Elevated levels of Ca(II) modulate the activity and inhibition of serine proteases: implication in the mechanism of apoptosis.

Elevated levels of intracellular Ca(II) are a prominent feature of apoptosis, a natural form of cell death involved in many physiological and pathological processes. Serine proteases play crucial roles in apoptosis and have been implicated in the genomic DNA degradation and the massive protein degradation that occur during apoptosis. In this study, the effects of the elevated level of Ca(II) on the activity and inhibition of serine proteases were examined by spectrophotometric methods. The effects of the elevated levels of Ca(II), Mg(II), K(I), and Na(I) on the activity and inactivation of three representative members of serine proteases were determined. The level of serine protease activity in CEM-C7-14 leukemic cells was also evaluated in the presence and absence of dexamethasone-induced apoptosis, and also in the presence of A23187, a Ca(II)-ionophore. Among the four metal-ions studied, only Ca(II) was found to significantly enhance the activity of mammalian serine proteases. Ca(II) was also found to significantly protect the enzymes from inhibition, while the other three metal-ions showed no significant effect on the inactivation of the enzymes. Compared to the control sample, the enzymic activity was found to be higher during apoptosis, and in the presence of the Ca(II)-ionophore. Results of this study indicate that Ca(II) can significantly enhance the catalytic efficiency of serine proteases during apoptosis.

13C-NMR investigation of protein synthesis during apoptosis in human leukemic cell lines.

In order to evaluate the role of protein synthesis in apoptosis, (13)C-NMR has been used to study the levels of protein synthesis in three different human leukemic cell lines in the presence and absence of dexamethasone-induced apoptosis. Measurements were done on one dexamethasone-sensitive (CEM-C7-14) and two different dexamethasone-resistant variants (CEM-4R4 and CEM-ICR27-4). The incorporation of (13)C-labeled amino acids into cellular proteins, which reflects the level of new protein synthesis, was monitored by (13)C-NMR spectroscopy. In the absence of dexamethasone, the level of protein synthesis was found to be significantly different among the three cell lines. Dexamethasone caused a significant reduction ( congruent with 60-87%) in the level of protein synthesis in dexamethasone-sensitive CEM-C7-14 cells, while having no significant effect on protein synthesis in dexamethasone-resistant CEM-4R4 cells. Dexamethasone treatment caused a significant enhancement of the level of protein synthesis in the CEM-ICR27-4 cells. Synthesis of proteins was found to occur during apoptosis, albeit at a low level, suggesting a role for the synthesis of specific proteins in the mechanism of apoptosis.

Phospholipid metabolism and resistance to glucocorticoid-induced apoptosis in a human leukemic cell line: a 31P-NMR study using a phosphonium analog of choline.

In order to evaluate any connection between phospholipid metabolism and the dexamethasone resistance in CEM-C1-15 leukemic cells, a phosphonium analog of choline was used to study the phospholipid metabolism in dexamethasone-sensitive and dexamethasone-resistant leukemic cells using 31P-NMR spectroscopy. Measurements were done on the two cell lines in the presence and absence of dexamethasone, a synthetic glucocorticoid steroid. Dexamethasone was found to cause a significant reduction in the phospholipid metabolism of the dexamethasone-sensitive CEM-C7-14 cells. In contrast, dexamethasone caused a significant enhancement of the phospholipid metabolism in the dexamethasone-resistant CEM-C1-15 cells, indicating a significant difference between the two cell lines in phospholipid metabolism during apoptosis. The results of this study suggest the involvement of phospholipid metabolism in the mechanism of dexamethasone resistance in the CEM-C1-15 cells.

Role of intracellular free Ca(II) and Zn(II) in dexamethasone-induced apoptosis and dexamethasone resistance in human leukemic CEM cell lines.

