RESUMEN
The multistage purposeful synthesis of 5,15-bis(4'-l-N-tyrosinylamidophenyl)-10,20-bis(N-methylpyridin-3'-yl)porphine diiodide was carried out, and the optimum synthesis conditions were determined. 5,15-Bis(4'-nitrophenyl)-10,20-bis(pyridin-3'-yl)porphine served as the starting porphyrin. The structure, individual character, and purity of the target compound were proved by electron spectroscopy, 1H NMR spectroscopy, mass spectrometry (MALDI TOF), and TLC. Specific features of the interaction of the synthesized porphyrin with S-protein of SARS-CoV-2 were studied using spectral and thermochemical methods, including conditions of photoirradiation. The photoirradiation of the synthesized porphyrin in a complex with the SARS-CoV-2 S-protein can result in the partial oxidation of amino acid residues of the protein and distort its primary and secondary structures. The photoirradiation of the S-protein complex with the porphyrin decreases its thermal resistance to melting by 15 °C compared to the free S-protein and causes porphyrin release.
RESUMEN
The results of experimental studies of the interaction of the S-protein with a monohetaryl-substituted porphyrin containing a benzimidazole residue are presented. It has been revealed that the S-protein forms high-affinity complexes with the specified porphyrin. The porphyrin binding by the SARS-CoV-2 S-protein has proceeded stepwise; at the first stage, the driving force of the complexation is electrostatic interaction between the surface negatively charged regions of the protein and cationic substituents of the porphyrin. At the second stage, the target complex of the S-protein with the porphyrin is formed. It has been established that the introduction of 5-[4'-(N-methyl-1,3-benzimidazol-2-yl)phenyl]-10,15,20-tri-(N-methyl-3'-pyridyl)porphyrin triiodide into a solution of the S-protein complex with the angiotensin-converting enzyme leads to the replacement of the latter with the porphyrin. Displacement of the angiotensin-converting enzyme from the complex with the S-protein under the action of 5-[4'-(N-methyl-1,3-benzimidazol-2-yl)phenyl]-10,15,20-tri-(N-methyl-3'-pyridyl)porphyrin triiodide is the experimental evidence for the porphyrin binding at the receptor-binding domain of the S-protein.
RESUMEN
Novel porphyrin compounds containing benzothiazole, benzoxazole, and benzimidazole moieties have been prepared and their structures have been confirmed. Molecular docking of non-symmetric hetaryl-substituted porphyrins and chlorin e6 with SARS-CoV-2 helicase has been carried out. The affinity of hetaryl-substituted porphyrins to this protein has been found significantly higher than that of the drugs approved by the FDA and chlorin e6. The structure of the complexes of SARS-CoV-2 helicase with the considered macroheterocyclic compounds has been analyzed. Possible ways to inhibit and photoinactivate SARS-CoV helicase have been suggested basing on the localization of porphyrins and chlorin e6 in the helicase domains.
RESUMEN
Asymmetrically substituted 5,10,15,20-tetraphenylporphyrin derivatives such as 5-(4-aminophenyl)10,15,20-triphenylporphine, 5-[4-(tyrosinylamino)phenyl]-10,15,20-triphenylporphine and 5-{4-[(N-tert-butoxycarbonyltyrosinyl)amino]phenyl}-10,15,20-triphenylporphine were synthesized. Their spectral and acid-base properties were studied. The acid-base interactions of the obtained compounds in the binary acetonitrile-perchloric acid and dimethyl sulfoxide-potassium cryptate (KOH[222]) systems were studied by spectrophotometry method. The effect of the nature of the solvent, concentration, pH value on the chemical activity of porphyrin was analyzed. Amino acid fragments affect the protolytic equilibrium of porphyrins in acidic and basic media.
RESUMEN
In present work interactions of bovine serum albumin with 5,10,15,20-tetrakis(4-N-methylpyridil) tetra iodide porphyrin have been studied by electron absorption and fluorescence spectroscopy. The studies were carried out in aqueous media at different pH and in water-dimethylformamide mixtures containing up to 0.19 M of the organic solvent. It has been demonstrated that the porphyrin forms stable complexes with BSA in which the porphyrin is located subdomains IB and IIA. The stability constants of the complexes is practically independent of pH.
