RESUMO
BACKGROUND: LIPH, a membrane-associated phosphatidic acid-selective phospholipase A1a, can produce LPA (Lysophosphatidic acid) from PA (Phosphatidic acid) on the outer leaflet of the plasma membrane. It is well known that LIPH dysfunction contributes to lipid metabolism disorder. Previous study shows that LIPH was found to be a potential gene related to poor prognosis with pancreatic ductal adenocarcinoma (PDAC). However, the biological functions of LIPH in PDAC remain unclear. METHODS: Cell viability assays were used to evaluate whether LIPH affected cell proliferation. RNA sequencing and immunoprecipitation showed that LIPH participates in tumor glycolysis by stimulating LPA/LPAR axis and maintaining aldolase A (ALDOA) stability in the cytosol. Subcutaneous, orthotopic xenograft models and patient-derived xenograft PDAC model were used to evaluate a newly developed Gemcitabine-based therapy. RESULTS: LIPH was significantly upregulated in PDAC and was related to later pathological stage and poor prognosis. LIPH downregulation in PDAC cells inhibited colony formation and proliferation. Mechanistically, LIPH triggered PI3K/AKT/HIF1A signaling via LPA/LPAR axis. LIPH also promoted glycolysis and de novo synthesis of glycerolipids by maintaining ALDOA stability in the cytosol. Xenograft models show that PDAC with high LIPH expression levels was sensitive to gemcitabine/ki16425/aldometanib therapy without causing discernible side effects. CONCLUSION: LIPH directly bridges PDAC cells and tumor microenvironment to facilitate aberrant aerobic glycolysis via activating LPA/LPAR axis and maintaining ALDOA stability, which provides an actionable gemcitabine-based combination therapy with limited side effects.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Frutose-Bifosfato Aldolase/genética , Frutose-Bifosfato Aldolase/metabolismo , Frutose-Bifosfato Aldolase/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Linhagem Celular Tumoral , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/patologia , Gencitabina , Proliferação de Células , Glicólise , Fenótipo , Regulação Neoplásica da Expressão Gênica , Microambiente TumoralRESUMO
BACKGROUND: Available therapeutics for visceral leishmaniasis (VL), a deadly parasitic infection, are usually associated with inadequate efficacy and adverse aftereffects. Further, the primary site of Leishmania parasite are host macrophages resulting in compromised immunity; ensuing marked T-cell immunosuppression. Such settings emphasize the exploration of chemo-immunotherapeutic strategies for improvising the infected person's immune status with better resolution of infection. METHODS: Present work employs the immunization of Leishmania-infected hamsters with Leishmania-derived recombinant aldolase (rLdAld) and enolase (rLdEno) proteins in consort with the sub-optimal dose of Ambisome (2.5 mg/kg). After the completion of immunization, hamsters were sacrificed on day 60 and 90 post infection and different organ samples were collected to perform immunological assay for evaluating the therapeutic efficacy and modulation in protective cellular immune responses. RESULTS: Combining these proteins, particularly rLdAld with Ambisome (2.5 mg/kg), has significantly reduced the parasitic load (â¼80%) with remarkable enhancement in DTH and lymphoproliferative responses compared to the infected control and only Ambisome treated groups. Moreover, cytokine levels at RNA and protein levels were noticed to be inclined towards Th-1 phenotype through up-regulation of IFN-γ and TNF-α with significant down-regulation in IL-10 and TGF-ß expression, an indication towards the generation of protective immunity against experimental VL. CONCLUSION: Our experimental findings demonstrated that the chemo-immunotherapeutic approach could be an effective way of controlling human VL infection at minimal dosages of antileishmanial with reduced side-effects and propensity of drug resistance emergence.