The levels of intracellular free Ca(II) and Zn(II) during dexamethasone (dex)-induced apoptosis in CEM cell lines were determined by 19F nuclear magnetic resonance (NMR), using the fluorinated intracellular chelator 1,2-bis-(2- amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5-FBAPTA). The effects of these divalent metal ions on growth rate and DNA degradation were evaluated. Measurements were done on one dex-sensitive (CEM-C7) and three different dex-resistant variants (CEM-C1, CEM-4R4, and CEM-ICR27). Dex caused a continuous increase in the Ca(II) level in dex-sensitive CEM-C7 cells, while in CEM-C1 cells dex caused an initial increase in the Ca(II) level which in approximately 36 h was restored to its normal value. The intracellular Ca(II) level in CEM-4R4 cells was not significantly affected by dex, while that of CEM-ICR27 cells decreased after dex incubation. Only the dex-sensitive CEM-C7 cells showed dex-induced DNA degradation. An intracellular free Zn(II) level of approximately 1 nM was measured for the dex-resistant CEM-C1 cells. No detectable level of intracellular Zn(II) was found in the other cell lines. Incubation with < 100 microM Zn(II) did not inhibit dex-induced apoptosis in CEM-C7 cells (e.g., DNA degradation). Treatment with approximately 250 microM Zn(II) caused significant decrease in growth rate in all cell lines and prevented dex-induced DNA degradation in CEM-C7 cells. A calibrated amount of Ca(II) ionophore (A23187), used to increase Ca(II) concentrations up to the dex-induced levels, did not induce DNA degradation in CEM-C7 or CEM-C1 cells. While elevation of intracellular Ca(II) by itself is not sufficient to initiate apoptosis in CEM-C7 cells, the results reported here suggest that Ca(II) is involved in the killing mechanism as a secondary factor. The combination of dex and ionophore caused significant DNA degradation in CEM-C1 cells, which normally showed resistance to each compound individually. The combination of dex and the Zn(II) chelator phenanthroline also caused extensive DNA degradation in the normally dex-resistant CEM-C1 cells, suggesting that Zn(II) plays a role in the dex resistance of these cells.

31P NMR characterization of cellular metabolism during dexamethasone induced apoptosis in human leukemic cell lines.

31P NMR has been used to study the effects of dexamethasone on phosphorus metabolism in one dexamethasone (dex)-sensitive (CEM-C7) and three different dex-resistant (CEM-C1, CEM-4R4, and CEM-ICR27) human leukemic cell lines. The use of these cell lines, containing widely varying amounts of glucocorticoid receptors, made it possible to evaluate the receptor-mediated contributions to the modes of action of dexamethasone in these cells. To evaluate the effects of dexamethasone without any significant contribution from experimental conditions, all the experiments were done with parallel controls. Results obtained showed: 1) significantly different levels of phosphorylethanolamine (PE) and phosphorylcholine (PC) among cell lines, suggesting significant differences in phospholipid metabolism; 2) the dexamethasone induced reduction of phosphomonoester (PE+PC), ATP, and metabolic rates probably through glucocorticoid receptor mediated mechanisms; 3) the dexamethasone induced stimulation of cellular metabolism in a process which seems to be independent of glucocorticoid receptors; and 4) the dexamethasone induced alkaline shift of intracellular pH in all the cell lines except ICR27. The reduction in PME levels seems to be an earlier step in dexamethasone-induced apoptosis than the reduction in ATP. The degree of alkaline shift was found to correlate with the number of glucocorticoid receptors present. The possible involvement of phospholipid metabolites as second messengers in dexamethasone-induced apoptosis is discussed.

Labeling of tyrosines in proteins with [15N]tetranitromethane, a new NMR reporter for nitrotyrosines.

