Conformational and physicochemical properties of quinoa proteins affected by different conditions of high-intensity ultrasound treatments.

Quinoa proteins (QP) have promise as a potential source of novel food ingredients, and it is of great interest to know how high-intensity ultrasound (HIUS) treatments affect the properties of QP. This work aimed to study the impact of on-off time-pulses of HIUS treatments on the structural and physicochemical properties of QP; samples were treated at 5, 10, 20, and 30 min with on-off pulses of 10 s/10 s, 5 s/1 s, and 1 s/5 s). Structural changes were evaluated using PAGE-SDS, circular dichroism, fluorescence spectroscopy, and differential scanning calorimetry. Meanwhile, physicochemical properties were also examined, including solubility, Z-average, polydispersity index PDI, and Z-potential. PAGE-SDS showed the appearance of polypeptides over 190 kDa in HIUS samples-treated. All samples presented 15.6% α-helices, 31.3% ß-sheets, 21.8% ß-rotations, and 31.4% random coils independent of the HIUS treatment. ß-Turn structures and "random coils" were not affected by HIUS. When US 10 s/10 s and 1 s/5 s were applied, an increase in the % α-helix and a decrease in ß-fold were observed, which could indicate a small conversion of ß-folds to α-helices. Fluorescence spectra for all HIUS showed a significant increase (23%) of average fluorescence intensity and a decrease of λmax in relation to that of the control (346 dnm and 340 nm average HIUS treatment). DSC showed one endotherm in all cases (81.6-99.8 °C), and an increase in Td was observed due to the effect of the HIUS treatment. HIUS caused a 48% increase in solubility. The Z-average of the HIUS samples compared to that of the controls showed an increase from 37.8 to 47.3 nm. PDI and Z-potential values from the QP controls and the HIUS samples did not show significance differences and presented average values of 0.466 ±â€¯0.021 (PDI) and -16.63 ±â€¯0.89 (Z-potential). It is possible to conclude that HIUS treatments affect the secondary and tertiary structure of quinoa proteins, and these changes resulted in an increase of solubility and particle size. HIUS treatment as a new and promising technology that can improve the QP solubility properties and in that way allow its use as an ingredient with a good source of protein to develop different types of beverages/protein sauces.

Surface freezing of water.

Freezing, melting, evaporation and condensation of water are essential ingredients for climate and eventually life on Earth. In the present work, we show how surface freezing of supercooled water in an open container is conditioned and triggered-exclusively-by humidity in air. Additionally, a change of phase is demonstrated to be triggered on the water surface forming surface ice crystals prior to freezing of bulk. The symmetry of the surface crystal, as well as the freezing point, depend on humidity, presenting at least three different types of surface crystals. Humidity triggers surface freezing as soon as it overpasses a defined value for a given temperature, generating a plurality of nucleation nodes. An evidence of simultaneous nucleation of surface ice crystals is also provided.

Orange Peel Oxidative Gasification on Ni Catalysts Promoted with CaO, CeO2 or K2O.

Orange peel can be considered as an attractive raw material to be gasified for hydrogen or syngas production. In this work, the catalytic evaluation of several silica-supported nickel catalysts in the oxidative degradation of waste orange peel is reported. It was found that the catalytic gasification with the K2O-Ni/silica catalyst produces more hydrogen than the non-catalytic route at 600 degrees C. Surprisingly, a significant amount of ethene was obtained with the CeO2-Ni/silica catalyst, which was explained in terms of an oxidative dehydrogenation reaction of ethane formed during biomass or tar decomposition.

A comparative study of photo-assisted deposition of silver nanoparticles on TiO2.

