RESUMO
Copper complexes with transformed methimazole ligand have been synthesized and characterized by elemental analysis, conductivity measurements, thermogravimetric analysis, EPR, FTIR and UV-Vis spectroscopies. Results support their stoichiometries and geometrical structures: [Cu(C4H5N2S)2Cl2]·2H2O(1), [Cu(C8H10N4S)SO4H2O](2) and [Cu(C8H10N4S)SO4](3). ((C4H5N2)2S: bis(l-methylimidazol-2-yl)sulfide; (C4H5N2S)2 = Bis[bis(l-methylimidazol-2-yl)disulfide]) Concurrently, the structurally distinct soluble species corresponding to complexes (1) and (2) were subsequently used in an in vitro investigation of their potential biological properties. In view of their possible pharmaceutical activity, the complexes were in vitro evaluated as phosphatase acid inhibitors. Their radical bio-protective effects were also studied measuring the effect against DPPH⢠and O2â¢- radicals. Additional catalytic properties as peroxidase mimics were evaluated using Michaelis-Menten kinetic model by means of phenol red and pyrogallol assays. The complexes exhibited catalytic bromination activity and the ability to oxidize pyrogallol substrate indicating that they can be considered as functional models. The relationships between the structures and the in vitro biological activities have also been considered. Serum protein albumin has attracted the greatest interest as drug carrier and the affinity of biological/pharmaceutical compound is relevant to the development of new medicine. In that sense, interaction studies by fluorescence and EPR spectroscopies were performed showing the binding capacity of the complexes.
Assuntos
Fosfatase Ácida/antagonistas & inibidores , Cobre/farmacologia , Osteoporose/tratamento farmacológico , Peroxidases/metabolismo , Substâncias Protetoras/uso terapêutico , Superóxido Dismutase/metabolismo , Fosfatase Ácida/metabolismo , Animais , Sítios de Ligação , Compostos de Bifenilo/química , Catálise , Bovinos , Espectroscopia de Ressonância de Spin Eletrônica , Cinética , Oxirredução , Picratos/química , Substâncias Protetoras/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Soroalbumina Bovina/metabolismo , Espectrometria de Fluorescência , Termodinâmica , Fatores de TempoRESUMO
A 39-amino acid peptide fragment that is derived from prostatic acidic phosphatase (PAP), PAP248-286 , is secreted in large amounts in human semen and forms amyloid fibrils. These fibrils can capture HIV virions and increase the attachment of virions to target cells; as such, they are called a "semen-derived enhancer of virus infection" (SEVI). Therefore, the inhibition of the formation of PAP248-286 amyloid fibrils is of great significance. Herein, we demonstrate that brazilin effectively inhibits PAP248-286 aggregation. The inhibitory effect increases with increasing brazilin concentration. Thioflavinâ T fluorescence assays and TEM observations confirmed that a few fibrils formed when brazilin was present with PAP248-286 in an equimolar concentration. Circular dichroism spectroscopy indicated that brazilin inhibited the secondary structural transitions from α-helices and random coils into ß-sheets. Cytotoxicity assays showed that brazilin significantly decreased the cytotoxicity of the fibrils at 0.01â mmol L-1 . Isothermal titration calorimetry revealed that hydrophobic interactions were the main driving force for the binding of brazilin to the PAP248-286 monomer (dissociation constant, 4.03â µmol L-1 ), and that the binding affinity of brazilin for the fibrils was at least three orders of magnitude lower than that for the monomer. These results indicate that brazilin holds great potential as a small-molecule agent against SEVIs.
Assuntos
Fosfatase Ácida/antagonistas & inibidores , Amiloide/antagonistas & inibidores , Amiloide/toxicidade , Benzopiranos/farmacologia , Fosfatase Ácida/metabolismo , Fosfatase Ácida/toxicidade , Amiloide/metabolismo , Animais , Benzopiranos/química , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Células PC12 , Agregados Proteicos/efeitos dos fármacos , Ratos , Relação Estrutura-AtividadeRESUMO
This study evaluated the effect of tin exposure on enzyme activity in the sea cucumber (Holothuria grisea Selenka, 1867). After exposure to 0 (control), 0.04, 0.08, or 0.12 mg L-1 tin, we tested the activities of total cholinesterase in longitudinal muscles, acid phosphatase in gonads and the respiratory tree, as well as alkaline phosphatase in the intestines during a 96-h bioassay. Regression analyses showed that all enzyme activities declined with increasing tin concentrations, except for acid phosphatase in the respiratory tree, which were similarly, inhibited at all tin concentrations. These results indicate that H. grisea is a potential bioindicator for seascape habitat monitoring programs, as its biochemical markers show sensitivity to trace elements that can indicate a rise in pollution levels.