Lysozyme and ribonuclease were used as model proteins to explore the feasibility of detecting protein-bound nitrotyrosines by 15N-NMR spectroscopy. The reporter group was introduced via synthesized [15N]tetranitromethane. Several experiments for detection of the 15N resonance in the model [3-15N]nitrotyrosine demonstrated a substantial pH-dependence of the chemical shift. When lysozyme was nitrated, either two or three 15N resonances were detected, depending on the extent of nitration. The pH-dependence of the detected resonances clearly described an apparent microscopic pK in accord with reported values, while addition of Gd(III) gave selective line broadening, indicating that the 15N reporter group could also monitor relative distances from paramagnetic sources. Nitration of ribonuclease showed five 15N resonances, of which three persisted in the purified monomer. The pH-dependence of these resonances also described apparent microscopic pK values. The [3-15N]nitrotyrosine model was reduced to the [3-15N]aminotyrosine and its 15N resonance was easily monitored by several methods, including selective population inversion. When the protein-bound nitrotyrosines were similarly reduced, much sample decomposition resulted, a possible result of photooxidation, and/or reduction of disulfide bond(s), thereby making interpretation difficult.

Bulk magnetic susceptibility induced broadening in the 19F NMR of suspended leukemic cells.

The relevance of bulk magnetic susceptibility (BMS) induced broadening to in vivo NMR studies of intact cells has been examined and the significance of the contribution of BMS difference to the resolution of intra- and extracellular resonances was demonstrated. BMS difference between intra- and extracellular compartments was found to limit the resolution of intra- and extracellular 19F resonances of fluoro compounds in leukemic cells.

19F NMR studies of changes in membrane potential and intracellular volume during dexamethasone-induced apoptosis in human leukemic cell lines.

The induction of apoptosis in leukemic cells by dexamethasone is well known, but the mechanism of this type of cell death and of dexamethasone resistance by some variants is still poorly understood. Apoptotic cell death is preceded by many changes in cellular properties, such as glucose metabolism, cell size, cell density, and others. In this study, 19F-NMR has been used to characterize changes in cell membrane potential and intracellular accessible volume during dexamethasone induced apoptosis. One dex-sensitive (CEM-C7) and three dex-resistant variants (CEM-C1, CEM-ICR27, and CEM-4R4) were examined. We have observed separate intracellular and extracellular resonances for trifluoroacetate and trifluoroacetamide added to suspended leukemic cells. From the equilibrium distribution of these fluoro-compounds between intra and extracellular spaces, the changes in membrane potential and intracellular accessible volume were calculated. The membrane potential for CEM-C7 cells was found to significantly decrease in the presence of dexamethasone (9-mV decrease within 18 h of dexamethasone treatment), while that of CEM-ICR27 was found in some samples to increase on dexamethasone incubation. The membrane potential for CEM-C1 decreased slightly, while that of CEM-4R4 was not appreciably affected by dexamethasone. The reduction of membrane potential seems to be an early step in the mechanism of dexamethasone induced apoptosis. Although the intracellular volume varied with cell type and dexamethasone incubation (for CEM-C7), the fractional intracellular volume (alpha = Vin/Vcell) was found to be the same (0.82 +/- 0.06) for all the cell lines in the presence and absence of dexamethasone.

Spectroscopic studies of lipids and biological membranes: carbon-13 and proton magic-angle sample-spinning nuclear magnetic resonance study of glycolipid-water systems.

We have obtained 1H and 13C magic-angle sample-spinning (MAS) nuclear magnetic resonance (NMR) spectra of three glycosyldiacylglycerol-water (1:1, weight ratio) mesophases, at 11.7 T, as a function of temperature, in order to probe lipid headgroup, backbone, and acyl chain dynamics by using natural-abundance NMR probes. The systems investigated were monogalactosyldiacyldiglyceride [MGDG; primarily 1,2-di[(9Z,12Z,15Z)octadec-9,12,15-trienoyl++ +]-3-beta-D-galactopyranosyl- sn-glycerol]; digalactosyldiacyldiglyceride [DGDG; primarily 1,2-di[(9Z,12Z,15Z)octadec-9,12,15-trienoyl++ +]-3- (alpha-D-galactopyranosyl-1-6-beta-D-glactopyranosyl)-sn-glycerol] ; and sulfoquinovosyldiacyldiglyceride [SQDG; primarily 1-[(9Z,12Z,15Z)octadec-9,12,15-trienoyl]-2 -hexadecanoyl-3-(6-deoxyl-6- sulfono-alpha-D-glucopyranosyl)-sn-glycerol]. At approximately 22 degrees C, all three lipid-water systems give well-resoled 13C and 1H MAS NMR spectra, characteristic of fluid, liquid-crystalline mesophases. 13C spin-lattice relaxation times of the headgroup and glycerol backbone carbons of all three materials give, within experimental error, the same NT1 values (approximately 400 ms), implying similar high-frequency motions, independent of headgroup size and charge. Upon cooling, pronounced line broadenings are observed, due to an increase in slow motional behavior. For each lipid, the onset of line broadening is seen with the glycosyl headgroup, glycerol backbone, and the first two or three carbons of the acyl chains. By approximately -20 degrees, all headgroup carbon resonances are broadened beyond detection. Both galactose moieties in DGDG "freeze out" together, implying a rigid-body motion of the disaccharide unit. Upon further cooling, the bulk polymethylene chain resonances in all three systems (in both 13C and 1H MAS) broaden greatly, followed by the olefinic and allylic carbon resonances.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon-13 nuclear magnetic resonance spectroscopy of lipids: differential line broadening due to cross-correlation effects as a probe of membrane structure.

