Acidic pH inhibits non-MHC-restricted killer cell functions.

Immunotherapeutic strategies in advanced stages of solid tumors have generally met with little success. Various mechanisms have been discussed permitting the escape of tumor cells from an effective antitumoral immune response. Solid tumors are known to develop regions with acidic interstitial pH. In a recent study performed in the human system, we were able to demonstrate that non-MHC-restricted cytotoxicity is inhibited by an acidic microenvironment. To get more insight into the mechanisms leading to this reduced cytotoxic activity, we have now investigated the influence of an acidic extracellular pH (pH(e)) on the killing process in detail. Unstimulated PBMC and LAK cells were used as effector cells. Both populations are able to kill tumor cells in a MHC-independent manner via perforin/granzymes or TNFalpha, whereas only IL-2-activated cells can use the killing pathway via Fas/FasL. We studied the influence of a declining pH(e) on the different killing pathways against TNFalpha-sensitive and -resistant, as well as Fas-positive and -negative, target cells. Experiments in the absence of extracellular Ca(2+) were used to discriminate the Ca(2+)-dependent perforin-mediated killing. Here we show that the release of perforin/granzyme-containing granules, the secretion of TNFalpha, and also the cytotoxic action of Fas/FasL interaction or of membrane-bound TNFalpha were considerably inhibited by declining pH(e). Furthermore, the secretion of the activating cytokine IFNgamma, as well as the release of the down-regulating cytokines IL-10 and TGF-beta(1), was strictly influenced by surrounding pH. As a pH(e) of 5.8 resulted in a nearly complete loss of cytotoxic effector cell functions without affecting their viability, we investigated the influence of pH(e) on basic cellular functions, e.g. , mitochondrial activity and regulation of intracellular pH. We found an increasing inhibition of both functions with declining pH(e). Therefore, an acidic pH(e) obviously impairs fundamental cellular regulation, which finally prevents the killing process. In summary, our data show a strict pH(e) dependence of various killer cell functions. Thus, an acidic microenvironment within solid tumors may contribute to the observed immunosuppression in vivo, compromising antitumoral defense and immunotherapy in general, respectively.

An acidic microenvironment inhibits antitumoral non-major histocompatibility complex-restricted cytotoxicity: implications for cancer immunotherapy.

Local immunosuppression may explain the failure of an effective immune response against solid tumors. Although it is well known that the interstitial pH is significantly lower in solid tumors than in normal tissue, only a few studies in the mouse system have investigated the influence of this acidic milieu on the anti-tumoral cytotoxic response. Here the authors report the suppression of human non-major histocompatibility complex (MHC)-restricted cytotoxicity against tumor cells by an acidic extracellular pH (pHe). Unstimulated peripheral blood mononuclear cells, lymphokine-activated killer (LAK) cells, and natural killer cell clones were used as effector cells. According to pH measurements in solid tumors, representative pH values of 7.2 to 5.3 were chosen during the cytotoxic assays. Target cell lysis was measured using two nonradioactive fluorometric methods, namely two-color flow cytometry and a modified calcein-release assay, which allowed cell-mediated cytotoxicity to be measured and compared with that in adherent targets. Using K562, Daudi, or Raji as suspended target cell lines, the cytotoxic activity of unstimulated peripheral blood mononuclear cells and of LAK cells was markedly reduced by a decreasing pHe. An extracellular pH of 5.8 to 5.3 resulted in a nearly complete loss of the cytotoxic response. This pHe-dependent impairment of the killing activity could also be shown for killer cells stimulated with interleukins-7 and -12, phytohemagglutinin, or lipopolysaccharide. The lytic potential of homogeneous natural killer cell clones as effectors was also strictly influenced by the surrounding pH. The pHe dependence of the non-MHC-restricted killer cell functions against tumor cells seems to be a general phenomenon, because the cytolytic activity of LAK cells against six human adherent tumor cell lines (HeLa, HepG2, LS174T, LS174Te, MCF-7, and RT112) was also clearly reduced under acidic conditions. To initiate the killing process, adhesion molecules play an important role in recognition and binding of the target cell. However, flow cytometric analysis revealed that the expression pattern of relevant adhesion molecules was unaffected by acidic pHe. In conclusion, these data clearly indicate an inhibition of non-MHC-restricted cytotoxicity against tumor cells by an acidic pHe, which may contribute to the failure of immunosurveillance against solid tumors. Consequently, efforts to enhance the anti-tumoral cytotoxicity by immunotherapies may have limited success.