Assuntos
Fosfatase Ácida/antagonistas & inibidores , Fosfatase Alcalina/antagonistas & inibidores , Inibidores da Colinesterase/farmacologia , Holothuria/enzimologia , Estanho/farmacologia , Animais , Relação Dose-Resposta a Droga , Gônadas/enzimologia , Intestinos/enzimologia , Músculos/enzimologia , Sistema Respiratório/enzimologiaRESUMO
It has been reported that various metal coordination compounds have improved some biological properties. A high activity of acid phosphatase (AcP) is associated to several diseases (osteoporosis, Alzheimer's, prostate cancer, among others) and makes it a target for the development of new potential inhibitors. Anti-thyroid agents have disadvantageous side effects and the scarcity of medicines in this area motivated many researchers to synthesize new ones. Several copper(II) complexes have shown antifungal activities. In this work we presented for a first time the inhibition of AcP and the anti-thyroid activity produced by methimazole-Cu(II) complexes. Cu-Met ([Cu(MeimzH)2(H2O)2](NO3)2·H2O) produces a weak inhibition action while Cu-Met-phen ([Cu(MeimzH)2(phen)(H2O)2]Cl2) shows a strong inhibition effect (IC50 = 300 µM) being more effective than the reported behavior of vanadium complexes. Cu-Met-phen also presented a fairly good anti-thyroid activity with a formation constant value, Kc=1.02 × 10(10)M(-1) being 10(6) times more active than methimazole (Kc = 4.16 × 10(4)M(-1)) in opposition to Cu-Met which presented activity (Kc=9.54 × 10(3)M(-1)) but in a lesser extent than that of the free ligand. None of the complexes show antifungal activity except Cu-phen (MIC = 11.71 µgmL(-1) on Candidaalbicans) which was tested for comparison. Besides, albumin interaction experiments denoted high affinity toward the complexes and the calculated binding constants indicate reversible binding to the protein.
Assuntos
Fosfatase Ácida/antagonistas & inibidores , Antifúngicos/farmacologia , Antitireóideos/farmacologia , Complexos de Coordenação/farmacologia , Cobre/farmacologia , Metimazol/farmacologia , Soroalbumina Bovina/metabolismo , Fosfatase Ácida/metabolismo , Animais , Antifúngicos/química , Antitireóideos/química , Candida/efeitos dos fármacos , Candidíase/tratamento farmacológico , Bovinos , Complexos de Coordenação/química , Cobre/química , Humanos , Metimazol/química , Conformação Proteica/efeitos dos fármacos , Soroalbumina Bovina/químicaRESUMO
The aim of this work was to investigate whether an alkaline ecto-phosphatase activity is present in the surface of Trypanosoma rangeli. Intact short epimastigote forms were assayed for ecto-phosphatase activity to study kinetics and modulators using ß-glycerophosphate (ß-GP) and p-nitrophenyl phosphate (pNPP) as substrates. Its role in parasite development and differentiation was also studied. Competition assays using different proportions of ß-GP and pNPP evidenced the existence of independent and non-interacting alkaline and acid phosphatases. Hydrolysis of ß-GP increased progressively with pH, whereas the opposite was evident using pNPP. The alkaline enzyme was inhibited by levamisole in a non-competitive fashion. The Ca(2+) present in the reaction medium was enough for full activity. Pretreatment with PI-PLC decreased the alkaline but not the acid phosphatase evidence that the former is catalyzed by a GPI-anchored enzyme, with potential intracellular signaling ability. ß-GP supported the growth and differentiation of T. rangeli to the same extent as high orthophosphate (Pi). Levamisole at the IC50 spared significantly parasite growth when ß-GP was the sole source of Pi and stopped it in the absence of ß-GP, indicating that the alkaline enzyme can utilize phosphate monoesters present in serum. These results demonstrate the existence of an alkaline ecto-phosphatase in T. rangeli with selective requirements and sensitivity to inhibitors that participates in key metabolic processes in the parasite life cycle.