Noble metals deposited on TiO2 act as electron traps facilitating electron-hole separation and promoting the interfacial electron transfer process. In particular, silver nanoparticles have the ability to absorb visible light due to localized surface plasmon resonance. Here we report a photochemical and photocatalytic method for depositing Ag nanoparticles (2-20 nm) on TiO2 by using UV light at room temperature. UV-Vis diffuse reflectance spectroscopy, transmission electron microscopy, X-ray photoelectron spectroscopy and time-resolved microwave conductivity were used as characterization techniques. The photocatalytic activity was investigated by measuring the decomposition of rhodamine B under UV and visible light irradiation. The fastest bleaching of RhB under visible-light irradiation has been obtained by Ag/TiO2 plasmonic photocatalyst prepared by the photocatalytic route. These results were explained in terms of the more efficient photon absorption due to the presence of the surface plasmon resonance.

In vivo fluctuation of Tax, Foxp3, CTLA-4, and GITR mRNA expression in CD4+CD25+ T cells of patients with human T-lymphotropic virus type 1-associated myelopathy

HTLV-1 Tax expression exerts an inhibitory effect on the Foxp3 transcription factor in CD4+CD25+ T-regulatory cells (Treg). For a better understanding of the role of Tax mRNA in the gene expression of cellular markers we measured Tax, Foxp3, CTLA-4, GITR, TGF-β, and IL-10 mRNA in Treg cells of 50 patients with human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP; 27 women and 23 men; mean age: 56.7 years). The control group consisted of 23 non-infected subjects (12 women and 11 men) with a mean age of 51.3 years. Real-time PCR was used to measure mRNA of Tax proteins and several cellular markers of Treg function. Determinations revealed a high level of Tax mRNA in HAM/TSP (124.35 copies/100 CD4+CD25+ T cells). Foxp3, GITR, and CTLA-4 mRNA levels were lower in HAM/TSP patients (mean ± SD, 22.07 ± 0.78, 9.63 ± 0.36, and 4.54 ± 0.39, respectively) than in non-infected controls (47.15 ± 12.94, 22.14 ± 1.91, and 21.07 ± 2.31). Both groups had similar levels of TGF-β and IL-10. An inverse relationship was found between Tax levels and Foxp3, CTLA-4, and GITR levels. Conversely, there was a direct correlation between levels of Foxp3, GITR, and CTLA-4. Disease severity and evolution time did not correlate with Tax or Foxp3 levels. The present results suggest that Tax and Foxp3 mRNA vary with the same degree of disease severity in HAM/TSP patients. Tax fluctuations may affect CTLA-4 and GITR expression via the Foxp3 pathway, causing virus-induced dysfunction of CD4+CD25+ T cells in HAM/TSP patients.

In vivo fluctuation of Tax, Foxp3, CTLA-4, and GITR mRNA expression in CD4(+)CD25(+) T cells of patients with human T-lymphotropic virus type 1-associated myelopathy.

HTLV-1 Tax expression exerts an inhibitory effect on the Foxp3 transcription factor in CD4(+)CD25(+) T-regulatory cells (Treg). For a better understanding of the role of Tax mRNA in the gene expression of cellular markers we measured Tax, Foxp3, CTLA-4, GITR, TGF-ß, and IL-10 mRNA in Treg cells of 50 patients with human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP; 27 women and 23 men; mean age: 56.7 years). The control group consisted of 23 non-infected subjects (12 women and 11 men) with a mean age of 51.3 years. Real-time PCR was used to measure mRNA of Tax proteins and several cellular markers of Treg function. Determinations revealed a high level of Tax mRNA in HAM/TSP (124.35 copies/100 CD4(+)CD25(+) T cells). Foxp3, GITR, and CTLA-4 mRNA levels were lower in HAM/TSP patients (mean ± SD, 22.07 ± 0.78, 9.63 ± 0.36, and 4.54 ± 0.39, respectively) than in non-infected controls (47.15 ± 12.94, 22.14 ± 1.91, and 21.07 ± 2.31). Both groups had similar levels of TGF-ß and IL-10. An inverse relationship was found between Tax levels and Foxp3, CTLA-4, and GITR levels. Conversely, there was a direct correlation between levels of Foxp3, GITR, and CTLA-4. Disease severity and evolution time did not correlate with Tax or Foxp3 levels. The present results suggest that Tax and Foxp3 mRNA vary with the same degree of disease severity in HAM/TSP patients. Tax fluctuations may affect CTLA-4 and GITR expression via the Foxp3 pathway, causing virus-induced dysfunction of CD4(+)CD25(+) T cells in HAM/TSP patients.