An acidic microenvironment impairs the generation of non-major histocompatibility complex-restricted killer cells.

The microenvironment within solid tumours has often been shown to exhibit an acidic local pH. In recent studies we could demonstrate that an acidic extracellular pH (pHe) inhibits the non-major histocompatibility complex (MHC) -restricted cytotoxicity of immunocompetent effector cells. However, within tumours the activation of cytotoxic cells may already be impaired by low pHe. Therefore, we investigated the influence of acidic conditions on the generation of active killer cells. The cytotoxic activity of natural killer (NK) as well as lymphokine-activated killer (LAK) cells against K562, Daudi and Raji cells was analysed after an activation period of 3 days at pHe 7.2-6.5. A minor reduction of pHe from 7.2 to 7.0 during the culture period resulted in a strong inhibition of the natural cytotoxicity of NK cells. Furthermore, acidic pHe below 7.2 prevented the generation of activated LAK cells by interleukin-2 (IL-2). The cytotoxic capacity could not be reconstituted if cells cultured at a pHe of 6.5 were returned to physiological pH for another 24 hr. Analysis of the cellular subtypes within the various cultures did not reveal differences regarding the frequencies of NK cells, CD8+ T cells, or CD4+ T cells. However, an acidic pHe clearly inhibited the activation-induced increase of relevant adhesion molecules. The production of cytokines which are involved in the regulation of the cytotoxic process (tumour necrosis factor-alpha, interferon-gamma, IL-10, IL-12 and transforming growth factor-beta1) was also affected by pHe, as their release was strongly inhibited at pHe 7.0. Furthermore, we observed a considerable decrease in the metabolic activity of effector cells at acidic pHe. In summary, our findings suggest that an acidic microenvironment impairs the induction of an anti-tumoral immune response within solid tumours.

Ca2+ release from the phosphorylated and the unphosphorylated sarcoplasmic reticulum Ca2+ ATPase results in parallel structural changes. An infrared spectroscopic study.

Structural changes of the sarcoplasmic reticulum Ca2+-ATPase occurring in the reaction step involving phosphoenzyme conversion and Ca2+ release (Ca2E1-P --> E2-P) were followed using time-resolved infrared spectroscopy in H2O and 2H2O. The difference spectra measured between 1800 and 1500 cm-1 were almost identical to those of Ca2+ release from the unphosphorylated ATPase (Ca2E1 --> E), implying that parallel structural changes occur in both steps. This suggests that characteristic structural features of the high affinity Ca2+ binding sites of Ca2E1 are still present in the ADP-sensitive phosphoenzyme Ca2E1-P. In both Ca2+ release steps at least two carboxyl groups become protonated, each of them experiencing the same strength of hydrogen bonding irrespective of whether or not the Ca2+ free ATPase is phosphorylated. This suggests that the same amino acid residues are involved and that they are most likely those that participate in high affinity Ca2+ binding and H+ countertransport. We propose that during Ca2+ release from the phosphoenzyme protons from the lumenal side have access to these residues. Our results are consistent with only one pair of Ca2+ binding sites on the ATPase that serves both Ca2+ translocation and H+ countertransport.

Time-resolved infrared spectroscopy of the Ca2+-ATPase. The enzyme at work.