Assuntos
Fosfatase Alcalina/metabolismo , Trypanosoma rangeli/enzimologia , Trypanosoma rangeli/crescimento & desenvolvimento , Fosfatase Ácida/antagonistas & inibidores , Fosfatase Ácida/metabolismo , Catálise , Cátions Bivalentes/farmacologia , Glicerofosfatos/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Levamisol/farmacologia , Nitrofenóis/metabolismo , Compostos Organofosforados/metabolismo , Especificidade por SubstratoRESUMO
BACKGROUND AND PURPOSE: Bones are widely innervated, suggesting an important role for the sympathetic regulation of bone metabolism, although there are controversial studies. We investigated the effects of propranolol in a model of experimental periodontal disease. EXPERIMENTAL APPROACH: Rats were assigned as follows: animals without ligature; ligated animals receiving vehicle and ligated animals receiving 0.1, 5 or 20 mg·kg(-1) propranolol. After 30 days, haemodynamic parameters were measured by cardiac catheterization. Gingival tissues were removed and assessed for IL-1ß, TNF-α and cross-linked carboxyterminal telopeptides of type I collagen (CTX) by elisa, or intercellular adhesion molecule 1 (ICAM-1), receptor activator of NF-κ B ligand (RANKL) and osteoprotegerin (OPG) by Western blot analysis. Sections from the mandibles were evaluated for bone resorption. Also, we analysed the ability of propranolol to inhibit osteoclastogenesis in vitro. RESULTS: Propranolol at 0.1 and 5 mg·kg(-1) reduced the bone resorption as well as ICAM-1 and RANKL expression. However, only 0.1 mg·kg(-1) reduced IL-1ß, TNF-α and CTX levels as well as increased the expression of OPG, but did not alter any of the haemodynamic parameters. Propranolol also suppressed in vitro osteoclast differentiation and resorptive activity by inhibiting the nuclear factor of activated T cells (NFATc)1 pathway and the expression of tartrate-resistant acid phosphatase (TRAP), cathepsin K and MMP-9. CONCLUSIONS AND IMPLICATIONS: Low doses of propranolol suppress bone resorption by inhibiting RANKL-mediated osteoclastogenesis as well as inflammatory markers without affecting haemodynamic parameters.
Assuntos
Reabsorção Óssea/tratamento farmacológico , Osteoclastos/efeitos dos fármacos , Propranolol/administração & dosagem , Fosfatase Ácida/antagonistas & inibidores , Fosfatase Ácida/genética , Perda do Osso Alveolar/prevenção & controle , Animais , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Catepsina K/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Colágeno Tipo I/metabolismo , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Gengiva/efeitos dos fármacos , Gengiva/metabolismo , Hemodinâmica/efeitos dos fármacos , Inflamação/prevenção & controle , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-1beta/metabolismo , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Masculino , Metaloproteinase 9 da Matriz/genética , Camundongos , Fatores de Transcrição NFATC/antagonistas & inibidores , Osteoclastos/patologia , Peptídeos/metabolismo , Ligante RANK/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Fosfatase Ácida Resistente a Tartarato , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: This study describes the expression of acidic ectophosphatase activity on twenty isolates of C. albicans from oral cavities of HIV-infected children (HIV+) and compares them with fifteen isolates from HIV-negative children (HIV-), as well as the fungal adhesion to epithelial cells and medical records. METHODS: The activities were measured in intact cells grown in BHI medium for 48 h at 37 degrees C. Phosphatase activity was assayed at pH 5.5 using 4-methylumbelliferyl phosphate. Yeast adhesion was measured using the MA 104 epithelial cell line. RESULTS: Mean values of ectophosphatase activity were 610.27 +/- 166.36 and 241.25 +/- 78.96 picomoles 4-methylumbelliferone/h/10(7) cells for HIV+ and HIV- group, respectively (P = 0.049). No correlation between C. albicans enzyme activity from HIV children with viral load and CD4 percentual was observed. Yeasts with high enzyme activity, isolated from HIV+ children showed greater adherence than yeasts with basal levels of ectophosphatases from HIV- (Spearman correlation, r = 0.8). Surface phosphatase activity was apparently involved in the adhesion to host cells, as the enhanced attachment of C. albicans to host epithelial cells was reversed by pretreatment of yeast with sodium orthovanadate (1 mM), an acid phosphatase inhibitor. CONCLUSION: These results show that C. albicans from HIV+ has an ectophosphatase activity significantly higher than the other isolates. Yeasts expressing higher levels of surface phosphatase activity showed greater adhesion to epithelial cells. So, the activity of acidic surface phosphatases on these cells may contribute to the early mechanisms required for disease establishment.
