Conformational properties of DNA fragments containing GAC trinucleotide repeats associated with skeletal displasias

The human gene for cartilage oligomeric matrix protein contains five tandem repeats of the GAC trinucleotide. Its expansion by one repeat causes multiple epiphyseal dysplasia, while expansion by two repeats or, remarkably, deletion of one repeat causes pseudoachondroplasia. Here we used CD spectroscopy, PAGE and UV absorption spectroscopy to compare conformational properties of the DNA strands containing four, five, six and seven repeats of the GAC trinucleotide. The (GAC)n strands were found to form four distinct ordered conformations, depending on the solution conditions. The first was a foldback, stable at slightly alkaline pH values and low and medium ionic strengths. Increasing salt concentration induced a transition of the foldback into an antiparallel right-handed homoduplex. Both the conformers contained the Watson-Crick G.C pairs while the intervening adenines contributed little to their B-like conformation. Thirdly, the strands associated into a parallel homoduplex stabilized by the hemiprotonated C+.C pairs and by the GpA steps that both favor the parallel DNA strand orientation. The parallel homoduplex was stable even at neutral pH. The fourth conformation was the left-handed Z-DNA, which formed easier with (GAC)n than with (GC)n of comparable length, indicating that the adenines of (GAC)n promoted the left-handed duplex. The paper shows that stability of the above four conformers strongly depends on the GAC repeat number.     

Circular dichroism spectra of DNA oligomers show that short interior stretches of C.C+ base pairs do not form in duplexes with A.T base pairs

Circular dichroism (CD) experiments were carried out on a series of DNA oligomers to determine if short internal stretches of protonated cytosine-cytosine (C.C+) base pairs could coexist with adenine-thymine (A.T) base pairs. (1) C.C+ base pairs did form in the absence of A.T base pairs in the individual oligomers d(AACC)5 and d(CCTT)5, as indicated by the appearance of a long-wavelength CD band centered at 282-284 nm, when the pH was lowered to 6 or 5 at 0.5 M Na+. A comparison of measured with calculated spectra showed that d(CCTT)5 at pH 5, 0.5 M Na+, 20 degrees C, likely adopted a structure with a central core of stacked C.C+ base pairs and looped-out thymines. Under the same conditions, it appeared that C.C+ base pairs also formed in d(AACC)5, but with the adenines remaining intrahelical. Each of these oligomers showed a cooperative transition for formation of C.C+ base pairs as the temperature was lowered, with C.C+ base pairs forming at a higher temperature in d(CCTT)5 than in d(AACC)5. A.T base formed in equimolar mixtures of d(AACC)5 plus d(CCTT)5 as monitored by an increase in the negative magnitude of the 250-nm CD band. However, a large increase did not appear at about 285 nm in CD spectra of the mixtures, showing that there were no stacked C.C+ base pairs in the d(AACC)5.d(CCTT)5 duplex even though they formed under the same conditions in the individual strands. Thus, in this duplex, A.T base pairs prevented the formation of neighboring internal C.C+ base pairs. (2) CD measurements were also made of d(A10C4T10).(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of base pairing in ribonucleic acid by Fourier-transform infrared spectrometry: yeast ribosomal 5S ribonucleic acid

Fourier-transform infrared (FT-IR) spectra of yeast ribosomal 5S RNA have been acquired at several temperatures between 30 and 90 degrees C. The difference spectrum between 90 (bases unstacked) and 30 degrees C (bases stacked) provides a measure of base stacking in the RNA. Calibration difference spectra corresponding to stacking of G-C or A-U pairs are obtained from "reference" FT-IR spectra of poly(rG) X poly(rC) minus 5'-GMP and 5'-CMP or poly(rA) X poly(rU) minus 5'-AMP and 5'-UMP. The best fit linear combination of the calibration G-C and A-U difference spectra to the 5S RNA (90-30 degrees C) difference spectrum leads to a total of 25 +/- 3 base pairs (17 G-C pairs + 8 A-U pairs) for the native yeast 5S RNA in the absence of Mg2+. In the presence of Mg2+, an additional six base pairs are detected by FT-IR (one G-C and five A-U). FT-IR melting curve midpoints show that A-U and G-C pairs melt together (65 and 63 degrees C) in the presence of Mg2+ but A-U pairs melt before G-C pairs (47 vs. 54 degrees C) in the absence of Mg2+.     