Changes in the vibrational spectrum of the sarcoplasmic reticulum Ca2+-ATPase in the course of its catalytic cycle were followed in real time using rapid scan Fourier transform infrared spectroscopy. In the presence of Ca2+, the cycle was induced by the photochemical release of ATP from a biologically inactive precursor (caged ATP, P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate). Absorbance changes arising from ATP binding to the ATPase were observed within the first 65 ms after initiation of ATP release. After ATP binding, up to two subsequent partial reactions of the ATPase reaction cycle were observed depending on the buffer composition (10 mM CaCl2 + 330 mM KCl or 1 mM CaCl2 + 20% Me2SO): (i) formation of the ADP-sensitive phosphoenzyme (kapp = 0.79 s-1 +/- 15% at 1 degrees C, pH 7.0, 10 mM CaCl2, 330 mM KCl) and (ii) phosphoenzyme conversion to the ADP-insensitive phosphoenzyme concomitant with Ca2+ release (kapp = 0.092 s-1 +/- 7% at 1 degrees C, pH 7.0, 1 mM CaCl2, 20% Me2SO). Each reaction step could well be described by a single time constant for all associated changes in the vibrational spectrum, and no intermediates other than those mentioned above were found. In particular, there is no evidence for a delay between the transition from ADP-sensitive to ADP-insensitive phosphoenzyme and Ca2+ release. In 2H2O a kinetic isotope effect was observed: both the phosphorylation reaction and phosphoenzyme conversion were slowed down by factors of 1.5 and 3.0, respectively. The small amplitudes of the observed changes in the infrared spectrum indicate that the net change of secondary structure is very small and of the same order of magnitude for ATP binding, phosphorylation, and phosphoenzyme conversion. Therefore, our results do not support a distinction between minor and major secondary structure changes in the catalytic cycle of the ATPase, which might be expected according to the classical E1-E2 model.

Molecular and crystal structure of an amphiphile: 4'-propoxybiphenyl-4-methyl-N,N-dimethylamineoxide dihydrate.

A novel amphiphile, 4'-propoxybiphenyl-4-methyl-N,N-dimethylamineoxide, has been synthesized, crystallized (P2(1)/a, a = 9.084 A, b = 8.911 A, c = 22.460 A, beta = 96.224 degrees) and its crystal structure was determined. The amphiphile forms a bilayer in which the amineoxide oxygen of each molecule binds two water molecules. In the hydrophobic part of the bilayer the biphenyls form edge-to-face contacts, in the polar layer there is a hydrogen bonding network. The potential use of the compound as a detergent for membrane proteins has been demonstrated and the relevance of the amineoxide hydrate for other detergents discussed.

Protonation of Glu L212 following QB- formation in the photosynthetic reaction center of Rhodobacter sphaeroides: evidence from time-resolved infrared spectroscopy.

The protonation events that occur upon QA-QB-->QAQB- electron transfer in photosynthetic reaction centers from Rhodobacter sphaeroides were investigated by time-resolved infrared spectroscopy using tunable diode lasers as previously described [Mäntele, W., Hienerwadel, R., Lenz, F., Riedel, E. J., Grisar, R., & Tacke, M. (1990) Spectrosc. Int. 2, 29-35; Hienerwadel, R., Thibodeau, D. L., Lenz, F., Nabedryk, E., Breton, J., Kreutz, W., & Mäntele, W. (1992) Biochemistry 31, 5799-5808]. In the mid-infrared region between 1695 and 1780 cm-1, transient signals associated with QA-QB-->QAQB- electron transfer were observed and characterized. The dominant transient absorbance changes are three positive signals at 1732, 1725, and 1706 cm-1 and two negative signals at 1716 and at 1698 cm-1. The 1725 cm-1-signal disappears upon 1H-->2H exchange as expected for an accessible COOH group and is absent in Glu L212 Gln mutant reaction centers. On this basis, we propose an assignment of this signal to the COOH group of Glu L212. The other signals could correspond to intensity changes and/or shifts of other carboxylic residues, although contributions from ester C = O groups of bacteriopheophytins cannot be ruled out. In native reaction centers at pH 7 and at 4 degrees C, biphasic kinetics of the transient components were observed at most frequencies. The major signal at 1725 cm-1 exhibits a fast kinetic component of t 1/2 = 0.18 ms (25% of the total amplitude) and a slow one of t1/2 = 1 ms (75% of the total amplitude). A global fit analysis of the signals between 1695 and 1780 cm-1 revealed that the spectral distributions of the fast and the slow components are different. Biphasic kinetics with comparable half-times were also observed for the Glu L212 to Gln mutant. The simplest model to explain these results is that the fast phase represents electron transfer and the slow phase represents proton transfer and/or conformational changes coupled to electron transfer. The difference spectra of the slow component from native reaction centers show that the 1725 cm-1 band corresponds to an absorbance increase and not to a shift of an existing band. The signal is therefore proposed to arise from the protonation of Glu L212. The amplitude of the 1725 cm-1 signal varies distinctly with pH as expected for protonation of a COO- group. With increasing pH, the amplitude of the slow component increases while that of the fast component decreases slightly.(ABSTRACT TRUNCATED AT 400 WORDS)