Asunto(s)
Yoduros/metabolismo , Porfirinas/metabolismo , Albúmina Sérica Bovina/metabolismo , Animales , Bovinos , Yoduros/química , Modelos Moleculares , Porfirinas/química , Estructura Terciaria de Proteína , Albúmina Sérica Bovina/química , Espectrometría de FluorescenciaRESUMEN
The immune-enzyme system of testing was developed to detect the level of Ca2+ ATPase of sarcoplasmic reticulum using monoclonal IgM and IgG antibodies to Ca2+ ATPase. The clinical approbation of the technique was carried out using the sample of 19 patients with acute cardiac infarction Q validated by the increase of content of such common markers of myocardium necrosis as troponin T and creatine phosphokinase MB. The increase of the level of Ca2+ ATPase of sarcoplasmic reticulum was detected in all patients. This occurrence is detected approximately in 4-6 hours after debut and it disappears in 144 hours (6 days). At the same time, Ca2+ ATPase was not detected in 10 Patients with acute traumatic damages of skeleton musculature, in 10 patients with chronic renal failure under hemodialysis and in 20 patients with acute coronary syndrome without rise of ST-segment of cardiogram. These facts testify rather high specificity and sensitivity of the developed technique of detection of the level of Ca2+ ATPase of sarcoplasmic reticulum in blood as biological marker of myocardium necrosis.
Asunto(s)
Infarto del Miocardio/diagnóstico , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/sangre , Adulto , Biomarcadores/sangre , Forma MB de la Creatina-Quinasa/sangre , Ensayo de Inmunoadsorción Enzimática , Humanos , Infarto del Miocardio/patología , Necrosis , Troponina/sangreRESUMEN
The novel metalloporphyrins (M=HH, Fe, Mn, Co, Cu, Zn) bearing 2,6-di-tert-butylphenol pendants as antioxidant substituents, and a long chain hydrocarbon palmitoyl group have been synthesized. The oxidation of compounds by PbO2 leads to the formation of the corresponding 2,6-di-tert-butylphenoxyl radicals studied by EPR. The activity of porphyrins in lipid peroxidation has been examined using (1) in vitro lipid peroxidation induced by tert-butylhydroperoxide in respiring rat liver mitochondria, (2) in vitro lipid peroxidation in liver homogenates of Wistar strain rats, and (3) a model process of peroxidation of (Z)-octadec-9-enic (oleic) acid as a structural fragment of lipids. The activity of these compounds depends dramatically on the nature of metal and might be changed from antioxidative (M=HH, Mn, Cu, Zn) to indifferent (M=Co), and to pro-oxidative one (M=Fe). The anti- or pro-oxidative action of these compounds may be derived from the concurrence between the involvement of 2,6-di-tert-butylphenol pendants acting as radical scavengers and redox active metal center promoting oxidation processes. The results of this study suggest that the polytopic compounds combining in one molecule 2,6-di-tert-butylphenol pendants, metalloporphyrin moiety, and a palmitoyl group, are membrane active compounds and might be studied in an effort to find novel pharmaceutical agents.
Asunto(s)
Antioxidantes/síntesis química , Antioxidantes/farmacología , Metaloporfirinas/síntesis química , Metaloporfirinas/farmacología , Ácidos Palmíticos/química , Fenoles/química , Animales , Cobalto/química , Cobre/química , Espectroscopía de Resonancia por Spin del Electrón , Hierro/química , Peroxidación de Lípido/efectos de los fármacos , Manganeso/química , Membranas Mitocondriales/efectos de los fármacos , Ácido Oléico/química , Ácidos Palmíticos/farmacología , Ratas , Ratas Wistar , Zinc/químicaRESUMEN
UNLABELLED: Previous study of the bleomycin-induced lung injury model suggested that (111)In-labeled antirat intercellular adhesion molecule-1 (aICAM-1) might be a useful acute respiratory distress syndrome (ARDS) diagnostic agent. We further investigated the ability of (111)In-aICAM-1 to detect inflammation in another ARDS lung injury model. METHODS: (111)In-labeled rat polymorphonuclear leukocytes (PMNs), (111)In-aICAM-1, (111)In-labeled normal mouse IgG (nmIgG), and (111)In-labeled rat serum albumin (RSA) were injected into rats 18-24 h before kill. Biodistributions, scintigraphic images, and lung ICAM-1 upregulation were obtained in uninjured rats and in rats after injury with oleic acid. RESULTS: (111)In-RSA and (111)In-nmIgG localized in inflamed lung at 5 min postinjury (PI). (111)In-PMN uptake increased significantly only at 24 h PI. (111)In-aICAM-1 localization increased significantly (30%-60%) at 1 h PI and remained elevated up to 24 h PI. Lung/blood ratios (L/B) at 1 and 4 h PI were very low (<0.6) for (111)In-nmIgG and (111)In-PMN rats; however, for (111)In-aICAM-1 rats, they were >1 and 25%-60% higher than those for the control samples. A low L/B suggests poor inflammation detection on the images. Images and region-of-interest analysis confirmed that only (111)In-aICAM-1 could distinguish inflamed lungs at 4 h PI. ICAM-1 was upregulated at 4 and 24 h PI. CONCLUSION: In this model, (111)In-aICAM-1 detected lung inflammation very early in the course of the disease. These results support the suggestion that (111)In-aICAM-1 could be a very early, highly specific ARDS diagnostic agent and may be useful to detect a wide range of inflammations.