Protonation of deoxycytidine residues in dC4 tetraloops: UV spectrophotometric study of dC10 and d(A14C4T14)

It is shown that component analysis could be applied to study the UV difference spectra of cytidine oligomers and hairpin oligonucleotides with cytidines in the loop region in order to account for the melting and titration results in terms of cytidine stacking and protonation. Upon acid titration, the dC(10) oligomer undergoes cooperative conformational transition at pH 6.3 accompanied by protonation and formation of the i-structure with half of the residues protonated. The stability of the hemiprotonated structure increases with decreasing pH, the i-structure persisting still in the region of pH相似文献   

Structure of the Glycyl-L-histidyl-L-lysine--copper(II) complex in solution

Optical, electron paramagnetic resonance, and electron spin-echo envelope spectroscopies were used to examine the structure of the Cu(II) complex of glycyl-L-histidyl-L-lysine (GHL) in solution. At neutral pH, GHL forms a mononuclear 1:1 Cu(II) compound having an EPR spectrum resembling that of Cu(II) equatorially coordinated by two or three nitrogen atoms. Electron spin-echo studies demonstrate that one of these is located in the histidyl imidazole ring. A pH titration of Cu(II)-GHL shows three optical transitions with apparent pKs of 3.6, 9.2 and 11.4 and molecularities, with respect to protons, of 2, 2, and 1, respectively. At the lowest pK, GHL binds Cu(II), forming the species present at physiological pH. At elevated pH, spectroscopic experiments suggest that an alteration of the Cu(II) structure occurs, yet the bound imidazole is retained. These solution studies are consistent with nitrogen coordination of Cu(II) in Cu(II)-GHL, but the solid-state polymeric structure, with oxygen-bridged Cu(II) pairs as previously determined by X-ray crystallographic analysis [Pickart, L., Freedman, J. H., Loker, W. J., Peisach, J., Perkins, C. M., Steinkamp, R. E., & Weinstein, B. (1980) Nature (London) 288, 715-717; C. M. Perkins, N. J. Rose, R. E. Steinkamp, L. H. Jensen, B. Weinstein, and L. Pickart, unpublished results], does not exist in solution.     

Examination of the premelting transition of DNA A-tracts using a fluorescent adenosine analogue

The fluorescent adenosine analogue 4-amino-8-(2-deoxy-beta-d-ribofuranosyl)-5'-O-dimethoxytrityl-6-methyl-7(8H)-pteridone (6MAP) has been used to perform residue specific analyses of DNA A-tracts during the premelting transition. DNA A-tracts, which exhibit sequence-induced curvature, adopt a B-DNA conformation as a function of increasing temperature. Fluorescence melting curves indicate that 6MAP is a more sensitive reporter of the premelting transition than UV absorption spectroscopy. Further, residue specific fluorescence analyses of A-tract and control duplexes reveal that some of the conformational changes associated with the premelting transition occur within A-tract regions. Analyses of the energetics of the premelting transition indicate that ApA steps make a larger enthalpic contribution to the premelting transition than ApT steps. To explore the effect of cations on the premelting transition, fluorescence melts were performed in the presence of NH(4)(+), Mg(2+), and low (0.05 M) and high (0.5 M) concentrations of Na(+). These studies show that the fluorescence intensity changes associated with the premelting transition are sensitive to cation type and concentration and are larger and more pronounced in the presence of 0.5 M Na(+), NH(4)(+), and Mg(2+). Incorporation of 6MAP into longer duplexes containing phased A-tracts shows that the local environment of adenosines in phased A-tracts is similar to that of individual A-tracts. Fluorescence quenching results indicate that ApA and ApT steps within A-tracts are less solvent exposed than their counterparts in control sequence isomers, possibly because of the narrowed minor groove of A-tract sequences.     

Solution conformation of an RNA hairpin loop.

The hairpin conformation adopted by the RNA sequence 5'GCGAUUUCUGACCGCC3' has been studied by one- and two-dimensional NMR spectroscopy. Exchangeable imino spectra in 60 mM Na+ indicate that the hairpin has a stem of six base pairs (indicated by boldface type) and a loop of three nucleotides. NOESY spectra of nonexchangeable protons confirm the formation of the stem region. The duplex has an A-conformation and contains an A.C apposition; a G.U base pair closes the loop region. The stem nucleotides have C3'-endo sugar conformations, as expected of an A-form duplex, whereas the three loop nucleotides adopt C2'-endo sugar puckers. Stacking within the loop, C8 upon the sugar of U7, stabilizes the structure. The pH dependence of both the exchangeable and nonexchangeable NMR spectra is consistent with the formation of an A+.C base pair, protonated at the N1 position of adenine. The stability of the hairpin was probed by using absorbance melting curves. The hairpin structure with the A+.C base pair is about +2 kcal/mol less stable in free energy at 37 degrees C than the hairpin formed with an A.U pair replacing the A+.C pair.     

