ABSTRACT
The amyloid peptides Aβ1–42 and Aβ25–35 strongly inhibited the activity of constitutive neuronal and endothelial nitric oxide synthases (i.e., NOS‐I and NOS‐III, respectively) in cell‐free ...assays. The molecular mechanism of NOS inhibition by Aβ fragments was studied in detail with Aβ25–35. The inhibitory ability was mostly NADPH‐dependent and specific for the soluble form of Aβ25–35. Optical, fluorescence, and NMR spectroscopy showed that the soluble, but not aggregated, Aβ25–35 interacted with NADPH, thus suggesting that a direct recruitment of NADPH may result in diminished availability of the redox cofactor for NOS functioning. To assess the physiological relevance of our findings, rat neuronal‐like PC12 and glioma C6 cell lines were used as cellular models. After Aβ25–35 internalization into cells was verified, the activity of constitutive NOS was measured using the DAF‐2DA detection system and found to be severely impaired upon Aβ25–35 uptake. Consistent with previous results on the molecular cross‐talk between NOS isoforms, repression of constitutive NOS by Aβ25–35 resulted in enhanced expression of inducible NOS (NOS‐II) mRNA in C6 cells. Our results represent the first evidence that amyloid fragments impair constitutive NOS activity in cell‐free and cellular systems, providing a possible molecular mechanism for the onset and/or maintenance of Alzheimer's disease.
Caged compounds in combination with protein crystallography represent a valuable tool in studies of enzyme reaction intermediates. To date, photochemical triggering of reactions has been performed ...close to room temperature. Synchronous reaction initiation has only been achieved with enzymes of relatively slow turnover (<0.1 s−1) and caged compounds of high quantum yield. Here X‐ray crystallography and microspectrophotometry were used to provide evidence that (nitrophenyl)ethyl (NPE) ester bonds can be photolyzed by UV light at cryotemperatures. NPE‐caged ATP in flash‐cooled crystals of Mycobacterium tuberculosis thymidylate kinase was photolyzed successfully at 100–150 K as assessed by the structural observation of ATP‐dependent enzymatic conversion of TMP to TDP after temporarily warming the crystals to room temperature. A new method is proposed in which cryo‐photolysis combined with temperature‐controlled protein crystallography can be used to trap reaction intermediates even in some of the fastest enzymes and/or when only compounds of low quantum yield are available. Raising the temperature after cryophotolysis may allow a transition barrier to be passed and an intermediate to accumulate in the crystal. A comparable method has only been used so far with proteins displaying endogenous photosensitivity. The approach described here opens the way to studying the reaction mechanisms of a much larger number of crystalline enzymes. Furthermore, it is shown that X‐ray‐induced radiolysis of caged compounds occurs if high‐intensity synchrotron beamlines are used. This caveat should be taken into account when deriving data‐collection protocols. It could also be used potentially as a way to trigger reactions.
The effect of temperature (between 5.0 and 45.0°C) on the catalytic activity of nitric oxide synthases types I, II, and III (NOS-I, NOS-II, and NOS-III, respectively) has been investigated, at pH ...7.5. The value of Vmax for NOS-I activity increases from 1.8 × 101 pmol min−1 mg−1, at 5.0°C, to 1.8 × 102 pmol min−1 mg−1, at 45.0°C; on the other hand, the value of Km (=4.0 × 10−6 M) is temperature independent. Again, the value of Vmax for NOS-II activity increases from 8.0 pmol min−1 mg−1, at 7.0°C, to 5.4 × 101 pmol min−1 mg−1, at 40.0°C, the value of Km (=1.8 × 10−5 M) being unaffected by temperature. Temperature exerts the same effect on NOS-I and NOS-II activity, as shown by the same values of ΔHVmax (=4.2 × 101 kJ mol−1), ΔHKm (=0 kJ mol−1), and ΔH(Vmax/Km) (=4.2 × 101 kJ mol−1). On the contrary, the value of Km for NOS-III activity decreases from 3.8 × 10−5 M, at 10.0°C, to 1.6 × 10−5 M, at 40.0°C, the value of Vmax (=6.8 × 101 pmol min−1 mg−1) being temperature independent. Present results indicate that temperature influences directly NOS-I and NOS-II activity independently of the substrate concentration, the values of Km being temperature independent. However, when l-arginine level is higher than 2 × 10−4 M, as observed under in vivo conditions, NOS-III activity is essentially unaffected by temperature, the substrate concentration exceeding the value of Km. As a whole, although further studies in vivo are needed, these observations seem to have potential physiopathologic implications.