Assuntos
Leishmania donovani , Leishmaniose Visceral , Cricetinae , Animais , Humanos , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Frutose-Bifosfato Aldolase/genética , Frutose-Bifosfato Aldolase/farmacologia , Frutose-Bifosfato Aldolase/uso terapêutico , Imunização , CitocinasRESUMO
Eimeria tenella (E. tenella) is a protozoal parasite that can cause severe cecal lesions and death in chickens, seriously harming the chicken industry. Conventional control strategies mainly rely on anticoccidial drugs. However, the emerging problems of anticoccidial resistance and drug residues necessitate exploring potential drug targets for developing new anticoccidial drugs. Fructose-1,6-bisphosphate aldolase (ALD) is an essential enzyme for parasite energy metabolism that has been considered a potential drug target. In this study, we analyzed the molecular and biochemical properties of E. tenella ALD2 (EtALD2). EtALD2 mRNA expression was highest in second-generation merozoites, whereas the protein level was highest in unsporulated oocysts. Indirect immunofluorescence showed that EtALD2 was mainly distributed in sporozoite' cytoplasm. The natural product inhibitor (morin) was screened by computer-aided drug screening. Enzyme kinetic and inhibition kinetic assays showed that morin had a good inhibitory effect on EtALD2 activity (IC50 = 10.37 µM, Ki = 48.97 µM). In vitro inhibition assay demonstrated that morin had an inhibitory effect on E. tenella development, with an IC50 value of 3.98 µM and drug selection index of 177.49. In vivo, morin significantly improved cecal lesions (p < 0.05) and reduced oocyst excretion (p < 0.05) in E. tenella-infected chickens compared with the untreated group. The anticoccidial index of the group receiving 450 mg morin per kg feed was 162, showing a good anticoccidial effect. These findings suggest that EtALD2 could be a novel drug target for E. tenella treatment, and morin should be further evaluated as a therapeutic candidate for chicken coccidiosis.
Assuntos
Coccidiose , Eimeria tenella , Doenças das Aves Domésticas , Animais , Eimeria tenella/genética , Frutose-Bifosfato Aldolase/genética , Frutose-Bifosfato Aldolase/metabolismo , Frutose-Bifosfato Aldolase/farmacologia , Galinhas/parasitologia , Doenças das Aves Domésticas/tratamento farmacológico , Doenças das Aves Domésticas/parasitologia , Doenças das Aves Domésticas/prevenção & controle , Coccidiose/tratamento farmacológico , Coccidiose/veterinária , Coccidiose/parasitologia , OocistosRESUMO
AIMS: High-fructose intake (HF) represents an inducible risk factor for non-alcoholic fatty liver disease (NAFLD). Present study aimed to illustrate the effect of HF diet (HFD) on the induction of NAFLD, hyperuricemia and role of ellagic acid as modulator. MAIN METHODS: Twenty-four adult male albino rats were randomly divided into four groups (6/each). The first group received normal chow diet only while the others received 60 % HFD for 4 weeks and subdivided later into 3 groups. The first and second groups received allopurinol and ellagic acid, respectively while the third group received HFD only for extra 4 weeks. KEY FINDINGS: Rats fed on HFD for 8 weeks displayed body weight gain, insulin resistance (IR), hyperglycemia, dyslipidemia, hyperuricemia with increased oxidative stress and hepatic lipogenic enzymes such as ATP citrate lyase (ACL), aldolase B, and fatty acid synthase (FAS), sterol regulatory element-binding protein 1 (SERBP-1c). C1q /tumor necrosis factor-related protein -3 (CTRP3), and phosphorylated AMP-activated protein kinase (p-AMPK) however showed significant decreases. Ellagic acid or allopurinol administration significantly decreased serum lipids, uric acid, glucose, insulin levels and hepatic contents of enzymes. Malondialdehyde (MDA), FAS, aldolase B, SERBP-1c, and xanthine oxidase (XO) hepatic contents showed significant decreases along with glutathione (GSH) increase as compared to fructose group where ellagic acid was more remarkable compared with allopurinol. SIGNIFICANCE: Our findings indicated that ellagic acid had alleviated HFD-induced hyperuricemia, its associated NAFLD pattern as mediated through activation of CTRP3 and inhibition of ACL activities in a pattern more remarkable than allopurinol.