Assuntos
Fosfatase Ácida/metabolismo , Candida albicans/enzimologia , Soronegatividade para HIV , Soropositividade para HIV/microbiologia , Fosfatase Ácida/antagonistas & inibidores , Animais , Contagem de Linfócito CD4 , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Linhagem Celular , Criança , Inibidores Enzimáticos/farmacologia , Células Epiteliais/microbiologia , HIV/isolamento & purificação , Soropositividade para HIV/virologia , Humanos , Concentração de Íons de Hidrogênio , Himecromona/análogos & derivados , Indicadores e Reagentes , Mucosa Bucal/microbiologia , Mucosa Bucal/patologia , Vanadatos/farmacologia , Carga ViralRESUMO
The presence of copper in water environment may have detrimental effects on aquatic organisms, including algae, where different enzymatic systems can be affected. Algae acid phosphatase plays important roles in metabolic processes such as decomposition of organic phosphate, autophagic digestive process, recycling cellular materials and zygote formation during reproduction. This work describes an in vitro activation effect of copper on the acid phosphatase of the green algae Pseudokirchneriella subcapitata (formely Selenastrum capricornutum) under preincubation condition. Apparent Michaelis constant values of 1.21 and 0.37 mM, and activation energy values of 26.8 and 13.6 kJ mol(-1) were determined in the absence and in the presence of 0.2 mM Cu(2+), respectively. The dissociation constant value for Cu(2+) binding to the enzyme was determined to be 22.04 microM. The decrease of the apparent Michaelis constant (Km) and activation energy values in the presence of Cu(2+) correlates well with its activating effect on the acid phosphatase activity. This propriety could be used as a sensitive bioindicator for copper in environmental samples.
Assuntos
Fosfatase Ácida/antagonistas & inibidores , Clorófitas/enzimologia , Sulfato de Cobre/farmacologia , Fosfatase Ácida/química , Fosfatase Ácida/metabolismo , Clorófitas/crescimento & desenvolvimento , Sulfato de Cobre/química , Ativação Enzimática/efeitos dos fármacos , Estabilidade Enzimática/efeitos dos fármacos , Cinética , Relação Estrutura-AtividadeRESUMO
Sewage sludge applied to soils as a fertilizer often contains metals and linear alkylbenzene sulphonate (LAS) as contaminants. These pollutants can be transported to the aquatic environment where they can alter the phosphatase activity in living organisms. The acid phosphatase of algae plays important roles in metabolism such as decomposing organic phosphate into free phosphate and autophagic digestive processes. The order of in vitro inhibition of Pseudokirchneriella subcapitata acid phosphatase at the highest concentration tested was LAS > Hg2+ = Al3+ > Se4+ = Pb2+ > Cd2+. A non-competitive inhibition mechanism was obtained for Hg2+ (Ki = 0.040 mM) and a competitive inhibition for LAS (Ki = 0.007 mM). In vivo studies with treated algae cultures showed that the inhibition of specific activity was observed in algae exposed during 7 days, in contrast to short term (24 h) treatments with both these chemicals. Our results suggest that the inhibition parameters in vitro did not markedly differ between the two chemicals. On the other hand, in vivo evaluations showed strong differences between both pollutants regarding the concentration values and the degree of response.
Assuntos
Fosfatase Ácida/antagonistas & inibidores , Proteínas de Algas/antagonistas & inibidores , Ácidos Alcanossulfônicos/química , Eucariotos/enzimologia , Metais Pesados/química , Poluentes Químicos da Água/química , Fosfatase Ácida/isolamento & purificação , Proteínas de Algas/isolamento & purificação , Ácidos Alcanossulfônicos/farmacologia , Eucariotos/efeitos dos fármacos , Concentração Inibidora 50 , Metais Pesados/farmacologia , Poluentes Químicos da Água/farmacologiaRESUMO
The etiological agent of Chagas disease, Trypanosoma cruzi, is consisted of two phylogenetic lineages. Using live epimastigotes, in this study we have characterized ecto-phosphatase activities of two strains of T. cruzi, one (Y strain) is a member of group T. cruzi I and the other (Colombiana) is a member of group T. cruzi II. About one-third of the total ecto-phosphatase activity from the Y strain was Mg(2+)-dependent, but no such activity was observed with Colombiana. The level of Mg(2+)-independent activity was dramatically different in the two strains, with Colombiana showing more than 15-fold higher activity. Experiments using classical inhibitors of acid phosphatases, as well as inhibitors of phosphotyrosine phosphatase, showed a decrease in these phosphatase activities, with different patterns of inhibition. The Mg(2+)-independent activities of the Colombiana and Y strains decreased inversely with pH, varying from 6.5 to 8.0. On the other hand, the Mg(2+)-dependent activity of the Y strain increased concomitantly with the increase in pH in the same range.