Clonidine, clinically used in the treatment of hypertension, is a central α
2-adrenergic agonist that reduces blood pressure and slows heart rate by reducing sympathetic stimulation. Considering the ...structural similarity between clonidine and hydrophobic heterocyclic nitric oxide synthase (NOS) inhibitors, the effect of clonidine on the nitric oxide (NO) pathway was investigated. This was verified by determination of NOS activity
in vitro and by analysis of inducible Ca
2+-independent NOS (NOS-II) mRNA expression and measurement of nitrite levels in rat C6 glioma cells, taken as a cellular model. Clonidine inactivated neuronal Ca
2+-dependent NOS (NOS-I) competitively without affecting NOS-II and endothelial Ca
2+-dependent NOS (NOS-III) activity. However, the value of
K
i
for clonidine binding to NOS-I depended on tetrahydrobiopterin (BH
4) concentration, as reported for NOS inhibition by other nitrogen heterocyclic compounds. In particular, the value of
K
i
for clonidine binding to NOS-I increased (from 7.9 ± 0.4 × 10
−5 M to 8.0 ± 0.4 × 10
−3 M) as BH
4 concentration was increased (between 3.0 × 10
−7 M and 1.0 × 10
−3 M), at pH 7.5 and 37.0°. In addition, clonidine (1.0 × 10
−4 M) enhanced NOS-II mRNA expression in rat C6 glioma cells, as induced by
Escherichia coli lipopolysaccharide (LPS) plus interferon-γ (IFN-γ). Finally, clonidine (1.0 × 10
−4 M to 1.0 × 10
−3 M) dose dependently increased the levels of LPS/IFN-γ-induced nitrites, the breakdown product of NO, in supernatants of rat C6 glioma cells. As reported for other NOS inhibitors, clonidine was also able to regulate NOS-I and NOS-II inversely.
Cys25‐nitrosylation inactivates papain Venturini, Giorgio; Fioravanti, Emanuela; Colasanti, Marco ...
Biochemistry and molecular biology international,
October 1998, Letnik:
46, Številka:
2
Journal Article
Recenzirano
Nitric oxide (NO) may modulate the catalytic activity of cysteine proteases. In the present study, the inhibitory effect of NO, released by the NO‐donors ...(±)‐(E)‐4‐ethyl‐2‐(E)‐hydroxyimino‐5‐nitro‐3‐hexenamide and nitroprusside, on papain action is reported. Papain inactivation via NO‐mediated nitrosylation of the Cys25 catalytic residue represents a molecular model for cysteine protease inhibition.
Performance of triple GEM prototypes in strong magnetic field has been evaluated bymeans of a muon beam at the H4 line of the SPS test area at CERN. Data have been reconstructedand analyzed offline ...with two reconstruction methods: the charge centroid and the micro-Time-Projection-Chamber exploiting the charge and the time measurement respectively. A combinationof the two reconstruction methods is capable to guarantee a spatial resolution better than 150{\mu}min magnetic field up to a 1 T.
The Beijing Electron Spectrometer III (BESIII) is a multipurpose detector that collects data provided by the collision in the Beijing Electron Positron Collider II (BEPCII), hosted at the Institute ...of High Energy Physics of Beijing. Since the beginning of its operation, BESIII has collected the world largest sample of J/{\psi} and {\psi}(2s). Due to the increase of the luminosity up to its nominal value of 10^33 cm-2 s-1 and aging effect, the MDC decreases its efficiency in the first layers up to 35% with respect to the value in 2014. Since BESIII has to take data up to 2022 with the chance to continue up to 2027, the Italian collaboration proposed to replace the inner part of the MDC with three independent layers of Cylindrical triple-GEM (CGEM). The CGEM-IT project will deploy several new features and innovation with respect the other current GEM based detector: the {\mu}TPC and analog readout, with time and charge measurements will allow to reach the 130 {\mu}m spatial resolution in 1 T magnetic field requested by the BESIII collaboration. In this proceeding, an update of the status of the project will be presented, with a particular focus on the results with planar and cylindrical prototypes with test beams data. These results are beyond the state of the art for GEM technology in magnetic field.
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