Asunto(s)
Anticuerpos Monoclonales , Radioisótopos de Indio , Molécula 1 de Adhesión Intercelular/inmunología , Pulmón/diagnóstico por imagen , Ácido Oléico , Síndrome de Dificultad Respiratoria/diagnóstico por imagen , Animales , Anticuerpos Monoclonales/farmacocinética , Técnica del Anticuerpo Fluorescente , Inmunoglobulina G , Radioisótopos de Indio/farmacocinética , Pulmón/química , Ratones , Neutrófilos , Cintigrafía , Ratas , Ratas Endogámicas F344 , Síndrome de Dificultad Respiratoria/inducido químicamente , Albúmina Sérica , Distribución TisularRESUMEN
Kinases mediating phosphorylation and activation of cytosolic phospholipase A2 (cPLA2) in intact cells remain to be fully characterized. Platelet-activating factor stimulation of human neutrophils increases cPLA2 phosphorylation. This increase is inhibited by PD 98059, a mitogen-activated protein (MAP)/extracellular signal-regulating kinase (erk) 1 inhibitor, but not by SB 203580, a p38 MAP kinase inhibitor, indicating that this action is mediated through activation of the p42 MAP kinase (erk2). However, platelet-activating factor-induced arachidonic acid release is inhibited by both PD 98059 and SB 203580. Stimulation by TNF-alpha increases cPLA2 phosphorylation, which is inhibited by SB 203580, but not PD 98059, suggesting a role for p38 MAP kinase. LPS increases cPLA2 phosphorylation and arachidonic acid release. However, neither of these actions is inhibited by either PD 98059 or SB 203580. PMA increases cPLA2 phosphorylation. This action is inhibited by PD 98059 but not SB 203580. Finally, FMLP increases cPLA2 phosphorylation and arachidonic acid release. Interestingly, while the FMLP-induced phosphorylation of cPLA2 is not affected by the inhibitors of the p38 MAP kinase or erk cascades, both inhibitors significantly decrease arachidonic acid release stimulated by FMLP. SB 203580 or PD 98059 has no inhibitory effects on the activity of coenzyme A-independent transacylase.