An estimate of the nearest neighbor base-pair content of 5S RNA using CD and absorption spectroscopy.

We analyzed the CD and uv absorption spectra of 5S RNA from Escherichia coli using the method developed in the preceding paper. The analysis of spectra of 5S RNA at 20 degrees C in 0.1M NaClO4, 2.5 mM Na+ (phosphate), pH 7.0, and 0.5 mM MgSO4 gave 7 +/- 3.6 A.U base pairs, 25 +/- 3.6 G.C base pairs, and 7.5 +/- 3.6 G.U base pairs. Estimates of nearest neighbor base pairs were more consistent with the Pieler-Erdmann and the Gewirth-Moore secondary structure models than with the Fox-Woese or the Burns-Luoma-Marshall models. We also examined the structure of 5S RNA as a function of temperature. The melting profile exhibited two transitions--one at about 35 degrees C and one above 50 degrees C. Our spectral data showed that helices I and II were stable during the first transition, and agreed with other data that helix III was the most likely helix to have melted. The results from this in-depth study of 5S RNA indicate that our method of analysis should be useful for studying the secondary structures of other small, unmodified RNAs.     

The influence of the thymine C5 methyl group on spontaneous base pair breathing in DNA

Sequences of four or more AT base pairs without a 5'-TA-3' step, so-called A-tracts, influence the global properties of DNA by causing curvature of the helix axis if phased with the helical repeat and also influence nucleosome packaging. Hence it is interesting to understand this phenomenon on the molecular level, and numerous studies have been devoted to investigations of dynamical and structural features of A-tract DNA. It was early observed that anomalously slow base pair-opening kinetics were a striking physical property unique to DNA A-tracts (Leroy, J. L., Charretier, E., Kochoyan, M., and Gueron, M. (1988) Biochemistry 27, 8894-8898). Furthermore, a strong correlation between DNA curvature and anomalously slow base pair-opening dynamics was found. In the present work it is shown, using imino proton exchange measurements by NMR spectroscopy that the main contribution to the dampening of the base pair-opening fluctuations in A-tracts comes from the C5 methylation of the thymine base. Because the methyl group has been shown to have a very limited effect on the DNA curvature as well as the structure of the DNA helix, the thymine C5 methyl group stabilizes the helix directly. Empirical potential energy calculations show that methylation of the tract improves the stacking energy of a base pair with its neighbors in the tract by 3-4 kcal/mol.     

Studying the Influence of the Pyrene Intercalator TINA on the Stability of DNA i-Motifs

Certain cytosine-rich (C-rich) DNA sequences can fold into secondary structures as four-stranded i-motifs with hemiprotonated base pairs. Here we synthesized C-rich TINA-intercalating oligonucleotides by inserting a nonnucleotide pyrene moiety between two C-rich regions. The stability of their i-motif structures was studied by using UV melting temperature measurements and circular dichroism spectra at different pH values under noncrowding and crowding conditions (20% poly(ethylene glycol)). When TINA ((R)-3-((4-(1-pyrenylethynyl)benzyl)oxy) propane-1,2-diol) is inserted, the oligonucleotides could form an i-motif at a higher pH than observed for the corresponding wildtype oligonucleotide.     