Assuntos
Hiperuricemia , Hepatopatia Gordurosa não Alcoólica , ATP Citrato (pro-S)-Liase/metabolismo , ATP Citrato (pro-S)-Liase/farmacologia , Alopurinol/farmacologia , Animais , Proteínas de Transporte/metabolismo , Complemento C1q/metabolismo , Dieta Hiperlipídica , Ácido Elágico/farmacologia , Frutose/toxicidade , Frutose-Bifosfato Aldolase/metabolismo , Frutose-Bifosfato Aldolase/farmacologia , Hiperuricemia/induzido quimicamente , Hiperuricemia/tratamento farmacológico , Fígado/metabolismo , Masculino , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Ratos , Ratos Wistar , Fatores de Necrose Tumoral/efeitos adversos , Fatores de Necrose Tumoral/metabolismoRESUMO
Neuronal apoptosis is one of the main pathophysiological events in the early brain injury (EBI) post subarachnoid hemorrhage (SAH). Wnt-3a, one of the endogenous wnt ligands crucial in neurogenesis, has been proven to be efficacious in neuroprotection in traumatic brain injury and ischemic stroke. The glycolytic enzyme aldolase C and ribosome biogenesis protein PPAN were revealed to be linked to wnt signaling pathway. The aim of the study was to explore the antiapoptotic effects of intranasal wnt-3a through Frizzled-1 (Frz-1)/aldolase C/PPAN pathway in SAH. Approaches for assessment included SAH grade, Garcia test, brain water content evaluation, rotarod test, Morris water maze test, Western blot, immunofluorescence and transmission electron microscopy. The results showed that wnt-3a improved the neurological scores, brain water content and long-term neurobehavioral functions after SAH. Wnt-3a increased the level of Frz-1, aldolase C, ß-catenin, PPAN and the Bcl-2/Bax ratio; and decreased the level of axin and cleaved caspase-3 (CC-3). The anti-apoptotic effect of wnt-3a was further evidenced by TUNEL staining and subcellular structure imaging. Frz-1 siRNA and aldolase C siRNA offset the effects of wnt-3a; and restoration of aldolase C by aldolase C CRISPR in Frz-1 siRNA preconditioned SAH rats salvaged the level of Frz-1, aldolase C, PPAN and reduced axin and CC-3. In summary, intranasal administration of wnt-3a alleviates neuronal apoptosis through Frz-1/aldolase C/PPAN pathway in the EBI of SAH rats. The feasible intranasal route and the long-lasting neuroprotective property of wnt-3a is of great clinical relevance.
Assuntos
Apoptose/efeitos dos fármacos , Lesões Encefálicas/tratamento farmacológico , Frutose-Bifosfato Aldolase/farmacologia , Hemorragia Subaracnóidea/tratamento farmacológico , Administração Intranasal , Animais , Lesões Encefálicas/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neuroproteção/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Hemorragia Subaracnóidea/metabolismoRESUMO
In addition to its roles in sugar metabolism, fructose-1,6-bisphosphate aldolase (aldolase) has been implicated in cellular functions independent from these roles, termed "moonlighting functions." These moonlighting functions likely involve the known aldolase-actin interaction, as many proteins with which aldolase interacts are involved in actin-dependent processes. Specifically, aldolase interacts both in vitro and in cells with Wiskott-Aldrich Syndrome Protein (WASP), a protein involved in controlling actin dynamics, yet the function of this interaction remains unknown. Here, the effect of aldolase on WASP-dependent processes in vitro and in cells is investigated. Aldolase inhibits WASP/Arp2/3-dependent actin polymerization in vitro. In cells, knockdown of aldolase results in a decreased rate of cell motility and cell spreading, two WASP-dependent processes. Expression of exogenous aldolase rescues these defects. Whether these effects of aldolase on WASP-dependent processes were due to aldolase catalysis or moonlighting functions is tested using aldolase variants defective in either catalytic or actin-binding activity. While the actin-binding deficient aldolase variant is unable to inhibit actin polymerization in vitro and is unable to rescue cell motility defects in cells, the catalytically inactive aldolase is able to perform these functions, providing evidence that aldolase moonlighting plays a role in WASP-mediated processes.