Stimulatory response of neutrophils from periodontitis patients with periodontal pathogens.

OBJECTIVE: Neutrophils play a crucial role in the defense of invading bacteria by releasing biologically active molecules. The response of peripheral blood neutrophils was studied in periodontitis-affected patients and in healthy controls towards stimulation to Porphyromonas gingivalis (Pg) and Actinobacillus actinomycetemcomitans (Aa) extracts. MATERIALS AND METHODS: Peripheral venous blood was drawn from 23 adult patients with moderate to advanced chronic periodontitis (probing depth >or=5 mm, attachment loss >or=3 mm), and 30 healthy volunteers. Neutrophil response followed by metalloproteinase-9 (MMP-9) and interleukin-8 (IL-8) secretion was assayed by zymography and enzyme-linked immunosorbent assay, respectively, on both whole blood and purified neutrophils. In addition to periodontal pathogen extracts, known stimulating agents were tested, such as Escherichia coli-lipopolysaccharide (LPS), phytohemagglutinin, and zymosan A. RESULTS: Neutrophil response, expressed as a secretion ratio under stimulated and non-stimulated conditions, measured in whole blood, showed no differences between periodontitis and healthy controls. Instead, in purified neutrophils from patients, MMP-9 exhibited a significantly higher secretion ratio with LPS and Pg (1.5- to 2-fold), whereas IL-8 showed a larger increase in secretion ratio (3- to 7-fold) in the presence of Pg, Aa, LPS, and zymosan A. CONCLUSION: Peripheral neutrophils of periodontitis-affected patients are more reactive as suggested by their significantly higher response toward periodontal pathogen extracts and other stimulating agents.

Potato tuber isoapyrases: substrate specificity, affinity labeling, and proteolytic susceptibility.

Apyrase/ATP-diphosphohydrolase hydrolyzes di- and triphosphorylated nucleosides in the presence of a bivalent ion with sequential release of orthophosphate. We performed studies of substrate specificity on homogeneous isoapyrases from two potato tuber clonal varieties: Desiree (low ATPase/ADPase ratio) and Pimpernel (high ATPase/ADPase ratio) by measuring the kinetic parameters K(m) and k(cat) on deoxyribonucleotides and fluorescent analogues of ATP and ADP. Both isoapyrases showed a broad specificity towards dATP, dGTP, dTTP, dCTP, thio-dATP, fluorescent nucleotides (MANT-; TNP-; ethene-derivatives of ATP and ADP). The hydrolytic activity on the triphosphorylated compounds was always higher for the Pimpernel apyrase. Modifications either on the base or the ribose moieties did not increase K(m) values, suggesting that the introduction of large groups (MANT- and TNP-) in the ribose does not produce steric hindrance on substrate binding. However, the presence of these bulky groups caused, in general, a reduction in k(cat), indicating an important effect on the catalytic step. Substantial differences were observed between potato apyrases and enzymes from various animal tissues, concerning affinity labeling with azido-nucleotides and FSBA (5'-p-fluorosulfonylbenzoyl adenosine). PLP-nucleotide derivatives were unable to produce inactivation of potato apyrase. The lack of sensitivity of both potato enzymes towards these nucleotide analogues rules out the proximity or adequate orientation of sulfhydryl, hydroxyl or amino-groups to the modifying groups. Both apyrases were different in the proteolytic susceptibility towards trypsin, chymotrypsin and Glu-C.

Longitudinal analysis of metalloproteinases, tissue inhibitors of metalloproteinases and clinical parameters in gingival crevicular fluid from periodontitis-affected patients.

