rich phase behavior of DPPC from the liquid-crystalline (LC) phase through various gel phases such as gel I (P beta'), gel II (L beta'), gel III, gel IV, gel X, and the interdigitated, Gi, gel phase. For pure DPPC bilayers, pressure had an ordering effect on the phospholipid headgroup within the same phase and induced an interdigitated Gi gel phase which was formed between the gel I (P beta') and gel II (L beta') phases. The 31P spin-lattice relaxation time measurements showed that the main phase transition (LC to gel I) was accompanied by the transition between the fast and slow correlation time regimes. Axially symmetric 31P NMR lineshapes were observed at pressures up to approximately 3 kbar but changed to characteristic axially asymmetric rigid lattice lineshapes at higher pressures (3.1-5.1 kbar).(ABSTRACT TRUNCATED AT 250 WORDS)     

31P NMR study of changes in the intracellular pH of Escherichia coli after freezing-thawing

Influence of two types of freezing, at -196 and -50 degrees C with following thawing of Escherichia coli cells at 37 degrees C on the value of intracellular pH has been studied by means of 31P NMR spectroscopy. All the cycles of freeze-thawing have been shown to result in acidification of intracellular medium on 1.0 unit pH apart from the freezing type. The extracellular medium (pHex) was acidified too, but the degree of pHex changes after freeze-thawing depended on the freezing depth.     

Active-site serine phosphate and histidine residues of phosphoglucomutase: pH titration studies monitored by 1H and 31P NMR spectroscopy

1H and 31P NMR pH titrations were conducted to monitor changes in the environment and protonation state of the histidine residues and phosphoserine group of rabbit muscle phosphoglucomutase on binding of metal ions at the activating site and of substrate (glucose phosphate) at the catalytic site. Imidazole C epsilon-H signals from 8 of the 10 histidines present in the free enzyme were observed in 1H NMR spectra obtained by a spin-echo pulse sequence at 470 MHz; their pH (uncorrected pH meter reading of a 2H2O solution measured with a glass electrode standardized with H2O buffer) titration properties (in 99% 2H2O) were determined. Three of these histidine residues, which have pKa values ranging from 6.5 to 7.9, exhibited an atypical pH-dependent perturbation of their chemical shifts with a pHmid of 5.8 and a Hill coefficient of about 2. Since none of the observed histidines has a pKa near 5.8, it appears that these three histidines interact with a cluster consisting of two or more groups which become protonated cooperatively at this pH. Binding of Cd2+ at the activating site of the enzyme abolishes the pH-dependent transition of these histidines; hence, the putative anion cluster may constitute the metal ion binding site, or part of it. Two separate 31P NMR peaks from phosphoserine-116 of the phosphoenzyme were observed between pH 6 and 9. Apparently, the metal-free enzyme exists as a pH-dependent mixture of conformers that provide two different environments, I and II, for the enzymic phosphate group; the transition of the phosphate group between these two environments is slow on the NMR time scale.(ABSTRACT TRUNCATED AT 250 WORDS)     

31P NMR saturation-transfer study of the in situ kinetics of the mitochondrial adenine nucleotide translocase

The exchange of intramitochondrial ATP (ATP(in)) for extramitochondrial ATP (ATP(out)) was measured by using 31P NMR spectroscopy over a range of temperatures in isolated rat liver mitochondria oxidizing glutamate and succinate in the presence of external ATP but no added ADP (state 4). The rate of this exchange is more than an order of magnitude faster than rates reported previously that were determined by using isotopic techniques in the presence of oligomycin, the potent ATPase inhibitor. Differences are ascribed in part to the low levels of matrix ATP present in oligomycin-treated mitochondria. The addition of oligomycin to mitochondrial suspensions decreases intramitochondrial ATP levels from 17 +/- 3 (SEM) nmol/mg of protein in state 4 to 1.51 +/- 0.1 nmol/mg of protein in the presence of inhibitor at 8 degrees C. Simultaneously, transporter flux falls from 960 +/- 55 nmol/min.mg to undetectable levels (less than 300 nmol/min.mg). Although transport rates are much faster when measured by saturation-transfer than by conventional isotopic methods, the enthalpy values obtained by determining the effect of temperature on flux are very similar to those reported in the past that were determined by using isotopic techniques. Intramitochondrial ATP content regulates the rate of the ATP(in)/ATP(out) exchange. At 18 degrees C, the concentration of internal ATP that produces half-maximal transport rate is 6.6 +/- 0.12 nmol/mg of mitochondrial protein. The relationship between substrate concentration and flux is sigmoidal and is 90% saturated at 11.3 +/- 0.18 nmol/mg of mitochondrial protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

A 31P NMR study of the thermal transition of dipalmitoyl lecithin vesicles.

Phosphorous-31 nuclear magnetic resonance (31P NMR) was used to study the liquid crystalline transition of sonicated dipalmitoyl lecithin vesicles in D2O. Linewidths were dependent upon temperature and changed dramatically through the transition region. The potential usefulness of 31P NMR spectroscopy for probing the thermal behavior of phospholipid membranes is evaluated and discussed.     