A-tract polarity dominate the curvature in flanking sequences

It is well-known, but little understood, that the nucleotide sequences between phased A(4-6)-tracts (at 10-11 bp intervals) have only a slight effect on overall curvature. To explore this phenomenon, we have examined the gel-migration properties of sequences containing both A-tracts as well as G-tracts (i.e., sequences of the form G(n)C(m) or C(n)G(m), n + m > 4) in various relative positioning. We show that the composite bend of these sequences depends on their relative arrangement. When G-tracts are placed between two A-tracts, such that both tracts are repeated in phase to themselves (e.g., G(5)A(6)G(5)A(5)), or adjacent to the 3'-side of A-tracts (e.g., A(6)G(5)N(10)), they have minimal influence on the extent of bending of the composite sequence. When G-tracts are placed one helical repeat away from A-tracts (e.g., G(5)N(5)A(6)N(6)), or are adjacent only to the 5'-side of A-tracts (e.g., G(5)A(6)N(10)) their influence on the composite bend is larger. The differential behavior of AG- versus GA-tracts means that A-tracts influence their flanking sequences in a polar manner. Whereas they suppress, or make constant, the intrinsic bending characteristics of any sequence placed immediately 3' to them (and hence by definition any sequence placed between two phased A-tracts), sequences adjoining them on their 5'-side are free to modulate the overall curvature. We interpret these results as evidence for the dominant nature of the unique and nonuniform structure adopted by tracts of four adenines or more. The effects of A-tracts extend at least five base pairs into the adjoining 3' region. This is further evidence for the complexity of DNA structure and the inadequacy of simple nearest-neighbor models to explain all its manifestations.     

Thermodynamics of triple helix formation: spectrophotometric studies on the d(A)10.2d(T)10 and d(C+3T4C+3).d(G3A4G3).d(C3T4C3) triple helices.

We have stabilized the d(A)10.2d(T)10 and d(C+LT4C+3).d(G3A4G3).d(C3T4C3) triple helices with either NaCl or MgCl2 at pH 5.5. UV mixing curves demonstrate a 1:2 stoichiometry of purine to pyrimidine strands under the appropriate conditions of pH and ionic strength. Circular dichroic titrations suggest a possible sequence-independent spectral signature for triplex formation. Thermal denaturation profiles indicate the initial loss of the third strand followed by dissociation of the underlying duplex with increasing temperature. Depending on the base sequence and ionic conditions, the binding affinity of the third strand for the duplex at 25 degrees C is two to five orders of magnitude lower than that of the two strands forming the duplex. Thermodynamic parameters for triplex formation were determined for both sequences in the presence of 50 mM MgCl2 and/or 2.0 M NaCl. Hoogsteen base pairs are 0.22-0.64 kcal/mole less stable than Watson-Crick base pairs, depending on ionic conditions and base composition. C+.G and T.A Hoogsteen base pairs appear to have similar stability in the presence of Mg2+ ions at low pH.     

Parallel-stranded linear homoduplexes of d(A+-G)n > 10 and d(A-G)n > 10 manifesting the contrasting ionic strength sensitivities of poly(A+.A+) and DNA.

In contrast to shorter homologs which only form a single-stranded nucleic acid alpha-helix in acid solution at [Na+]</=0.02 M Na+, d(A-G)20,30 form in addition a parallel-stranded duplex with (A+.A+) and (G.G) base pairs and interstrand dA+...PO2-ionic and dA+NH2... O=P H-bonds. Under conditions where duplex prevails over alpha-helix, the contribution of the base-backbone interactions to stability varies directly with [H+] and inversely with [Na+], just as in poly(A+.A+). These duplexes are characterized by intense circular dichroism and a large cooperative thermally-induced hyperchromic transition that is dependent on oligomer concentration. Dimethylsulfate reactivity of the dG residues indicates G.G and therefore dA+.dA+rather than dA+.G base pairs. At much higher ionic strength (Na+>/=0.2 M) the protonated base-backbone interactions are so weakened that duplex stability becomes increasingly dependent upon H-bonded base pairing and stacking and almost independent of pH. Between pH 6 and 8 this duplex structure is devoid of protonated dA residues and shows positive dependence of T m on ionic strength similar to that of DNA.     

Secondary structure of calsequestrin in solutions and in crystals as determined by Raman spectroscopy

Calsequestrin has been precipitated with calcium into five different crystal forms: cruciform twins, flat rectangles, thin needles, bipyramids, rectangular prisms, and a sixth precrystalline form, spheres. Raman spectra of the spheres and the cruciform twins are the same. The Raman spectrum of a physiological concentration (10%) of calsequestrin in calcium-free solution is the same as the spectrum of calcium precipitated calsequestrin in the amide I region, and in the C-C stretching region, but these spectra are different in the amide III region. The Raman spectrum of unfolded calsequestrin in 5 M guanidine hydrochloride is quite different from the other spectra, but it is not similar to the spectra of other unfolded proteins. Estimates of secondary structure from the amide I region indicate that calsequestrin in calcium-free solution and calcium-precipitated forms has 40 +/- 5% helix, 30 +/- 4% beta-strand, and 18 +/- 2% reverse turn. Secondary structure estimates calculated from the amide III region are not significantly different. They indicate 41 +/- 5% helix and 36 +/- 6% beta-strand for the precipitated forms, and 32 +/- 5% helix and 39 +/- 6% beta-strand for solutions. Calsequestrin unfolded in 5 M guanidine hydrochloride at 100 mg/ml gives 24 +/- 5% helix and 48 +/- 6% beta-strand.     