BACKGROUND: The aim of this work was to improve the assessment of the periodontal disease status through measurements of extracellular matrix metalloproteinases (MMPs) and their tissular inhibitors (TIMPs) in the gingival crevicular fluid from patients diagnosed with chronic periodontitis. METHODS: Gingival crevicular fluid samples from patients (n = 13) were taken from 60 sites initially, and from 51 and 41 sites, respectively, 3 and 6 months after scaling and root planing. Gingival crevicular fluid samples were also taken from healthy subjects (n = 11, 24 sites). The presence of MMP-9 and MMP-8 was assessed by zymography and immunowestern blotting, respectively. The actual MMP activity (gelatinase and collagenase) was measured using the fluorogenic substrate assay. TIMP-1 and -2 levels were measured by immunodot blot. RESULTS: The fluorogenic substrate assay determinations showed higher MMP activity in sites with probing depth > or = 4 mm, with significant reduction post-treatment. Gelatinase activity followed by zymography consisted mainly of MMP-9. A different pattern of MMP-8 in control and patient sites was found. Controls only showed species of a partially active form (69 kDa), whereas patient sites showed a high frequency of the active form (56 kDa), and in some cases the latent form (85 kDa) was also observed. The active form reduced its frequency in sites with probing depth > or = 4 mm. TIMP-1 and -2 levels in patients were significantly lower than in controls, and after treatment the recovery of TIMP-1 level similar to control was observed. CONCLUSION: Significant correlations between the severity of the periodontal disease and the actual MMP activity, the active form of MMP-8 and the low level of both TIMP-1 and TIMP-2 were found.

Extracellular matrix protein expression in cerebrospinal fluid from patients with tropical spastic paraparesis associated with HTLV-I and Creutzfeldt-Jakob disease.

The cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the CNS, thus biochemical processes in the CNS could potentially be reflected in the CSF. Changes in extracellular matrix (ECM) proteins can be studied through their analysis in the CSF. ECM plays an essential role in CNS homeostasis and several proteins such as laminin (LN), fibronectin (FN), thrombospondin (TS) and heparan sulphate proteoglycan (HS, perlecan) form part of its structure. Possible changes in the levels of these proteins were investigated in two different pathologies--tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM) (n=25) and Creutzfeldt-Jakob disease (CJD) (n=19)--and compared with those in a control group with or without neurological disease (n=25). CSF analyses were carried out using monoclonal or monospecific polyclonal antibodies. In comparison with the control group, it was found that TSP/HAM patients presented significantly higher levels of LN, TS and HS, while in CJD patients the levels of FN, TS and HS were increased. In CJD patients the HS level was almost double that of the TSP/HAM patients. These results suggest a distinct pattern of ECM proteins in CSF in relation to the type of neurological disease. TSP/HAM is a chronic motor disease that affects the white matter of the spinal cord, while CJD is a subacute dementia that affects cerebral neurons and their synapsis.

Matrix metalloproteinase profile in patients with Creuztfeldt-Jakob disease.

Preliminary findings suggest that abnormalities in matrix metalloproteinase (MMP) activity may be found in the cerebrospinal fluid (CSF) of patients with Creutzfeldt-Jakob disease (CJD). In this study of 16 subjects with CJD and 16 age-, and sex-matched controls, we determined the presence of MMP-2 and MMP-9 in their active and proenzyme forms, the relative levels of MMP-3 and four inhibitors of MMP activity (TIMP-1, TIMP-2, TIMP-3 and TIMP-4), and the concentration of 4-3-3 protein. The methodology used involved zymography and immunological techniques. The results indicate that, compared with controls, CJD patients have a significantly higher positive frequency of pro-MMP-9 and of the active form of MMP-2, along with significantly higher levels of TIMP-1 and TIMP-2, classical inhibitors of MMP-9 and MMP-2, respectively. We also found a positive correlation between 14-3-3 protein concentration and that of TIMP-1 and TIMP-2 levels (correlation coefficients of 0.793 and 0.798, respectively). These results suggest that abnormalities in MMP and TIMP profiles may be helpful in the biochemical characterisation of CJD.

Differences in nucleotide-binding site of isoapyrases deduced from tryptophan fluorescence.