Structural changes of sarcoplasmic reticulum Ca(2+)-ATPase upon Ca2+ binding studied by simultaneous measurement of infrared absorbance changes and changes of intrinsic protein fluorescence.

Ca2+ binding to sarcoplasmic reticulum Ca(2+)-ATPase was investigated by Fourier transform infrared (FTIR) spectroscopy using the photolytic release of Ca2+ from the photolabile Ca2+ chelator 1-(2-nitro-4,5-dimethoxy)-N,N,N',N',- tetrakis[(oxycarbonyl)]methyl-1,2-ethandiamine (DM-nitrophen). IR absorbance changes in 1H2O and 2H2O were detected in the spectral region from 1800 cm-1 to 1200 cm-1, reflecting photolysis of DM-nitrophen and Ca2+ binding to the Ca(2+)-ATPase. As an independent probe for protein conformational changes, intrinsic fluorescence changes upon Ca2+ release were monitored simultaneously to the FTIR measurements. Both the IR absorbance changes and the fluorescence intensity changes correlated well with the Ca2+ binding activity of the ATPase in this specific step. Ca2+ binding caused IR difference bands mainly in the region of amide I absorption of the polypeptide backbone, reflecting conformational changes of the protein. The small amplitude of the signals indicates that only a few residues perform local structural changes such as changes of bond angles or hydrogen bonding. Other absorbance changes appearing above 1700 cm-1 can be assigned to Ca2+ binding to Glu or Asp side chain carboxyl groups and concomitant deprotonation of these residues. This assignment is strengthened by downshifts of these bands by 4 cm-1 to 6 cm-1 upon 1H2O/2H2O exchange. This is in line with results of mutagenesis studies where such residues containing carboxyl groups were associated with the high affinity Ca2+ binding site (Clarke, D.M., Loo, T.W. and MacLennan, D.H. (1990) J. Biol. Chem. 265, 6262-6267).

Changes of protein structure, nucleotide microenvironment, and Ca(2+)-binding states in the catalytic cycle of sarcoplasmic reticulum Ca(2+)-ATPase: investigation of nucleotide binding, phosphorylation and phosphoenzyme conversion by FTIR difference spectroscopy.