We have obtained proton-coupled carbon-13 nuclear magnetic resonance (NMR) spectra of a variety of lipid-water and lipid-drug-water systems, at 11.7 T, as a function of temperature, using the "magic-angle" sample-spinning (MAS) NMR technique. The resulting spectra show a wide range of line shapes, due to interferences between dipole-dipole and dipole-chemical shielding anisotropy interactions. The differential line-broadening effects observed are particularly large for aromatic and olefinic (sp2) carbon atom sites. Coupled spectra of the tricyclic antidepressants desipramine and imipramine, in 1,2-dimyristoyl-sn-glycero-3-phosphocholine-water mesophases, show well-resolved doublets having different line shapes for each of the four aromatic methine groups, due to selective averaging of the four C-H dipolar interactions due to rapid motion about the director (or drug C2) axis. 2H NMR spectra of [2,4,6,8-2H4]desipramine (and imipramine) in the same 1,2-dimyristoyl-sn-glycero-3-phosphocholine-water mesophase exhibit quadrupole splittings of approximately 0-2 and approximately 20 kHz, indicating an approximate magic-angle orientation of the C2-2H(1H) and C8-2H(1H) vectors with respect to an axis of motional averaging, in accord with the 13C NMR results. Selective deuteration of imipramine confirms these ideas. Spectra of digalactosyl diglyceride [primarily 1,2-di[(9Z,12Z,15Z)-octadeca-9,12,15-trienoyl ]-3- (alpha-D-galactopyranosyl-1-6-beta-D-galactopyranosyl)-sn-glycerol]-H2O (in the L alpha phase) show a large differential line broadening for C9 but a reduced effect for C10, consistent with the results of 2H NMR of specifically 2H-labeled phospholipids [Seelig, J., & Waespe-Saracevic, N. (1978) Biochemistry 17, 3310-3315].(ABSTRACT TRUNCATED AT 250 WORDS)

Distal and proximal ligand interactions in heme proteins: correlations between C-O and Fe-C vibrational frequencies, oxygen-17 and carbon-13 nuclear magnetic resonance chemical shifts, and oxygen-17 nuclear quadrupole coupling constants in C17O- and 13CO-labeled species.