Comparative studies of intrinsic and extrinsic fluorescence of apyrases purified from two potato tuber varieties (Pimpernel and Desirée) were performed to determine differences in the microenvironment of the nucleotide binding site. The dissociation constants (K(d)) of Pimpernel apyrase for the binding of different fluorescent substrate analogs: methylanthranoyl (MANT-), trinitrophenyl (TNP-), and epsilon -derivatives of ATP and ADP were determined from the quenching of Trp fluorescence, and compared with K(d) values previously reported for Desirée enzyme. Binding of non-fluorescent substrate analogues decreased the Trp emission of both isoapyrases, indicating conformational changes in the vicinity of these residues. Similar effect was observed with fluorescent derivatives where, in the quenching effect, the transfer of energy from tryptophan residues to the fluorophore moiety could be additionally involved. The existence of energy transfer between Trp residues in the Pimpernel enzyme was demonstrated with epsilon -analogues, similar to our previous observations with the Desirée. From these results we deduced that tryptophan residues are close to or in the nucleotide binding site in both enzymes. Experiments with quenchers like acrylamide, Cs(+) and I(-), both in the presence and absence of nucleotide analogues, suggest the existence of differences in the nucleotide binding site of the two enzymes. From the results obtained in this work, we can conclude that the differences found in the microenvironment of the nucleotide binding site can explain, at least in part, the kinetic behaviour of both isoenzymes.

ATPase and Adpase activities in synovial membrane of equine metacarpophalangeal joint.

ATPase and ADPase activities capable of hydrolyzing nucleoside di- and triphosphates in the presence of Ca2+ are present in synovial membrane of metacarpophalangeal joint mainly associated to membrane fractions. These hydrolytic activities have been considered involved in the inflammatory process where ATP and ADP are inflammatory mediators while adenosine counteracts this effect. Both, subcellular localization and kinetic properties of these nucleotidase activities, suggest that could correspond to single enzyme called ATP-diphosphohydrolase or apyrase. The comparison of the activity on ATP-Ca and ADP-Ca from normal and pathological equine synovial membrane did not show significant differences either in the subcellular fraction distribution or in the enrichment of each subcellular fraction. Neither differences on 5'-nucleotidase activity present in the microsomal fraction were observed.

[Radioguided surgery of hyperparathyroidism. A case report]. / Cirugía radioguiada en hiperparatiroidismo primario. Caso clínico.

Primary hyperparathyroidism is the main cause of hypercalcemia in ambulatory patients. Classic surgical resolution of this disease includes bilateral cervical exploration with exhibition of the four parathyroid glands and evaluation of the suspicious gland with a rapid biopsy. We report a 79 years old female with a primary hyperparathyroidism in whom the adenoma causing the disease was located with the help of an intraoperative gamma-probe that detected the zone of higher radiation counting of Tc99m Sestamibi, that was injected preoperatively. The patient was discharged with normal serum calcium and phosphate levels.

Nitric oxide synthase activity in brain tissues from llama fetuses submitted to hypoxemia.

The fetal llama (Lama glama; a species adapted to live in chronic hypoxia in the highlands of the Andes) did not increase cerebral blood flow and reduce the brain oxygen uptake during hypoxemia. Although nitric oxide (NO) is a normal mediator in the regulation of vascular tone and synaptic transmission, NO overproduction by hypoxemia could produce neuronal damage. We hypothesized that nitric oxide synthase (NOS) activity is either maintained or reduced in the central nervous system of the llama fetuses submitted to chronic hypoxemia. Approximately 85% of the Ca(2+)-dependent NOS activity was soluble, at least 12% was associated with the mitochondrial fraction, and less than 5% remains associated with microsomes. To understand the role of NO in chronic hypoxemia, we determined the effect of 24-h hypoxemia on NOS activity in the central nervous system. No changes in activity or the subcellular distribution of NOS activity in brain tissues after hypoxemia were found. We proposed that the lack of changes in NOS activity in the llama under hypoxemia could be a cytoprotective mechanism inherent to the llama, against possible toxic effects of NO.