A study of the conformation of 5S RNA by 31P NMR.

Only a small number of resolved, single phosphorous, phosphodiester resonances are observed in the 31P spectrum of the 5S rRNA from E. coli. Its spectrum is much simpler than that of a tRNA (Gueron, M. and Shulman, R.G. (1975) Proc. Natl. Acad. Sci. 72, 3482-3484), which suggests that 5S RNA does not have a tightly folded, tRNA-like, tertiary structure. The resolved resonances in the 5S spectrum originate in loops D and E, near bases 88 and 76, respectively.     

Butylated hydroxytoluene and inorganic phosphate plus Ca2+ increase mitochondrial permeability via mutually exclusive mechanisms

Mitochondria undergo a permeability transition (PT), i.e., become nonselectively permeable to small solutes, in response to a wide range of conditions/compounds. In general, opening of the permeability transition pore (PTP) is Ca²⁺- and P_i-dependent and is blocked by cyclosporin A (CsA), trifluoperazine (TFP), ADP, and butylated hydroxytoluene (BHT). Gudz and coworkers have reported [7th European Bioenergetics Conference, EBEC Short Reports (1992)7, 125], however, that, under some conditions, BHT increases mitochondrial permeability via a process that may not share all of these characteristics. Specifically, they determined that the BHT-induced permeability transition was independent of Ca²⁺ and was insensitive to CsA. We have used mitochondrial swelling to compare in greater detail the changes in permeability induced by BHT and by Ca²⁺ plus P_i with the following results. (1) The dependence of permeability on BHT concentration is triphasic: there is a threshold BHT concentration (ca. 60 nmol BHT/ mg mitochondrial protein) below which no increase occurs; BHT enhances permeability in an intermediate concentration range; and at high BHT concentrations (> 120 nmol/mg) permeability is again reduced. (2) The effects of BHT depend on the ratio of BHT to mitochondrial protein. (3) Concentrations of BHT too low to induce swelling block the PT induced by Ca²⁺ and P_i. (4) The dependence of the Ca²⁺-triggered PT on P_i concentration is biphasic. Below a threshold of 50–100 M, no swelling occurs. Above this threshold swelling increases rapidly. (5) P_i levels too low to support the Ca²⁺-induced PT inhibit BHT-induced swelling. (6) Swelling induced by BHT can bestimulated by agents and treatments that block the PT induced by Ca²⁺ plus P_i. These data suggest that BHT and Ca²⁺ plus P_i, increase mitochondrial permeability via two mutually exclusive mechanisms.     

Effect of Ca2+, peroxides, SH reagents, phosphate and aging on the permeability of mitochondrial membranes

The mechanism by which a number of agents such as hydroperoxides, inorganic phosphate, azodicarboxylic acid bis(dimethylamide) (diamide), 2-methyl-1,4-naphthoquinone (menadione) and aging, induce Ca2+ release from rat liver mitochondria has been analyzed by following Ca2+ fluxes in parallel with K+ fluxes, matrix swelling and triphenylmethylphosphonium fluxes (as an index of transmembrane potential). Addition of hydroperoxides causes a cycle of Ca2+ efflux and reuptake and an almost parallel cycle of delta psi depression. The hydroperoxide-induced delta psi depression is biphasic. The first phase is rapid and insensitive to ATP and is presumably due to activation of the transhydrogenase reaction during the metabolization of the hydroperoxides. The second phase is slow and markedly inhibited by ATP and presumably linked to the activation of a Ca2+-dependent reaction. The slow phase of delta psi depression is paralleled by matrix K+ release and mitochondrial swelling. Nupercaine and ATP reduce or abolish also K+ release and swelling. Inorganic phosphate, diamide, menadione or aging also cause a process of Ca2+ efflux which is paralleled by a slow delta psi depression, K+ release and swelling. All these processes are reduced or abolished by Nupercaine and ATP. The slow delta psi depression following addition of hydroperoxide and diamide is largely reversible at low Ca2+ concentration but tends to become irreversible at high Ca2+ concentration. The delta psi depression increases with the increase of hydroperoxide, diamide and menadione concentration, but is irreversible only in the latter case. Addition of ruthenium red before the hydroperoxides reduces the extent of the slow but not of the rapid phase of delta psi depression. Addition of ruthenium red after the hydroperoxides results in a slow increase of delta psi. Such an effect differs from the rapid increase of delta psi due to ruthenium-red-induced inhibition of Ca2+ cycling in A23187-supplemented mitochondria. Metabolization of hydroperoxides and diamide is accompanied by a cycle of reversible pyridine nucleotide oxidation. Above certain hydroperoxide and diamide concentrations the pyridine nucleotide oxidation becomes irreversible. Addition of menadione results always in an irreversible nucleotide oxidation. The kinetic correlation between Ca2+ efflux and delta psi decline suggests that hydroperoxides, diamide, menadione, inorganic phosphate and aging cause, in the presence of Ca2+, an increase of the permeability for protons of the inner mitochondrial membrane. This is followed by Ca2+ efflux through a pathway which is not the H+/Ca2+ exchange.