Assuntos
Proteína 3 Relacionada a Actina/metabolismo , Actinas/metabolismo , Angiopoietinas/metabolismo , Movimento Celular/fisiologia , Frutose-Bifosfato Aldolase/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteína 3 Relacionada a Actina/genética , Actinas/genética , Proteína 2 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/genética , Animais , Movimento Celular/efeitos dos fármacos , Frutose-Bifosfato Aldolase/genética , Frutose-Bifosfato Aldolase/farmacologia , Técnicas de Silenciamento de Genes , Camundongos , Células NIH 3T3 , Coelhos , Proteína da Síndrome de Wiskott-Aldrich/genéticaRESUMO
These studies relate to a working hypothesis that glycogen storage is facilitated in resting muscle by inhibiting glycolysis via inhibition of LDH, AK, and PFK-1 by ascorbate; when muscle is active, these isozymes combine with muscle proteins and are released and protected from inhibition by ascorbate and glycolysis proceeds. Focus in these studies is on the ability of G-actin and aldolase to prevent PFK-1 inhibition by ascorbate. We found that inhibition by ascorbate was PFK-1 concentration dependent; ascorbate does not inhibit above 200 nM PFK-1. We conclude that ascorbate inhibits PFK-1 dimers (and perhaps monomers) but not PFK-1 tetramers. Separation of PFK-1 dimers from tetramers was achieved with centrifugal filter devices and differences in their sensitivity to ascorbate inhibition were demonstrated. Some comparisons are made with attributes of AK inhibitions by ascorbate that, like PFK-1, are also enzyme concentration dependent. Discussions relate findings to cellular infrastructure and the role of ascorbate in glycogen synthesis.
Assuntos
Ácido Ascórbico/farmacologia , Fosfofrutoquinase-1/antagonistas & inibidores , Actinas/farmacologia , Animais , Dimerização , Relação Dose-Resposta a Droga , Frutose-Bifosfato Aldolase/farmacologia , Glicólise , Fosfofrutoquinase-1/química , CoelhosRESUMO
Monoclonal antibody (mAb) 38C2 belongs to a group of catalytic antibodies that were generated by reactive immunization and contains a reactive lysine. 38C2 catalyzes aldol and retro-aldol reactions, using an enamine mechanism, and mechanistically mimics natural aldolase enzymes. In addition, mAb 38C2 can be redirected to target integrins alpha(v)beta(3) and alpha(v)beta(5) through the formation of a covalent bond between a beta-diketone derivative of an arginine-glycine-aspartic acid (RGD) peptidomimetic and the reactive lysine residue in the antibody combining site to provide the chemically programmed mAb cp38C2. In this study, we investigated the potential of enhancing the activity of receptor-binding small molecule drug (SCS-873) through antibody conjugation. Using a M21 human melanoma xenograft model in nude mice, cp38C2 inhibited the growth of the tumor by 81%. The chemically programmed antibody was shown to be highly active at a low concentration while SCS-873 alone was ineffective even at dosages 1,000-fold higher than those used for the chemically programmed antibody. In vitro programming of the catalytic antibody was shown to be as effective as in vivo programming. In an experimental metastasis assay, treatment with mAb cp38C2 significantly prolonged overall survival of tumor-bearing severe combined immuno-deficient (SCID) mice when compared to treatment with unprogrammed mAb 38C2, SCS-873 alone or the integrin-specific monoclonal antibody LM609. In vitro, cp38C2 inhibited human and mouse endothelial and human melanoma cell adhesion, migration and invasion. Additionally, cp38C2 inhibited human and mouse endothelial cell proliferation and was active in complement-dependent cytotoxicity assays. These studies establish the potential of chemically programmed monoclonal antibodies as a novel and effective class of immunotherapeutics that combine the merits of traditional small molecule drug design with immunotherapy.
Assuntos
Anticorpos Monoclonais , Antineoplásicos/química , Antineoplásicos/farmacologia , Frutose-Bifosfato Aldolase/farmacologia , Fragmentos Fab das Imunoglobulinas/farmacologia , Integrinas/efeitos dos fármacos , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Citometria de Fluxo , Humanos , Integrinas/metabolismo , Melanoma/patologia , Camundongos , Invasividade Neoplásica , Neoplasias Cutâneas/patologiaAssuntos
Doxorrubicina/química , Lógica , Modelos Biológicos , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Antibióticos Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Ensaios de Seleção de Medicamentos Antitumorais , Frutose-Bifosfato Aldolase/química , Frutose-Bifosfato Aldolase/farmacologia , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/farmacologia , Penicilina Amidase/química , Penicilina Amidase/farmacologiaRESUMO
Muscle-type LDH (LDH-m4) activity is critical for efficient anaerobic glycolysis. The results here show that rabbit LDH-M4 is inhibited by concentrations of ascorbate normally found in tissues. Aldolase and muscle G-actin were found to protect and to reverse inhibitions of LDH-m4 by ascorbate. G-actins showed some species specificity. Myosin, tropomyosin and troponin from rabbit muscle and muscle proteins from other animal sources had no affect on the inhibitions by ascorbate. The substrate inhibition of LDH-m4 by pyruvate is partially relieved by the presence of aldolase and lowers the Km without affecting the Vm. G-actin under similar conditions has no affect. It is believed that these studies reflect some of the resting properties of glycolytic enzymes that bind and unbind to contractile elements. It is proposed that ascorbate facilitates the storage of glycogen in muscle at rest by inhibiting glycolysis.