[Risk factors for morbidity and mortality in patients with essential hypertension, followed for 25 years]. / Factores predictores de riesgo de morbimortalidad en hipertensos esenciales seguidos 25 años.

BACKGROUND: Among hypertensive patients, other risk factors for mortality and morbidity, besides blood pressure, must be considered when therapeutic decisions are done. AIM: To assess the incidence and relevance of cardiovascular risk factors in a cohort of patients with essential hypertension. MATERIAL AND METHODS: A cohort of 1,072 treated patients with essential hypertension was followed for a period up to 25 years. Four hundred eighty six were men and 586 were women, age ranged from 31 to 70 years. At entry, 56% of subjects did not have any organic complications associated to hypertension (stage I WHO criteria), 30% had mild alterations (Stage II) and 14% had major complications (myocardial infarction, stroke, heart failure or renal failure). Likewise, 43.8% had mild, 14.5%, moderate and 41.7%, severe hypertension. Patients were treated with monotherapy or combined therapy based on diuretics, beta blockers, calcium antagonists and angiotensin converting enzyme inhibitors. Goal of therapy was 140/90 mm Hg. Risk factors associated diseases and complications were registered carefully. Causes of death were obtained from hospital records and death certificates. Mortality was analyzed using life tables (intention to treat method included). RESULTS: Blood pressure dropped significantly during follow up from a mean of 182/110 to 154/92 mm Hg. During follow up, 143 patients died and 429 complications (lethal or non lethal) were recorded. Twenty four percent of patients smoked, 24% reported alcohol intake, 56% had hypercholesterolemia, 11% were obese, 13% had diabetes and 3% had gout. The proportional hazard model showed that the existence of previous complications, the presence of more than 3 risk factors, and age over 60 and mean systolic and diastolic pressure during therapy, were independent and significant risk factors for mortality. CONCLUSIONS: The incidence of risk factors among our hypertensive patients is very similar to that of other national or international cohorts.

Fluorescence studies of ATP-diphosphohydrolase from Solanum tuberosum var. Desirée.

Chemical modification of potato apyrase suggests that tryptophan residues are close to the nucleotide binding site. Kd values (+/- Ca2+) for the complexes of apyrase with the non-hydrolysable phosphonate adenine nucleotide analogues, adenosine 5'-(beta,gamma-methylene) triphosphate and adenosine 5'-(alpha,beta-methylene) diphosphate, were obtained from quenching of the intrinsic enzyme fluorescence. Other fluorescent nucleotide analogues (2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate, 2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-diphosphate. 1,N6-ethenoadenosine triphosphate and 1,N6-ethenoadenosine diphosphate) were hydrolysed by apyrase in the presence of Ca2+, indicating binding to the active site. The dissociation constants for the binding of these analogues were calculated from both the decrease of the protein (tryptophan) fluorescence and enhancement of the nucleotide fluorescence. Using the sensitised acceptor (nucleotide analogue) fluorescence method, energy transfer was observed between enzyme tryptophans and ethene-derivatives. These results support the view that tryptophan residues are present in the nucleotide-binding region of the protein, appropriately oriented to allow the energy transfer process to occur.

[Increased activity of metalloproteinases and their inhibitors in cerebrospinal fluid of patients with tropical spastic paraparesis]. / Incremento de la actividad metaloproteinásica y de sus inhibidores en el líquido cefalorraquídeo, en pacientes con paraparesia espástica tropical.