Assuntos
Monoéster Fosfórico Hidrolases/metabolismo , Trypanosoma cruzi/classificação , Trypanosoma cruzi/enzimologia , Fosfatase Ácida/antagonistas & inibidores , Fosfatase Ácida/metabolismo , Animais , Cátions Bivalentes/farmacologia , Inibidores Enzimáticos/farmacologia , Concentração de Íons de Hidrogênio , Magnésio/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/metabolismo , Especificidade da Espécie , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Acid phosphatase activity (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) increased during the first 24 h of maize (Zea mays) seed germination. The enzyme displayed a pH optimum of 4.5-5.5. Catalytic activity in vitro displayed a linear time course (60 min) and reached its half maximum value at 0.47 mM p-nitrophenyl phosphate (pNPP). Phosphatase activity towards phosphoamino acids was greatest for phosphotyrosine. The phosphatase activity was strongly inhibited by ammonium molybdate, vanadate and NaF and did not require divalent cations for the catalysis. The temperature optimum for pNPP hydrolysis was 37 degrees C. Under the same conditions, no enzyme activity was detected with phytic acid as substrate. Western blotting of total homogenates during seed germination revealed proteins/polypeptides that were phosphorylated on tyrosine residues; a protein of approximately 14 kDa is potentially a major biological substrate for the phosphatase activity. The results presented in this study suggest that the acid phosphatase characterized under the tested conditions is a member of the phosphotyrosine phosphatase family.
Assuntos
Fosfatase Ácida/metabolismo , Germinação/fisiologia , Proteínas de Plantas/metabolismo , Sementes/enzimologia , Zea mays/enzimologia , Fosfatase Ácida/antagonistas & inibidores , Cátions Bivalentes/farmacologia , Ativação Enzimática , Germinação/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Cinética , Molibdênio/farmacologia , Nitrofenóis/metabolismo , Compostos Organofosforados/metabolismo , Fosforilação , Ácido Fítico , Sementes/efeitos dos fármacos , Fluoreto de Sódio/farmacologia , Temperatura , Tirosina/metabolismo , Vanadatos/farmacologia , Zea mays/efeitos dos fármacosRESUMO
Vitellin (VT) is a lipoglycophosphoprotein stored inside the eggs of every oviparous organism during oogenesis. In the blood-sucking bug Rhodnius prolixus, VT is deposited inside growing oocytes together with two acid hydrolases: acid phosphatase (AP) and cathepsin D (CD). Egg fertilization triggers AP activity and VT proteolysis in vivo [Insect Biochem. Mol. Biol. 2002 (32) 847]. Here, we show that CD is the main protease targeting VT proteolysis during egg development. CD activity in total egg homogenates is blocked by the classical aspartyl protease inhibitor, pepstatin A. Surprisingly, AP inhibitors such as NaF, Na+/K+ tartrate, and inorganic phosphate also block VT proteolysis, whereas this effect is not observed when tyrosine phosphatase inhibitors such as vanadate and phenylarsine oxide or an inhibitor of alkaline phosphatases such as levamisole are used in a VT proteolysis assay. NaF concentrations that block isolated AP activity do not affect the activity of partially purified CD. Therefore, a specific repressor of VT proteolysis must be dephosphorylated by AP in vivo. In conclusion, these results demonstrate for the first time that acid hydrolases act cooperatively to promote yolk degradation during egg development in arthropods.