Assuntos
Actinas/farmacologia , Ácido Ascórbico/farmacologia , Frutose-Bifosfato Aldolase/farmacologia , L-Lactato Desidrogenase/metabolismo , Músculos/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Glicólise , L-Lactato Desidrogenase/antagonistas & inibidores , Músculos/enzimologia , Miosinas/metabolismo , Substâncias Protetoras/farmacologia , Ácido Pirúvico/metabolismo , Coelhos , Tropomiosina/metabolismo , Troponina/metabolismoRESUMO
Muscle electrotransfer has recently become a promising tool for efficient delivery of plasmids and transgene expression in skeletal muscle. This technology has been mainly applied to use of muscle as a bioreactor for production of therapeutic proteins. However, it remains to be determined whether muscle electrotransfer may also be accurately used as an alternative tool to transgenesis for studying aspects of muscle-specific gene control that must be explored in fully mature muscle fibers in vivo, such as fiber specificity and nerve dependence. It was also not known to what extent the initial electrical stimulations alter muscle physiology and gene expression. Therefore, optimized conditions of skeletal muscle electroporation were first tested for their effects on muscles of transgenic mice harboring a pM310-CAT transgene in which the CAT reporter gene was under control of the fast IIB fiber-specific and nerve-dependent aldolase A pM promoter. Surprisingly, electrostimulation led to a drastic but transient shutdown of pM310-CAT transgene expression concomitant with very transient activation of MyoD and, mostly, with activation of myogenin, suggesting profound alterations in transcriptional status of the electroporated muscle. Return to a normal transcriptional state was observed 7-10 days after electroporation. Therefore, we investigated whether a reporter construct placed under control of pM could exhibit fiber-specific expression 10 days after electrotransfer in either fast tibialis anterior or slow soleus muscle. We show that not only fiber specificity, but also nerve dependence, of a pM-driven construct can be reproduced. However, after electrotransfer, pM displayed a less tight control than previously observed for the same promoter when integrated in a chromatin context.
Assuntos
Eletroporação/métodos , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/inervação , Regiões Promotoras Genéticas/fisiologia , Animais , Denervação , Estimulação Elétrica/métodos , Feminino , Frutose-Bifosfato Aldolase/genética , Frutose-Bifosfato Aldolase/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Músculo Esquelético/fisiologia , Transgenes/fisiologiaRESUMO
7,8-Dihydroneopterin aldolase catalyzes the formation of the tetrahydrofolate precursor, 6-hydroxymethyl-7,8-dihydropterin, and is a potential target for antimicrobial and anti-parasite chemotherapy. The last step of the enzyme-catalyzed reaction is believed to involve the protonation of an enol type intermediate. In order to study the stereochemical course of that reaction step, [1',2',3',6,7-13C5]dihydroneopterin was treated with aldolase in deuterated buffer. The resulting, partially deuterated [6alpha,6,7-13C3]6-hydroxymethyl-7,8-dihydropterin was converted to partially deuterated 6-(R)-[6,7,9,11-13C4]5,10-methylenetetrahydropteroate by a sequence of three enzyme-catalyzed reactions followed by treatment with [13C]formaldehyde. The product was analyzed by multinuclear NMR spectroscopy. The data show that the carbinol group of enzymatically formed 6-hydroxymethyl-dihydropterin contained 2H predominantly in the pro-S position.