BACKGROUND: Proteolytic modifications of neuronal surfaces and the surrounding extracellular matrix are very important in neuronal development and regeneration. Increased activity of matrix metalloproteinases (MMPs) and their tissue inhibitors, due to secretion by macrophages and lymphocytes, occur in inflammatory processes that disrupt the blood brain barrier. However, neurons and microglia can also secrete these enzymes. AIM: To identify the type of MMP present in the cerebrospinal fluid (CSF) and changes in the expression of tissue inhibitors of metalloproteinases (TIMPs) in patients with HTLV-1 associated tropical spastic paraparesis. PATIENTS AND METHODS: CSF samples from 12 patients with HTLV-1 associated tropical spastic paraparesis and 12 healthy controls were obtained by an atraumatic lumbar puncture. The presence of MMPs was measured by zymography and the relative amounts of TIMPs were measured by immunowestern blot. RESULTS: In the CSF of both controls and patients, a similar gelatinolytic band corresponding to proMMP-2 (latent form) was observed. In 83.3% of patients with HTLV 1 associated tropical spastic paraparesis, the MMP-9 was also present. TIMP-1, TIMP-2 and TIMP-3 were elevated 2.24 +/- 0.72, 3.85 +/- 1.38 and 5.89 +/- 3.4 fold, respectively, in the CSF of patients as compared to controls. CONCLUSIONS: Patients with HTLV-1 associated tropical spastic paraparesis have elevated activity of MMP-9 and levels of TIMPs in the CSF, when compared to healthy controls.

Studies on ATP-diphosphohydrolase nucleotide-binding sites by intrinsic fluorescence.

Potato apyrase, a soluble ATP-diphosphohydrolase, was purified to homogeneity from several clonal varieties of Solanum tuberosum. Depending on the source of the enzyme, differences in kinetic and physicochemical properties have been described, which cannot be explained by the amino acid residues present in the active site. In order to understand the different kinetic behavior of the Pimpernel (ATPase/ADPase = 10) and Desirée (ATPase/ADPase = 1) isoenzymes, the nucleotide-binding site of these apyrases was explored using the intrinsic fluorescence of tryptophan. The intrinsic fluorescence of the two apyrases was slightly different. The maximum emission wavelengths of the Desirée and Pimpernel enzymes were 336 and 340 nm, respectively, suggesting small differences in the microenvironment of Trp residues. The Pimpernel enzyme emitted more fluorescence than the Desirée apyrase at the same concentration although both enzymes have the same number of Trp residues. The binding of the nonhydrolyzable substrate analogs decreased the fluorescence emission of both apyrases, indicating the presence of conformational changes in the neighborhood of Trp residues. Experiments with quenchers of different polarities, such as acrylamide, Cs+ and I- indicated the existence of differences in the nucleotide-binding site, as further shown by quenching experiments in the presence of nonhydrolyzable substrate analogs. Differences in the nucleotide-binding site may explain, at least in part, the kinetic differences of the Pimpernel and Desirée isoapyrases.

Studies on ATP-diphosphohydrolase nucleotide-binding sites by intrinsic fluorescence

Potato apyrase, a soluble ATP-diphosphohydrolase, was purified to homogeneity from several clonal varieties of Solanum tuberosum. Depending on the source of the enzyme, differences in kinetic and physicochemical properties have been described, which cannot be explained by the amino acid residues present in the active site. In order to understand the different kinetic behavior of the Pimpernel (ATPase/ADPase = 10) and Desirée (ATPase/ADPase = 1) isoenzymes, the nucleotide-binding site of these apyrases was explored using the intrinsic fluorescence of tryptophan. The intrinsic fluorescence of the two apyrases was slightly different. The maximum emission wavelengths of the Desirée and Pimpernel enzymes were 336 and 340 nm, respectively, suggesting small differences in the microenvironment of Trp residues. The Pimpernel enzyme emitted more fluorescence than the Desirée apyrase at the same concentration although both enzymes have the same number of Trp residues. The binding of the nonhydrolyzable substrate analogs decreased the fluorescence emission of both apyrases, indicating the presence of conformational changes in the neighborhood of Trp residues. Experiments with quenchers of different polarities, such as acrylamide, Cs+ and I- indicated the existence of differences in the nucleotide-binding site, as further shown by quenching experiments in the presence of nonhydrolyzable substrate analogs. Differences in the nucleotide-binding site may explain, at least in part, the kinetic differences of the Pimpernel and Desirée isoapyrases.