Resonance Raman microprobe spectroscopy of rhodopsin mutants: effect of substitutions in the third transmembrane helix.

A microprobe system has been developed that can record Raman spectra from as little as 2 microL of solution containing only micrograms of biological pigments. The apparatus consists of a liquid nitrogen (l-N2)-cooled cold stage, an epi-illumination microscope, and a substractive-dispersion, double spectrograph coupled to a l-N2-cooled CCD detector. Experiments were performed on native bovine rhodopsin, rhodopsin expressed in COS cells, and four rhodopsin mutants: Glu134 replaced by Gln (E134Q), Glu122 replaced by Gln (E122Q), and Glu113 replaced by Gln (E113Q) or Ala (E113A). Resonance Raman spectra of photostationary steady-state mixtures of 11-cis-rhodopsin, 9-cis-isorhodopsin, and all-trans-bathorhodopsin at 77 K were recorded. The Raman spectra of E134Q and the wild-type are the same, indicating that Glu134 is not located near the chromophore. Substitution at Glu122 also does not affect the C = NH stretching vibration of the chromophore. The fingerprint and Schiff base regions of the Raman spectra of the 380-nm, pH 7 forms of E113Q and E113A are characteristic of unprotonated retinal Schiff bases. The C = NH modes of the approximately 500-nm, pH 5 forms of E113Q and E113A in H2O (D2O) are found at 1648 (1629) and 1645 (1630) cm-1, respectively. These frequencies indicate that the protonated Schiff base interacts more weakly with its protein counterion in the Glu113 mutants than it does in the native pigment. Furthermore, perturbations of the unique bathorhodopsin hydrogen out-of-plane (HOOP) vibrations in E113Q and E113A indicate that the strength of the protein perturbation near C12 is weakened compared to that in native bathorhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nigericin-mediated H+, K+ and Na+ transports across vesicular membrane: T-jump studies.

The decay of delta pH across vesicular membranes by nigericin-mediated H+ and metal ion (M+) transports has been studied at 25 degrees C after creating delta pH by temperature jump (T-jump). In these experiments K+ or Na+ were chosen as M+ for the compensating flux. Theoretical expressions derived to analyse these data suggest a method for estimating the intrinsic rate constants for the translocation of nig-H (k1) and for the translocation of nig-M (k2) across membrane, from the pH dependence of the delta pH decay. The following could be inferred from the analysis of data. (a) At pH approximately 7.5 and 250 mM ion concentrations, nigericin-mediated H+ and M+ transport rates are lower in a medium of K+ than in a medium of Na+, although ionophore selectivity of nigericin towards K+ is 25-45-times higher than that towards Na+. However, at lower [M+] (approximately 50 mM) the transport rates are higher in a medium of K+ than in a medium of Na+. Such behaviours can be understood with the help of parameters determined in this work. (b) The intrinsic rate constants k1 and k2 associated with the translocations of nig-H and nig-K or nig-Na across membrane are similar in magnitude. (c) At pH approximately 7.5 translocation of nig-H is the dominant rate-limiting step in a medium containing K+. In contrast with this, at this pH, translocation of nig-M is the dominant rate-limiting step when metal ion is Na+. (d)k1 approximately k2 approximately 6.10(3) s-1 could be estimated at 25 degrees C in vesicles prepared from soyabean phospholipid, and lipid mixtures of 80% phosphatidylcholine (PC) + 20% phosphatidylethanolamine and 92% PC + 8% phosphatidic acid. (e) The apparent dissociation constants of nig-M in vesicles were estimated to be approximately 1.5.10(-3) M for K+ and 6.4.10(-2) M for Na+ (at 50 mM ion concentrations) using approximately 10(-8.45) M for the apparent dissociation constant of nig-H.     