Changes of infrared absorbance of sarcoplasmic reticulum Ca(2+)-ATPase (EC 3.6.1.38) associated with partial reactions of its catalytic cycle were investigated in the region from 1800 to 950 cm-1 in H2O and 2H2O. Starting from Ca2E1, 3 reaction steps were induced in the infrared cuvette via photolytic release of ATP and ADP: (a) nucleotide binding, (b) formation of the ADP-sensitive phosphoenzyme (Ca2E1P) and (c) formation of the ADP-insensitive phosphoenzyme (E2P). All reaction steps caused distinct changes of the infrared spectrum which were characteristic for each reaction step but comparable for all steps in the number and magnitude of the changes. Most pronounced were absorbance changes in the amide I spectral region sensitive to protein secondary structure. However, they were small--less than 1% of the total protein absorbance--indicating that the reaction steps are associated with small and local conformational changes of the polypeptide backbone instead of a large conformational rearrangement. Especially, there is no outstanding conformational change associated with the phosphoenzyme conversion Ca2E1P-->E2P. ADP-binding induces conformational changes in the ATPase polypeptide backbone with alpha-helical structures and presumably beta-sheet or beta-turn structures involved. Phosphorylation is accompanied by the appearance of a keto group vibration that can tentatively be assigned to the phosphorylated residue Asp351. Phosphoenzyme conversion and Ca(2+)-release produce difference signals which can be explained by the release of Ca2+ from carboxylate groups and a change of hydrogen bonding or protonation state of carboxyl groups.

pH-dependent LAK cell cytotoxicity.

In the microenvironment of many solid tumors the pH is considerably lower (mean pH between 6.6 to 7.2) than the pH in normal tissue (pH 7.0-7.5). Therefore, the influence of acidic pH on the cytotoxic activity of lymphokine-activated killer cells (LAK cells) after different culture periods was tested. K-562 human erythroleukemia cells were selected as target cells. Cell killing was measured using a two-color flow cytometric method. At physiological pH of 7.4, LAK cell-mediated cytotoxicity ranged from 15 to 48% (E:T ratio = 50:1). The specific lysis of target cells was considerably reduced (up to 70% inhibition of specific lysis) under acidic conditions (pH 6.8, 6.3, 5.8). This effect was independent of donors, duration of the culture period, and the E:T ratio in the cytotoxic assay. As pH gradients surrounding tumor cells may reach values below pH 6.0 at the cell surface, the pH-dependence of LAK cell cytotoxicity could at least partially explain the inhibition of the natural immune response in solid tumors. Therapeutic immunological strategies concerning the enhancement of the natural immune response like LAK cell and IL-2 immunotherapy including IL-2 gene therapy may only be successful if a simultaneous inhibition of the acidification process and an elevation of tumor pH is achieved.

Crystallization and preliminary X-ray diffraction analysis of ScrY, a specific bacterial outer membrane porin.

The sucrose-specific outer membrane porin ScrY of Salmonella typhimurium was isolated from Escherichia coli K-12 strain KS 26 containing the plasmid pPSO112. The protein was purified to homogeneity by differential extraction of the cell envelope in the presence of the detergents sodium dodecyl sulfate and lauryl (dimethyl)-amine oxide (LDAO). The porin had apparent molecular weights of 58 kDa and 120 kDa for the monomer and for the trimer, respectively, on SDS/PAGE. The purified trimers were crystallized using poly(ethylene glycol) 2000 and the detergents octylglucoside (OG) and hexyl-(dimethyl)-amine oxide (C6DAO). X-ray diffraction of the crystals showed reflections to 2.3 A. The space group of the crystals was R3 and the lattice constants of the hexagonal axes were a = b = 112.85 A and c = 149.9 A. The crystal volume per unit of protein molecular weight was 3.47 A3/Da.

Localization of Ca(2+)-stores and tissue compartments with a Ca(2+)-binding capacity in the organ of Corti of the guinea-pig by electron energy-loss spectroscopy.

The addition of 10 mM CaCl2 to glutaraldehyde fixative leads to the formation of small electron-dense deposits in the organ of Corti of the guinea-pig. These precipitates are mainly attached to cell membranes in contact with different extracellular lymphatic fluids. A higher number of precipitates is localized in the acellular parts of tectorial and basilar membrane. Electron energy-loss spectroscopy (EELS) was used to determine the elemental composition of the deposits formed. The spectra showed a prominent signal at the Ca2+ L2,3 ionization edge. Oxygen could also be detected in all the precipitates analysed. EELS analysis of mitochondria of the inner and outer hair cells after conventional fixation (glutaraldehyde followed by post-fixation in OsO4) revealed a small but significant calcium signal.