Asunto(s)
Ácido Araquidónico/metabolismo , Citosol/enzimología , Proteínas Quinasas Activadas por Mitógenos , Neutrófilos/enzimología , Fosfolipasas A/metabolismo , Aciltransferasas/antagonistas & inhibidores , Aciltransferasas/metabolismo , Ácido Araquidónico/agonistas , Ácido Araquidónico/antagonistas & inhibidores , Ácido Araquidónico/sangre , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Flavonoides/farmacología , Humanos , Imidazoles/farmacología , Lipopolisacáridos/farmacología , Microsomas/enzimología , Proteína Quinasa 1 Activada por Mitógenos , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Fosfolipasas A/sangre , Fosfolipasas A2 , Fosforilación , Factor de Activación Plaquetaria/farmacología , Proteína Quinasa C/fisiología , Piridinas/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Tirosina/metabolismo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
UNLABELLED: We have investigated whether an (111)In-labeled mouse monoclonal antibody to rat intercellular adhesion molecule-1 ((111)In*aICAM-1) could detect lung injury early in rats treated with bleomycin. METHODS: Rats received an intravenous injection of either (111)In*aICAM-1 or (111)In-labeled normal mouse IgG ((111)In*nmIgG) and were imaged and killed 24 hr later. Lung injury was induced by an intratracheal injection of bleomycin 4 or 24 hr before the rats were killed. After death, tissue was removed and activity was measured, lungs were cryostat-sectioned to detect the presence of ICAM-1 by immunofluorescence, and the up-regulation of LFA-1alpha was examined on blood polymorphonuclear leukocytes (PMNs) using fluorescence-activated cell-sorter (FACS) analysis. RESULTS: In rats injected with (111)In*aICAM-1, the percent injected dose/organ in lungs both at 4 and 24 hr postbleomycin increased significantly compared to the values in either uninjured rats or rats that received (111)In*nmIgG. At 4 and 24 hr postinjury, the target-to-blood (T/B) ratio was 8/1 and 6/1, respectively. For (111)In*nmIgG, the T/B ratio at 4 hr was 0.5/1 and 0.4/1 at 24 hr. In (111)In*aICAM-1 rats injured at 4 or 24 hr, images could easily be distinguished from uninjured rats. All images of (111)In*nmIgG rats showed only cardiac blood-pool and liver activity with little lung activity. Lung ICAM-1 immunofluorescence intensity increased in the bleomycin-treated samples compared to uninjured lungs. Expression of LFA-1alpha on PMNs increased 19% and 210% at 4 hr and 24 hr postinjury, respectively, compared to control values. CONCLUSION: Biodistribution and imaging data demonstrate that (111)In*aICAM-1 can detect early acute bleomycin-induced lung injury. Immunofluorescence and FACS data suggest that (111)In*ICAM-1 uptake is a specific process. This antibody has potential as an early radionuclide detector of acute inflammations.
Asunto(s)
Anticuerpos Monoclonales , Radioisótopos de Indio , Molécula 1 de Adhesión Intercelular/inmunología , Enfermedades Pulmonares/diagnóstico por imagen , Radioinmunodetección , Animales , Anticuerpos Monoclonales/farmacocinética , Bleomicina , Citratos , Técnica del Anticuerpo Fluorescente , Galio , Inmunoglobulina G/inmunología , Radioisótopos de Indio/farmacocinética , Molécula 1 de Adhesión Intercelular/análisis , Pulmón/química , Pulmón/diagnóstico por imagen , Enfermedades Pulmonares/inducido químicamente , Enfermedades Pulmonares/metabolismo , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Masculino , Ratas , Ratas Endogámicas F344 , Distribución TisularAsunto(s)
Anexina A2/genética , Anexina A5/genética , Aorta Abdominal/fisiología , Cardiomegalia/metabolismo , Regulación de la Expresión Génica , Miocardio/metabolismo , Animales , Anexina A4/genética , Northern Blotting , Masculino , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Transcripción GenéticaAsunto(s)
Anexina A5/fisiología , Anticuerpos Monoclonales/farmacología , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Insuficiencia Cardíaca/enzimología , Miocardio/enzimología , Retículo Sarcoplasmático/enzimología , Anexina A5/inmunología , Fraccionamiento Celular , Humanos , Microsomas/enzimologíaRESUMEN
UNLABELLED: We examined the biodistribution in normal rats of an 111In-labeled mouse monoclonal antibody to rat intercellular adhesion molecule-1 (111In-alCAM-1), as a potential detector of inflammation. METHODS: Indium-111-alCAM-1 or 111In-labeled normal mouse polyclonal immunoglobulin G (111In-nmIgG) was injected into rats. Groups of three to four rats were killed up to 18 hr after injection, and activity was measured in various tissues. Rats were also imaged at 1 and 18 hr after injection. RESULTS: Uptake of 111In-alCAM-1 was greatest in the lung (approximately 10% injected dose [ID]/g at 15 min) and then declined steadily (to approximately 2% ID/g at 18 hr). Lung uptake of 111In-nmIgG was eightfold less than that for 111In-alCAM-1 and did not change throughout the 18 hr. At all time points, blood activity for 111In-alCAM-1 was only 30% to 40% of that for 111In-nmIgG, whereas the percent injected dose per gram was increased more than twofold in the major organs. Compared with 111In-nmIgG, the 111In of alCAM-1 was shifted from the blood and was distributed among the lung kidney, spleen and liver. CONCLUSION: Indium-111-alCAM-1 may be useful as an early inflammation detection agent. Intercellular adhesion molecule-1 upregulation is a very early event in inflammation and rapid removal from the blood of this antibody provides low background in contrast to the usual high background with whole antibodies.