Assuntos
Aldeído Liases/química , Tetra-Hidrofolatos/biossíntese , Tetra-Hidrofolatos/química , Cromatografia Líquida de Alta Pressão , Di-Hidropteroato Sintase/química , Di-Hidropteroato Sintase/isolamento & purificação , Difosfotransferases/química , Difosfotransferases/isolamento & purificação , Frutose-Bifosfato Aldolase/farmacologia , Espectroscopia de Ressonância Magnética , Modelos Químicos , Plasmídeos/metabolismo , Pterinas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , EstereoisomerismoRESUMO
This paper presents a modified method of enzymatic assay for Fructose-1,6-diphosphate(FDP). FDP is split to dihydroxyacetone phosphate (DAP) and glyceraldehyde-3-phosphate (GAP) by the action of aldolase. DAP is hydrolyzed at room temperature to free triose. Under alkaline conditions, the free triose is reacted with 2,4-dinitrophenylhydrazine (DNPH), yielding a 2,4-dinitrophenylhydrazine derivative which dissolve in alkali forming a purple color mixture, with maximum absorption at 540.nm. It is proportional to the contents of FDP. Because the method depends on the colorimetric determination of triose formed from fructose-1,6-diphosphate only by aldolase, glycerophosphate dehydrogenase/triosephosphate isomerase (GDH/TIM) and reduced nicotinamide adenine dinucleotide (NADH) which usually applied in multienzymatic method, are omitted in the modified method. The method is specific, convenient and accuracy for the determination of FDP.
Assuntos
Colorimetria/métodos , Frutosedifosfatos/análise , Frutose-Bifosfato Aldolase/farmacologia , Hidrazinas/farmacologiaRESUMO
A low-affinity fluorescent Ca2+ indicator Indo-1FF was used to measure cytoplasmic Ca2+ increments in single smooth muscle cells isolated from the urinary bladder of the guinea-pig. The in vitro Kd of Indo-1FF for Ca2+ measured at the microscope stage was 21 microM. Calibration parameters measured in the cell differed substantially from respective in vitro values suggesting that the properties of the cytoplasmic dye had been altered. Addition of proteins (aldolase or albumin) increased the in vitro F405/F495 ratio close to the range observed intracellularly. Emission spectra of Ca(2+)-free Indo-1FF demonstrated a blue-shift of 29 nm with 10 mg/ml aldolase and 60 nm with 10 mg/ml albumin. The Kd value of Indo-1FF for Ca2+ in vitro was not changed by addition of aldolase (up to 20 mg/ml) and was approximately doubled in the presence of 20 mg/ml albumin. Intracellular calibration either by skinning the cells with beta-escine, 'opening' the cell or by intracellular perfusion of 100 microM free Ca2+ (40 mM DPTA-Ca2+ buffer) suggest that the affinity of intracellular Indo-1FF for Ca2+ is not markedly changed. The Indo-1FF concentration of 20 microM in the patch-pipette was found to be a reasonable compromise between acceptable signal-to-noise ratio and increased cytoplasmic Ca2+ buffering. This is because neither the amplitude nor the time-course of depolarization-induced micromolar Ca2+ increments were significantly changed during cell loading with this concentration of the dye. In contrast to Indo-1 loaded cells where rapid changes of [Ca2+]i were buffered, in Indo-1FF loaded cells ICa evoked rapid (rate of rise 150 microM/s) and large (4-6 microM in 35-60 ms) increments of free Ca2+. This results suggest that [Ca2+]i increments in smooth muscle cells are fast and large.
Assuntos
Cálcio/metabolismo , Corantes Fluorescentes/metabolismo , Indóis/metabolismo , Músculo Liso Vascular/metabolismo , Animais , Calibragem , Compartimento Celular , Citoplasma/metabolismo , Fluorometria , Frutose-Bifosfato Aldolase/farmacologia , Cobaias , Transporte de Íons , Cinética , Técnicas de Patch-Clamp , Albumina Sérica/farmacologia , Bexiga Urinária/citologiaRESUMO
The effect of calmodulin on the associative properties of D-glyceraldehyde-3-phosphate dehydrogenase was investigated by means of a covalently attached fluorescent probe. We found that calmodulin shifts the equilibrium between the different forms of glyceraldehyde-3-phosphate dehydrogenase and binds to the subunits with an apparent dissociation constant of 1.8 microM. Within this heterologous complex calmodulin has no effect on the catalytic activity of the enzyme. The formation of the heterocomplex can be modulated by the specific anti-calmodulin drug, trifluoperazine, as well as by aldolase. The possible role of these associations is that they influence the interaction of both glyceraldehyde-3-phosphate dehydrogenase and calmodulin with other soluble proteins or structural elements.