Time-resolved infrared spectroscopy of electron transfer in bacterial photosynthetic reaction centers: dynamics of binding and interaction upon QA and QB reduction.

Light-induced forward electron transfer in the bacterial photosynthetic reaction center from Rhodobacter sphaeroides was investigated by time-resolved infrared spectroscopy. Using a highly sensitive kinetic photometer based on a tunable IR diode laser source [Mäntele, W., Hienerwadel, R., Lenz, F., Riedel, W. J., Grisar, R., & Tacke, M. (1990a) Spectrosc. Int. 2, 29-35], molecular processes concomitant with electron-transfer reactions were studied in the microsecond-to-second time scale. Infrared (IR) signals in the 1780-1430-cm-1 spectral region, appearing within the instrument time resolution of about 0.5 microseconds, could be assigned to molecular changes of the primary electron donor upon formation of a radical cation and to modes of the primary quinone electron acceptor QA and its environment upon formation of QA-. These IR signals are consistent with steady-state FTIR difference spectra of the P+Q- formation [Mäntele, W., Nabedryk, E., Tavitian, B. A., Kreutz, W., & Breton, J. (1985) FEBS Lett. 187, 227-232; Mäntele, W., Wollenweber, A., Nabedryk, E., & Breton, J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8468-8472; Nabedryk, E., Bagley, K. A., Thibodeau, D. L., Bauscher, M., Mäntele, W., & Breton, J. (1990) FEBS Lett. 266, 59-62] and with time-resolved FTIR studies [Thibodeau, D. L., Nabedryk, E., Hienerwadel, R., Lenz, F., Mäntele, W., & Breton, J. (1990) Biochim. Biophys. Acta 1020, 253-259]. At given wavenumbers, kinetic components with a half-time of approximately 120 microseconds were observed and attributed to QA----QB electron transfer. The time-resolved IR signals, in contrast to steady-state experiments where full protein relaxation after electron transfer can occur, allow us to follow directly the modes of QA and QB and their protein environment under conditions of forward electron transfer. Apart from signals attributed to the primary electron donor, signals are proposed to arise not only from the C = O and C = C vibrational modes of the neutral quinones and from the C-O and C-C vibrations of their semiquinone anion form but also from amino acid groups forming their binding sites. Some of the signals appearing with the instrument rise time as well as the transient 120-microseconds signals are interpreted in terms of binding and interaction of the primary and secondary quinone electron acceptor in the Rb. sphaeroides reaction center and of the conformational changes in their binding site.(ABSTRACT TRUNCATED AT 400 WORDS)

Collagen stabilization induced by natural humic substances.

Humic substances are polyphenolic compounds. They have antiviral as well as desmutagenic effects and react with biopolymers such as collagen; thereby they have no toxic side effects by oral administration. In vitro incubation with humic substances raises the breaking point of the tail tendon of the rat by about 75%. The chemical resistance of the collagen fibres in tail tendon collagen is also increased by in vitro incubation with humic substances, at least insofar as the ultrastructurally and biophysically measurable destruction of the collagen fibres by 4 M guanidinium chloride is inhibited. As humic substances increase the mechanical and chemical resistance of collagen fibres and promote their "maturity", it seems likely that this effect of humic substances depends upon their interaction with the hydrogen bonding and covalent bonding of the collagen fibres. Such a conclusion is confirmed by the results of X-ray diffraction analysis.

Progress in electron microscopic diagnostics: semi-quantitative determination of precipitable calcium in different cell types of the organ of Corti in the guinea-pig.