Asunto(s)
Anticuerpos Monoclonales , Radioisótopos de Indio , Molécula 1 de Adhesión Intercelular/inmunología , Radioinmunodetección , Animales , Inmunoglobulina G , Pulmón/diagnóstico por imagen , Masculino , Ratones , Ratas , Ratas Endogámicas F344 , Factores de Tiempo , Distribución TisularRESUMEN
The oleic acid (OA) model of rat lung injury was originally developed as a model of fat embolism syndrome. A single intravenous dose of pure OA causes an acute diffuse lung injury, which, in its initial stages, histologically and physiologically resembles human ARDS. Rat lungs acutely injured by intravenous OA manifest increased levels of ICAM-1 within 30-60 min. This study shows that human umbilical vein endothelial cells (HUVEC) can be used in a bioassay to reveal some of the adhesion molecule stimulating activities present in bronchoalveolar lavage fluid (BALF) from post-OA-injected rats: (1) There is an as yet unidentified ICAM-1 and ELAM (E Selectin) inducing activity in BALF within 15 min of OA injection that is not TNF alpha; there is very little measurable tumor necrosis factor-alpha (TNF alpha) in 15 min BALF (BALF15). (2) BALF15 also stimulates cultured macrophage derived from BALF of normal rat lungs to produce TNF alpha. (3) By 60 min after OA injection, 50-75% of the ICAM-1 and ELAM inducing activity in BALF (BALF60) is TNF alpha; BALF60 contains about 250-280 pg/mL TNF alpha. The other 25-50% adhesion molecule-inducing activity in BALF60 is unidentified. (4) The ICAM-1-inducing activity of pure TNF alpha was equal to that of BALF60 containing equivalent concentrations of TNF alpha. The ELAM inducing activity of pure TNF alpha, however, was about 1/2 that of BALF60 containing equivalent concentrations of TNF alpha. The time courses for ICAM and ELAM stimulation with pure TNF alpha or BALF60 containing equivalent levels of TNF alpha were the same. The identity of the mediators in BALF15 and BALF60 that are not TNF alpha and the mechanisms by which OA injection stimulates cytokine production remain to be elucidated.
Asunto(s)
Líquido del Lavado Bronquioalveolar/química , Mediadores de Inflamación/análisis , Enfermedades Pulmonares/inducido químicamente , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Selectina E/biosíntesis , Inyecciones Intravenosas , Molécula 1 de Adhesión Intercelular/biosíntesis , Enfermedades Pulmonares/metabolismo , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/metabolismo , Masculino , Modelos Biológicos , Ácido Oléico , Ratas , Ratas Endogámicas F344 , Factores de Tiempo , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Levels of immunoreactive ICAM-1 in rat lung were followed during the kinetic development of acute oleic acid-induced lung injury in the rat by the ELISA assay. Significant increases in ICAM-1 immunoreactivity were found on rat lung membranes within 30 min of oleic acid injection. The increased immunoreactive ICAM-1 persisted for the duration of the study (4 h) and paralleled lung injury as measured by decreased lung compliance. Enhanced ICAM-1 immunofluorescence was also observed on cryostat sections of lungs from oleic acid-treated rats. No direct effect of oleic acid on ICAM-1 levels of cultured human umbilical vein endothelial cells or rat lung microvascular endothelial cells was observed. This suggests that either oleic acid raises rat lung ICAM-1 levels on endothelial cells by an indirect mechanism or that oleic acid increases ICAM-1 levels on other cell types, such as fibroblasts or lung epithelial cells, by direct or indirect mechanisms. Some of the increased ICAM-1 may also be due to the accumulation of ICAM-1 containing circulating leukocytes in the lung. The role of ICAM-1 in the pathophysiology of oleic acid-induced lung injury and the mechanism by which oleic acid increases ICAM-1 expression in the lung therefore remain to be defined by future experimentation.