We have obtained the oxygen-17 nuclear magnetic resonance (NMR) spectra of a variety of C17O-labeled heme proteins, including sperm whale (Physeter catodon) myoglobin, two synthetic sperm whale myoglobin mutants (His E7----Val E7; His E7----Phe E7), adult human hemoglobin, rabbit (Oryctolagus cuniculus) hemoglobin, horseradish (Cochlearia armoracia) peroxidase (E.C. 1.11.1.7) isoenzymes A and C, and Caldariomyces fumago chloroperoxidase (E.C. 1.11.1.10), in some cases as a function of pH, and have determined their isotropic 17O NMR chemical shifts, delta i, and spin-lattice relaxation times, T1. We have also obtained similar results on a picket fence prophyrin, [5,10,15,20-tetrakis(alpha, alpha, alpha, alpha, alpha-pivalamidophenyl)porphyrinato]iron(II) (1-MeIm)CO, both in solution and in the solid state. Our results show an excellent correlation between the infrared C-O vibrational frequencies, v(C-O), and delta i, between v(C-O) and the 17O nuclear quadrupole coupling constant (e2qQ/h, derived from T1), and as expected between e2qQ/h and delta i. Taken together with the work of others on the 13C NMR of 13CO-labeled proteins, where we find an excellent correlation between delta i(13C) and v(Fe-C), our results suggest that IR and NMR measurements reflect the same interaction, which is thought to be primarily the degree of pi-back-bonding from Fe d to CO pi* orbitals, as outlined previously [Li, X.-Y., & Spiro, T.G. (1988) J. Am. Chem. Soc. 110, 6024]. The modulation of this interaction by the local charge field of the distal heme residue (histidine, glutamine, arginine, and possibly lysine) in a variety of species and mutants, as reflected in the NMR and IR measurements, is discussed, as is the effect of cysteine as the proximal heme ligand.

Intracellular sodium content of a wall-less strain of Neurospora crassa and effects of insulin: a 23Na-NMR study.

23Na-NMR has been used to investigate some factors influencing the sodium content of a wall-less strains of Neurospora crassa. The shift reagent Tm(DOTP)H2(NH4)3 proved useful for this purpose, while several other reagents, previously used by others, were found to be unsuitable for use with these cells. When the cells were grown, washed and resuspended in medium containing sodium (25.3 mM), the intracellular sodium concentration was calculated to be 11.9 +/- 1.4 mM. This value rose within two minutes of addition of glucose (100 mM), to greater than 14 mM. Preincubation of cells with insulin (100 nM) had a significant effect on the subsequent rate of sodium accumulation during the period 3-12 minutes following glucose addition. Insulin-treated cells showed a slow, continued accumulation of sodium during this period (+1.14 +/- 0.39%/min), while control cells lost sodium very slowly (-0.63 +/- 0.29%/min; P of difference = 0.005).

Multinuclear magnetic resonance studies on the calcium (II) binding site in trypsin, chymotrypsin, and subtilisin.

Two different nuclear magnetic resonance experiments were conducted to elucidate the properties of the Ca(II) binding locus on serine proteases in solution. Trypsin, alpha-chymotrypsin, and subtilisin were inactivated with diisopropyl fluorophosphate, and the distance of the phosphorus from Gd(III) in place of Ca(II) was determined from the lanthanide-induced relaxation on the 31P resonance. The distances found (between 20 and 21 A) were in excellent agreement with those reported in the X-ray crystallographic structures of trypsin and subtilisin, demonstrating that the method has wide applicability to systems for which no X-ray structure is available. Subsequently, the 113Cd spectra [in place of Ca(II)] were examined in the presence of the native enzymes. At ambient temperatures only a single 113Cd resonance could be observed, presumably representing the weighted average of the variously weakly bound ions and the free ion. At 280 K for trypsin and chymotrypsin, and at 268 K for subtilisin there was observed a resonance at ca. 65-70 ppm higher field than the previous averaged resonance that could be attributed to tightly bound Cd. The chemical shift of the resonance was consistent with its assignment to an octahedral environment around Cd with oxygen ligands.

Multinuclear magnetic resonance studies on serine protease transition state analogues.