Assuntos
Catepsina D/metabolismo , Proteínas do Ovo/metabolismo , Fosfatase Ácida/antagonistas & inibidores , Fosfatase Ácida/química , Fosfatase Ácida/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Catepsina D/química , Catepsinas/química , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Hidrolases/química , Insetos/metabolismo , Oócitos/metabolismo , Oogênese , Fosfoproteínas/química , Monoéster Fosfórico Hidrolases/química , Fosforilação , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/metabolismo , Rhodnius/metabolismo , Sódio/química , Tartaratos/química , Temperatura , Fatores de Tempo , Vitelinas/químicaRESUMO
In this work, we describe the ability of living Tritrichomonas foetus to hydrolyze extracellular ATP. The addition of MgCl(2) to the assay medium increased the ecto-ATPase activity in a dose-dependent manner. At 5mM ATP, half maximal stimulation of ATP hydrolysis was obtained with 0.46mM MgCl(2). The ecto-ATPase activity was also stimulated by MnCl(2) and CaCl(2), but not by SrCl(2). The Mg(2+)-dependent ATPase presents two apparent K(m) values for Mg-ATP(2-) (K(m1)=0.03 mM and K(m2)=2.01 mM). ATP was the best substrate for this enzyme, although other nucleotides such as ITP, CTP, UTP also produced high reaction rates. GTP produced a low reaction rate and ADP was not a substrate for this enzyme. The Mg(2+)-dependent ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A(1), ouabain, furosemide, vanadate, molybdate, sodium fluoride and levamizole. The acid phosphatase inhibitors (vanadate and molybdate) inhibited about 60-70% of the Mg(2+)-independent ecto-ATPase activity, suggesting that the ATP hydrolysis measured in the absence of any metal divalent could, at least in part, also be catalyzed by an ecto-phosphatase present in this cell. In order to confirm the observed Mg(2+)-dependent activity as an ecto-ATPase, we used an impermeant inhibitor, 4,4'-diisothiocyanostylbene-2',2'-disulfonic acid (DIDS) as well as suramin, an antagonist of P(2) purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. This ecto-ATPase was stimulated by more than 90% by 50mM D-galactose. Since previous results showed that D-galactose exposed on the surface of host cells is involved with T. foetus adhesion, the Mg(2+)-dependent ecto-ATPase may be involved with cellular adhesion and possible pathogenicity.
Assuntos
Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Cloreto de Magnésio/farmacologia , Tritrichomonas foetus/enzimologia , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Fosfatase Ácida/antagonistas & inibidores , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Galactose/farmacologia , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Especificidade por Substrato , Suramina/farmacologia , Tritrichomonas foetus/efeitos dos fármacos , Tritrichomonas foetus/patogenicidade , Tripanossomicidas/farmacologiaRESUMO
In a preceding article, we described alterations occurring in rat pancreas acinar cells at successive post-mortem (PM) intervals. In ultra-thin sections from samples obtained from 0.5, 1, 2, 4, 8 and 12 h, we observed in the Golgi apparatus the appearance of an anomalous membrane bound structure. Such structures are formed by tubules and vesicles that we have called tubular vesicular structure (TVS), and they are frequently located in the position corresponding to the 4th cisterna of the Golgian cisternal pile. Lobules of rat pancreas, incubated in vitro with metabolic inhibitors (such as antimycin A, sodium fluoride, sodium azide and potassium cyanide), were processed in order to be compared with the PM samples of the rat acinar cells. In sliced pieces of lobules, acid phosphatase (AcPase) and tiaminopirophosphatase (TPPase) activity were evaluated. Except for the potassium cyanide treatment, we frequently observed the TVS located at the position corresponding to the 4th cisternae (similar to those observed in the PM acinar cells). These TVS's are predominantly TPPase positive. Based on this result and the fact that the TVS's are surrounded by a membrane (as confirmed by the freeze-fracture replica results) with no structural elements inside, they seem not to correspond to autophagosomes. The TVS's, observed either at PM consecutive times or incubated with metabolic inhibitors, seem to be structures formed in response to ATP deprivation. In 0,5 h PM cells and in cells incubated for 30 and 60 min with metabolic inhibitors, the subcellular structures reacted for AcPase in the rigid lamellae, CV and lysosomes.