Parallel stranded DNA with AT base pairing

The concentration and temperature dependences of the UV and CD spectra of the oligonucleotide 3'-d(ApTpApTpApTpApTpApTp)-O(CH2)6O-5'-d(pApTpApTpApTpApT pApT) (eicosamer) in aqueous solution at pH 7 in the presence of 0.5 M NaCl were studied. At less than 10(-6) M, the eicosamer was shown to form in solution a hairpin with parallel orientation of chains (parallel hairpin). From thermal denaturation profiles [A260(T)] the thermodynamic parameters, delta H degrees, delta S degrees and Tm for parallel hairpin formation were calculated to be -90 +/- 8 kJ/mol. -300 +/- 20 J.mol-1.K-1 and 40.5 degrees C, respectively. The CD spectra of the parallel double helix differed from those of B-form DNA and had characteristic features: decreasing magnitude of the positive maximum at 265 nm and a negative peak at 285 nm.     

Chemical modification of pyrimidine TFOs: effect on i-motif and triple helix formation

In order to form more stable triple helical structures or to prevent their degradation in cells, oligonucleotide analogs are routinely used, either in the backbone or among the bases. The target sequence chosen for this study is a 16-base-long oligopurine-oligopyrimidine region present in the human neurotrophin 4/5 gene. Seven different chemical modifications were tested for their effect on (i) triple helix formation and (ii) i-DNA stability. i-DNA is a tetrameric structure involving hemiprotonated C x C+ base pairs, which may act as a competing structure for triplex formation, especially in the case of a cytosine-rich third strand. At acid pH, oligophosphoramidates formed the most stable triple helix, whereas oligonucleotides including 5-propynyl-dU formed a stable i-motif which precluded triplex formation. Only two candidates stabilized triple helices at neutral pH: oligonucleotides with phosphoramidate linkage and phosphodiester oligonucleotides containing 5-methyl-dC and 5-propynyl-dU.     

Conformational stability changes of the amino terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system produced by substituting alanine or glutamate for the active-site histidine 189: implications for phosphorylation effects

The amino terminal domain of enzyme I (residues 1-258 + Arg; EIN) and full length enzyme I (575 residues; EI) harboring active-site mutations (H189E, expected to have properties of phosphorylated forms, and H189A) have been produced by protein bioengineering. Differential scanning calorimetry (DSC) and temperature-induced changes in ellipticity at 222 nm for monomeric wild-type and mutant EIN proteins indicate two-state unfolding. For EIN proteins in 10 mM K-phosphate (and 100 mM KCl) at pH 7.5, deltaH approximately 140 +/- 10 (160) kcal mol(-1) and deltaCp approximately 2.7 (3.3) kcal K(-1) mol(-1). Transition temperatures (Tm) are 57 (59), 55 (58), and 53 (56) degrees C for wild-type, H189A, and H189E forms of EIN, respectively. The order of conformational stability for dephospho-His189, phospho-His189, and H189 substitutions of EIN at pH 7.5 is: His > Ala > Glu > His-PO3(2-) due to differences in conformational entropy. Although H189E mutants have decreased Tm values for overall unfolding the amino terminal domain, a small segment of structure (3 to 12%) is stabilized (Tm approximately 66-68 degrees C). This possibly arises from an ion pair interaction between the gamma-carboxyl of Glu189 and the epsilon-amino group of Lys69 in the docking region for the histidine-containing phosphocarrier protein HPr. However, the binding of HPr to wild-type and active-site mutants of EIN and EI is temperature-independent (entropically controlled) with about the same affinity constant at pH 7.5: K(A)' = 3 +/- 1 x 10(5) M(-1) for EIN and approximately 1.2 x 10(5) M(-1) for EI.     

TPA induces cytoplasmic alkalinization in human monoblastic U-937 cells without activation of Na+/H+ exchange

The effect of the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on cytoplasmic pH (pHi) and H+ extrusion was studied in the human monoblastic cell line U-937. About 2 min after addition of TPA, pHi started to increase and reached a steady state 10-15 min later. The resulting alkalinization corresponded to 0.03 and 0.09 pH units at 10(-10) and 10(-7) M TPA, respectively. The TPA-induced increase in pHi was independent of the presence of extracellular Na+. Moreover, TPA did not affect the H+ extrusion from the U-937 cells. Together these observations indicate the presence of a novel mechanism for TPA-induced cytoplasmic alkalinization. This mechanism is independent of Na+/H+ exchange across the plasma membrane, but may involve organelle sequestration of H+.