31P Nuclear Magnetic Resonance (NMR) studies were performed on mono- and diisopropylphosphoryl derivatives of alpha-chymotrypsin, trypsin, and subtilisin. Questions addressed included the pKa of the active center Asp...His...Ser triad in both species. While the pKa in the diisopropylphosphoryl derivatives is near 7.4 (found in this and other laboratories earlier) and reflects a nearly normal imidazolium titration curve, the apparent pKa in the monoisopropylphosphoryl enzymes (obtained by "aging" of the diisopropylphosphoryl derivatives and monitored by 31P NMR) is between 9.7 and 11.4 depending on the protease. This latter "titration" of the 31P NMR signal is reversible and presumably reflects the interaction of the imidazolium positive charge with the monoanionic phosphodiester. Of the two tetrahedral intermediates, the properties of the monoisopropylphosphoryl enzyme are probably more representative of the tetrahedral oxyanionic intermediate invoked during peptide hydrolysis. The same NMR technique was used to determine the action of PAM (pyridine-2-aldoxime methiodide, a known "antidote" for acetylcholinesterase inactivated by diisopropylfluorophosphate), on the inactivated enzymes. It was clear that the "antidote" could reverse the diisopropylphosphorylation but was ineffective on the monoisopropylphosphoryl ("aged") enzyme. 11B NMR studies were performed on phenylboronic (PBA) acid and 3,5-bis-trifluoromethylphenylboronic acid in the absence and presence of chymotrypsin and subtilisin. At 22 degrees C the former, but not the latter, compound was in fast exchange between the free and enzyme bound states. The relaxation parameters could be calculated for the bound PBA in chymotrypsin and the fluorinated analogue in subtilisin and clearly indicated that the boron nucleus was tetrahedral in the active centers, a good analogue for the tetrahedral oxyanionic intermediate.

Metabolism of D-glucose in a wall-less mutant of Neurospora crassa examined by 13C and 31P nuclear magnetic resonances: effects of insulin.

13C NMR and 31P NMR have been used to investigate the metabolism of glucose by a wall-less strain of Neurospora crassa (slime), grown in a supplemented nutritionally defined medium and harvested in the early stationary stage of growth. With D-[1-13C]- or D-[6-13C]glucose as substrates, the major metabolic products identified from 13C NMR spectra were [2-13C]ethanol, [3-13C]alanine, and C1- and C6-labeled trehalose. Several observations suggested the existence of a substantial hexose monophosphate (HMP) shunt: (i) a 70% greater yield of ethanol from C6- than from C1-labeled glucose; (ii) C1-labeled glucose yielded 19% C6-labeled trehalose, while C6-labeled glucose yielded only 4% C1-labeled trehalose; (iii) a substantial transfer of 13C from C2-labeled glucose to the C2-position of ethanol. 31P NMR spectra showed millimolar levels of intracellular inorganic phosphate (Pi), phosphodiesters, and diphosphates including sugar diphosphates and polyphosphate. Addition of glucose resulted in a decrease in cytoplasmic Pi and an increase in sugar monophosphates, which continued for at least 30 min. Phosphate resonances corresponding to metabolic intermediates of both the glycolytic and HMP pathways were identified in cell extracts. Addition of insulin (100 nM) with the glucose had the following effects relative to glucose alone: (i) a 24% increase (P less than 0.01) in the rate of ethanol production; (ii) a 38% increase (P less than 0.05) in the rate of alanine production; (iii) a 27% increase (P less than 0.05) in the rate of glucose disappearance. Insulin thus increases the rates of production of ethanol and alanine in these cells, in addition to increasing production of CO2 and glycogen, as previously shown.

Stimulation by mammalian insulin of glycogen metabolism in a wall-less strain of Neurospora crassa.

Addition of bovine insulin to cells of the wall-less variant FGSC4761 of Neurospora crassa ("slime") produced several significant effects on glycogen metabolism. 1) Intracellular levels of the glycogen precursor UDP-glucose decreased 17-18% (P less than 0.01) within 30 min of insulin addition. 2) Cells grown with insulin possessed 40% more glycogen than did control cells. 3) The incorporation of 14C-labeled glucose into glycogen increased 41% after 30-min treatment with 100 nM bovine insulin (P less than 0.01). 4) Insulin treatment of the cells caused activation of the enzyme glycogen synthase from a glucose-6-phosphate-dependent form to an independent form. Half-maximum activation occurred with 2 nM insulin. These are similar to insulin-induced effects in some mammalian cells. In contrast, no insulin-induced effect on glucose transport could be demonstrated in these cells.