Assuntos
Complexo de Golgi/enzimologia , Proteínas de Membrana Transportadoras/metabolismo , Pâncreas/enzimologia , Fosfatase Ácida/análise , Fosfatase Ácida/antagonistas & inibidores , Animais , Antimetabólitos/farmacologia , Antimicina A/farmacologia , Inibidores Enzimáticos/farmacologia , Técnica de Fratura por Congelamento , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/ultraestrutura , Histocitoquímica , Técnicas In Vitro , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Pâncreas/efeitos dos fármacos , Pâncreas/ultraestrutura , Cianeto de Potássio/farmacologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Ratos , Ratos Wistar , Azida Sódica/farmacologia , Fluoreto de Sódio/farmacologia , Tiamina Pirofosfatase/análise , Tiamina Pirofosfatase/antagonistas & inibidores , Fatores de TempoRESUMO
The four soybean seed acid phosphatase isoforms AP1, AP2, AP3A and AP3B were competitively inhibited by phosphate, vanadate, fluoride and molybdate, using p-nitrophenylphosphate as substrate. The four isoforms were not significantly affected by compounds that can interact with SH residues or by pyridoxal phosphate. These results indicated that cysteine and lysine residues are not present in the active site of the four soybean seed acid phosphatase isoforms. The inhibition constant values for phosphate, vanadate, fluoride and molybdate at pH 5.0 were respectively: API (250, 12.8, 1.7, 0.05 microM). AP2 (800, 10, 500, 0.025 microM), AP3A (250, 24.2,250, 0.032 microM ), AP3B (2400 36.9, 750, 0.05 microM).
Assuntos
Fosfatase Ácida/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Glycine max/enzimologia , Isoenzimas/antagonistas & inibidores , Sementes/enzimologia , Fosfatase Ácida/isolamento & purificação , Fosfatase Ácida/metabolismo , Fluoretos/farmacologia , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Cinética , Molibdênio/farmacologia , Nitrofenóis/metabolismo , Compostos Organofosforados/metabolismo , Fosfatos/farmacologia , Vanadatos/farmacologiaRESUMO
Acid phosphatase (AP) is readily inactivated when exposed to the free radicals generated in the pyrolysis of 2, 2'-azobis(2-amidinopropane) hydrochloride (AAPH) under aerobic conditions. On average, a large number of tryptophan groups are modified by each protein molecule that loses its catalytic activity. In spite of this, the enzyme inactivation takes place without induction times, a result that indicates either that damage is progressive or that damage of a critical target is needed to inactivate the enzyme (all-or-nothing mechanism). A Lineweaver-Burk plot of the enzyme activity measured at pH 4.8 is not compatible with an all-or-nothing mechanism, showing that after exposure of the native protein ensemble to the free radical source there are partially damaged molecules whose affinity for the substrate is widely different from that of the native molecules. On the other hand, the partially damaged ensemble shows a normal Michaelis-Menten behavior when the activity is measured at pH 7.0, with only a reduced value of V(M), relative to that of the unmodified ensemble. These results show that the native protein and modified proteins that remain active constitute different populations, with different responses to pH changes. Comparative heat denaturation studies of the native and pretreated proteins support this proposal.
Assuntos
Fosfatase Ácida/antagonistas & inibidores , Fosfatase Ácida/metabolismo , Peróxidos/metabolismo , Peróxidos/farmacologia , Solanum tuberosum/enzimologia , Fosfatase Ácida/química , Amidinas/farmacologia , Catálise/efeitos dos fármacos , Estabilidade Enzimática/efeitos dos fármacos , Fluorescência , Radicais Livres/metabolismo , Radicais Livres/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Oxidantes/farmacologia , Desnaturação Proteica/efeitos dos fármacos , Temperatura , Triptofano/metabolismo , Ureia/farmacologiaRESUMO
To study the role of parasite protein kinase C (PKC) activity in the uptake of Leishmania amazonensis by mononuclear phagocytes we treated the parasites with 12-O-tetradecanoyl phorbol-13-acetate (TPA) and/or sphingosine, before interaction assays. Promastigotes of Leishmania amazonensis were incubated with 20 ng/ml TPA and/or 50 ng/ml sphingosine before the interaction with murine peritoneal macrophages. The short treatment enhanced about 200% the parasite association with the host cells, whereas the sphingosine treatment reduced about 50% the promastigote binding, as did the prolonged TPA treatment. The binding of cells treated with both drugs was not significantly altered. Biochemical and cytochemical data indicate that the protein kinase C agonists TPA and sphingosine, respectively, increased and decreased acid phosphatase (AcP) activity. The addition of sodium tartrate, a secreted AcP inhibitor, suppressed the TPA enhancing effects, but did not affect the basal parasite binding observed in control cells. The supernatants of TPA-treated L. amazonensis promastigotes increased the parasite association by about the same extent as the TPA treatment, and this effect was also abolished by tartrate. Although TPA did not enhance the association of L. major, a species that does not secrete AcP, the supernatants of TPA-treated L. amazonensis increased it in a tartrate-sensitive manner. The results suggest that Leishmania amazonensis PKC activity may modulate its interaction with macrophages via secreted AcP.