Potassium antimonate was used to precipitate calcium in the cochlea of the guinea-pig. The distribution of the calcium antimonate precipitates was analysed by electron microscopy. The precipitate density was determined in different cell types in the organ of Corti by counting the number of calcium binding sites in a 10-micron 2 area. The size of the precipitates varied considerably, and thus the relative amount of the precipitable calcium was estimated only semi-quantitatively. As the prominent carbon signal is superimposed over the nearby small Ca(2+)-edge signals, the combined signal of the antimony M4,5-edge and the oxygen K-edge of the calcium antimonate salt formed was chosen for the semi-quantitative estimation. Images of the inelastically scattered electrons of the precipitates at delta E = 570 eV were recorded by electron spectroscopic imaging. The area covered by the calcium precipitates within a given cell type was determined in different ultrathin sections of the same organ of Corti by an image processing system.

Elemental composition of pyroantimonate precipitates analysed by electron spectroscopic imaging (ESI) and electron energy-loss spectroscopy (EELS) in vitellogenic ovarian follicles of Drosophila.

Ca2+ was precipitated with potassium antimonate in vitellogenic follicles of the fruit fly Drosophila melanogaster and the distribution of the precipitates formed was studied by electron microscopy. The microvilli of the oolemma in mid- and late vitellogenic follicles were lined with precipitates. The chemical composition of the precipitates was analysed by electron spectroscopic imaging (ESI). The images produced by inelastically scattered electrons at specific ionization edges were compared, and the non-specific background signals were subtracted by an image processing system. The presence of Ca2+, antimony and oxygen in the precipitates formed could be demonstrated. The elemental composition of the precipitates and of yolk spheres was also analysed by electron energy-loss spectroscopy (EELS). With respect to the precipitates, signals at the calcium L2,3-edge, the oxygen K-edge and the antimony M4,5-edge were recorded without deconvolution and background subtraction. The yolk spheres, which were free of precipitates, gave the characteristic signal of the nitrogen K-edge. The applied techniques combine good ultrastructural resolution with the possibility of analysing the elemental composition of histochemical reaction products and cellular structures.

Infrared spectroscopic signals arising from ligand binding and conformational changes in the catalytic cycle of sarcoplasmic reticulum calcium ATPase.

Fourier transform infrared spectroscopy was used to investigate ligand binding and conformational changes in the Ca2(+)-ATPase of sarcoplasmic reticulum during the catalytic cycle. The ATPase reaction was started in the infrared sample by release of ATP from the inactive, photolabile ATP derivative P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate (caged ATP). Absorption spectroscopy in the visible spectral region using the Ca2(+)-sensitive dye Antipyrylazo III ensured that the infrared samples were able to transport Ca2+ in spite of their low water content, which is required for mid-infrared measurements (1800-950 cm-1). Small, but characteristic and highly reproducible infrared absorbance changes were observed upon ATP release. These infrared absorbance changes exhibit different kinetic properties. Comparison with model compound infrared spectra indicates that they are related to photolysis of caged ATP, hydrolysis of ATP in consequence of ATPase activity and to molecular changes in the active ATPase. The absorbance changes due to alterations in the ATPase were observed mainly in the region of Amide I and Amide II protein absorbance and presumably reflect the molecular processes upon phosphoenzyme formation. Since the absorbance changes were small compared to the overall ATPase absorbance, no major rearrangement of ATPase conformation as the result of catalysis could be detected.

Molecular changes in the sarcoplasmic reticulum calcium ATPase during catalytic activity. A Fourier transform infrared (FTIR) study using photolysis of caged ATP to trigger the reaction cycle.

Fourier transform infrared spectroscopy was used to study ligand binding and conformational changes in the Ca2(+)-ATPase of sarcoplasmic reticulum. Novel in infrared difference spectroscopy, the catalytic cycle in the IR sample was started by photolytic release of ATP from an inactive, photolabile ATP-derivative (caged ATP). Small, but characteristic infrared absorbance changes were observed upon ATP release. On the basis of model spectra, the absorbance changes corresponding to the trigger and substrate reactions, i.e. to photolysis of caged ATP and hydrolysis of ATP, were separated from the absorbance changes due to the active ATPase reflecting formation of the phosphorylated Ca2E1P enzyme form. A major rearrangement of ATPase conformation as the result of catalysis can be excluded.