Assuntos
Calmodulina/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Animais , Encéfalo/metabolismo , Calmodulina/isolamento & purificação , Calmodulina/farmacologia , Bovinos , Fluoresceína-5-Isotiocianato , Polarização de Fluorescência , Corantes Fluorescentes , Frutose-Bifosfato Aldolase/metabolismo , Frutose-Bifosfato Aldolase/farmacologia , Gliceraldeído-3-Fosfato Desidrogenases/isolamento & purificação , Cinética , Músculo Esquelético/enzimologia , Coelhos , Trifluoperazina/farmacologiaRESUMO
Previous reports showed that the presence of proteins shifts the apparent dissociation constant (Kd) of a fluorescent dye indicator to Ca2+. To elucidate the sensitivity of Kd of an NMR-sensitive Ca2+ indicator, 5-fluoro-1,2-bis(2-amino-phenoxy)ethane N,N,N'N'-tetraacetic acid (5F-BAPTA) to proteins, and compare with that of a dye indicator, Fura-2, we measured Kd of Fura-2 or 5F-BAPTA using Ca-EGTA buffer with or without proteins. Aldolase (ALD) or bovine cardiac protein (BCP) extracted from bovine hearts was used at concentrations of 10, 25, or 50 mg/ml. ALD significantly increased the apparent Kd of Fura-2 to Ca2+ from 164.1 +/- 5.6 nM (mean +/- SE, N = 8) to 757.2 +/- 2.1 nM (n = 4, P < 0.05) at the concentration of 50 mg/ml. In contrast, Kd of 5F-BAPTA was not markedly changed by ALD (298.4 +/- 3. nM without ALD (n = 8), 385.1 +/- 2.7 nM (n = 4) with 50 mg/ml ALD). BCP (50 mg/ml) also significantly increased Kd of Fura-2 (928.5 +/- 3.3 nM, n = 4, P < 0.05), but did not change Kd of 5F-BAPTA (316.0 +/- 2.9 nM, n = 4). These results indicate that Kd of 5F-BAPTA is much less sensitive to the presence of proteins than Fura-2, and that 19F-NMR coupled with 5F-BAPTA is a more robust method to measure intracellular Ca2+ concentration than a fluorescent method with Fura-2.
Assuntos
Cálcio/análise , Frutose-Bifosfato Aldolase/farmacologia , Espectroscopia de Ressonância Magnética/métodos , Proteínas Musculares/farmacologia , Espectrometria de Fluorescência/métodos , Animais , Bovinos , Ácido Egtázico/análogos & derivados , Corantes Fluorescentes , Frutose-Bifosfato Aldolase/química , Fura-2 , Cinética , Peso Molecular , Proteínas Musculares/química , Miocárdio/química , CoelhosRESUMO
When assayed in vitro, the activity of the photosynthetic enzyme ribulose 1,5 bisphosphate carboxylase oxygenase is both enhanced and protected from spontaneous decay by exogenous proteins such as hemoglobin, serum albumin, and aldolase. Other proteins and amino acids tested are either ineffective (lysozyme, ferritin, lysine, and cysteine) or afford only partial protection (catalase, glycine, and phenylalanine). Protective proteins do not bind to, or exchange disulfides with, ribulose 1.5 bisphosphate carboxylase/oxygenase. Since their effect can be mimicked by reductively treated detergents such as Triton X-100, it appears that proteins protect from decay by quenching the spontaneous oxidative degradation and inhibiting surface adsorption which could lead to enzyme unfolding. Release of adsorbed molecules from the container surface is likely to be the cause of carboxylase activity enhancement.