Assuntos
Fosfatase Ácida/metabolismo , Leishmania mexicana/patogenicidade , Macrófagos Peritoneais/parasitologia , Proteína Quinase C/metabolismo , Fosfatase Ácida/antagonistas & inibidores , Animais , Membrana Celular/enzimologia , Flagelos/enzimologia , Interações Hospedeiro-Parasita , Leishmania mexicana/efeitos dos fármacos , Leishmania mexicana/enzimologia , Camundongos , Proteína Quinase C/agonistas , Esfingosina/farmacologia , Tartaratos/farmacologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Like many other bacteria, Nocardia sp. possess acid phosphatase activity. In N. brasiliensis, a human and animal pathogen, this activity was resolved into two enzyme forms by native gel electrophoresis. One (isozyme I) was partially purified and characterized. It exhibited an estimated molecular weight on SDS-PAGE of 50 kDa, a pH optimum of 5.2, and a Km value of 1.25 mM for p-nitrophenylphosphate. The N. brasiliensis enzyme was stable at 4 degrees C for at least 24 h, but readily inactivated at 60 degrees C. Ammonium molybdate, sodium fluoride and L-(+)-tartrate were found to be potent inhibitors of the enzyme. Although its function is presently unknown, by analogy to other bacterial systems it could be envisioned to play an important role in the physiology and pathogenicity of the microorganism.
Assuntos
Fosfatase Ácida/isolamento & purificação , Isoenzimas/isolamento & purificação , Nocardia/enzimologia , Fosfatase Ácida/antagonistas & inibidores , Fosfatase Ácida/química , Fosfatase Ácida/metabolismo , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Peso Molecular , Nocardia/patogenicidadeRESUMO
In Pseudomonas aeruginosa, the effect of different cations on the acid phosphatase activity was studied in order to acquire more information related to a previously proposed mechanism, involving the coordinated action of this enzyme with phospholipase C. Although the natural substrate of this enzyme is phosphorylcholine, in order to avoid the possible interaction of its positive charge and those of the different cations with the enzyme molecule, the artificial substrate p-nitrophenylphosphate was utilized. Kinetic studies of the activation of acid phosphatase (phosphorylcholine phosphatase) mediated by divalent cations Mg2+, Zn2+ and Cu2+ revealed that all these ions bind to the enzyme in a compulsory order (ordered bireactant system). The Km values obtained for p-NPP in the presence of Mg2+, Zn2+ and Cu2+ were 1.4 mM, 1.0 mM and 3.5 mM, respectively. The KA values for the same ions were 1.25 mM, 0.05 mM and 0.03 mM, respectively. The Vmax obtained in the presence of Cu2+ was about twofold higher than that obtained in the presence of Mg2+ or Zn2+. The inhibition observed with Al3+ seems to be a multi-site inhibition. The K'app and n values, from the Hill plot, were about 0.25 mM and 4.0 mM, respectively, which were independent of the metal ion utilized as activator. It is proposed that the acid phosphatase may exert its action under physiological conditions, depending on the availability of either one of these metal ions.
Assuntos
Fosfatase Ácida/metabolismo , Alumínio/farmacologia , Pseudomonas aeruginosa/enzimologia , Fosfatase Ácida/antagonistas & inibidores , Cátions Bivalentes , Cobre/farmacologia , Ativação Enzimática/efeitos dos fármacos , Cinética , Magnésio/farmacologia , Zinco/farmacologiaRESUMO
In this work the action of the following compounds upon Ps. aeruginosa acid phosphatase has been studied: 1) alkylammonium compounds; 2) aminoalcohols and aminoacids with different substituents (-H, -CH2OH and -CH3) attached to the nitrogen atom; 3) alcohols analogous to some compounds of the above series, but without the amino group. It was found that the enzyme inhibition was more effective with N-trimethylated compounds than with the triethylated ones. The degree of inhibition depended on the number of methyl groups bound to the nitrogen atom. Taking into account the choline and betaine series the hydroxyl derivatives showed more affinity for the enzyme than the carboxylated ones. In each series the Ki values increased with the decrease of methyl groups bound to the nitrogen atom. The presence of a positively charged nitrogen atom in the molecule of the effector was essential. These results enable us to confirm that in the molecule of Ps. aeruginosa acid phosphatase there exists an anionic site with one subsite with affinity for methyl groups.