Assuntos
Plantas/enzimologia , Proteínas/farmacologia , Ribulose-Bifosfato Carboxilase/metabolismo , Detergentes/farmacologia , Dissulfetos/metabolismo , Frutose-Bifosfato Aldolase/farmacologia , Hemoglobinas/farmacologia , Cinética , Octoxinol , Ovalbumina/farmacologia , Polietilenoglicóis/farmacologia , Albumina Sérica/farmacologia , Tireoglobulina/farmacologia , ÁrvoresRESUMO
The inhibition of rat hepatic glutathione (GSH) S-transferase 1-2 by bilirubin exhibited pseudo first-order kinetics with k(obs) values of 0.0214 +/- 0.0005 and 0.040 +/- 0.008 sec-1 at 4 and 8 microM bilirubin, when followed to 72 and 84% completion respectively. These correspond to calculated second-order rate constants of 5.3 +/- 0.1 x 10(3) and 5.0 +/- 1.0 x 10(3)/M.sec. The extent of inhibition of the transferase increased with bilirubin concentration, with half-maximal inhibition at 4 microM bilirubin. Inhibition was reversed by 10-fold dilution of bilirubin or by increasing the pH from 6.0 to 7.4. Premixing 0.2 to 0.5 microM albumin, hemoglobin or aldolase with bilirubin prevented inhibition of GSH S-transferase 1-2. Protection by these proteins occurred at a selected high concentration (0.2 to 0.4 microM) at which they reduced free bilirubin to concentrations (less than 0.5 microM) that did not inhibit isoenzyme 1-2 significantly. No protection was afforded by a selected low protein concentration (0.001 to 0.01 microM) which did not strikingly reduce bilirubin levels in solution. We conclude that bilirubin inhibition of GSH S-transferase 1-2 appears to be a second-order process; the reaction is clearly first-order with respect to GSH S-transferase and appears also to be first-order with respect to bilirubin. It is proposed that (a) inhibition of GSH S-transferase 1-2 results from slow, reversible bilirubin binding, and (b) added proteins appear to prevent GSH S-transferase inhibition by binding high molar ratios of bilirubin.
Assuntos
Bilirrubina/farmacologia , Glutationa Transferase/antagonistas & inibidores , Fígado/enzimologia , Albuminas/farmacologia , Animais , Bilirrubina/antagonistas & inibidores , Frutose-Bifosfato Aldolase/farmacologia , Hemoglobinas/farmacologia , Cinética , RatosRESUMO
Phosphofructokinase from the liver fluke, Fasciola hepatica, was phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase isolated from this organism. Phosphorylated fluke phosphofructokinase had a sevenfold lower apparent Km for its substrate, Fru-6-P, and an eightfold higher 0.5 Vopt for ATP, the enzyme's primary inhibitor, than native phosphofructokinase. Activation of fluke phosphofructokinase following phorphorylation by a mammalian protein kinase catalytic subunit was previously reported (E. S. Kamemoto and T. E. Mansour (1986) J. Biol. Chem. 261, 4346-4351). The catalytic subunit of protein kinase isolated from the liver fluke phosphorylated sites on fluke phosphofructokinase similar to those phosphorylated by the mammalian enzyme. Maximal phosphate incorporation was 0.3 mol P/mol of protomer. The native enzyme was found to contain 1.3 mol P/mol of protomer. In contrast to fluke phosphofructokinase, activity of the mammalian heart enzyme was slightly decreased following phosphorylation. The dependence of allosteric interaction on an acidic pH observed with the mammalian phosphofructokinase was not observed with the fluke enzyme. Unlike mammalian phosphofructokinase, allosteric kinetics of the fluke enzyme was observed at alkaline pH (8.0). Fluke phosphofructokinase was found to be relatively insensitive to inhibition by citrate, a known potent inhibitor of the mammalian enzyme. Fru-2,6-P2, a potent modifier of phosphofructokinase from a variety of sources, was found to activate both native and phosphorylated fluke phosphofructokinase. The most potent activators of fluke phosphofructokinase were found to be Fru-2,6-P2, AMP, and phosphorylation. The endogenous level of Fru-2,6-P2 in the flukes was determined to be 29 +/- 1.3 nmol/g wet wt, a level that may well modulate enzyme activity. Fru-6-P,2-kinase, the enzyme responsible for synthesis of Fru-2,6-P2, was found to be present in the flukes. Our results suggest physiological roles for phosphorylation and Fru-2,6-P2 in regulation of fluke phosphofructokinase.