Asunto(s)
Molécula 1 de Adhesión Intercelular/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Ácidos Oléicos/toxicidad , Animales , Anticuerpos Monoclonales , Membrana Celular/metabolismo , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunohistoquímica , Molécula 1 de Adhesión Intercelular/análisis , Molécula 1 de Adhesión Intercelular/inmunología , Cinética , Rendimiento Pulmonar/efectos de los fármacos , Lesión Pulmonar , Masculino , Ácido Oléico , Ratas , Ratas Endogámicas F344RESUMEN
Amputation of the extremity was performed in 26 of 52 patients with the IV stage obliterating atherosclerosis. Clinical severity of the disease was confronted with results of special investigations. Three groups of such patients are described: with traumatic necroses, with ischemic necroses and with necrotic ischemia (criteria of its irreversibility are presented). Surgical tactics of treatment in the first group of patients may include sympathectomy with necrectomy. The outcome of treatment of the second group patients is determined by the efficiency of reconstructive operations. Amputation of the extremity is indicated to patients of the third group.
Asunto(s)
Arteriosclerosis Obliterante/cirugía , Isquemia/cirugía , Pierna/irrigación sanguínea , Amputación Quirúrgica , Arteriosclerosis Obliterante/patología , Arteriosclerosis Obliterante/fisiopatología , Gangrena , Humanos , Isquemia/patología , Isquemia/fisiopatología , Región Lumbosacra , Necrosis , Flujo Sanguíneo Regional , Simpatectomía/métodos , Supervivencia Tisular/fisiologíaRESUMEN
An experimental investigation in 40 rabbits and 13 dogs has shown that arrest of blood flow in tissues is accompanied by activation of lipid peroxidation processes with reduced activity of the antioxidant system. Tissue anoxia during 4h sharply activates lipid peroxidation processes which is considerably aggravated after early performance of oxygenobarotherapy sessions (1h after revascularization). Relatively late sessions of oxygenobarotherapy (10h after the reestablishment of bloodflow) are not accompanied by activation of lipid peroxidation and decrease of the level of antioxidants of lipid nature, alpha-tocopherol acetate has a marked antioxidation effect and enhances tissue survival after anoxia.
Asunto(s)
Oxigenoterapia Hiperbárica , Hipoxia/metabolismo , Isquemia/metabolismo , Peroxidación de Lípido/fisiología , Músculos/irrigación sanguínea , Superóxido Dismutasa/deficiencia , Vitamina E/análogos & derivados , alfa-Tocoferol/análogos & derivados , Animales , Antioxidantes , Perros , Miembro Posterior/irrigación sanguínea , Hipoxia/etiología , Hipoxia/terapia , Isquemia/complicaciones , Peroxidación de Lípido/efectos de los fármacos , Músculos/metabolismo , Músculos/trasplante , Conejos , Tocoferoles , Vitamina E/administración & dosificaciónRESUMEN
A monoclonal antibody (mAb 4B4) was raised against purified sarcoplasmic reticulum vesicles from canine myocardium, and shown to inhibit Ca2+ uptake by microsomes isolated from cardiac, skeletal, and smooth muscle. The amount of mAb 4B4 needed to inhibit the Ca2+ uptake 50% at a given membrane concentration correlated with the amount of Ca2+ pump protein in the microsomal preparation. This is consistent with the observation the mAb 4B4 binds specifically to the sarcoplasmic/endoplasmic reticulum Ca2+ pump (Mr 100 kDa), but has no effect on the T-tubule Mg2+-ATPase. Changes in the binding of mAb 4B4 to crude microsomes isolated from dog heart after various durations of global ischemia showed that the decrease in microsomal Ca2+ transport during the first 15 min of ischemia correlated with a loss of active Ca2+ pump molecules. The monoclonal antibody mAb 4B4 may therefore serve as a specific marker for the sarcoplasmic/endoplasmic reticulum Ca2+ pump system in various cells, and can provide quantitative information about the loss of active Ca2+ pump proteins under pathological conditions.
Asunto(s)
Anticuerpos Monoclonales , Miocardio/inmunología , Retículo Sarcoplasmático/inmunología , Animales , Canales de Calcio/inmunología , Canales de Calcio/metabolismo , ATPasas Transportadoras de Calcio/inmunología , ATPasas Transportadoras de Calcio/metabolismo , Perros , Retículo Endoplásmico/inmunología , Retículo Endoplásmico/metabolismo , Miocardio/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMEN
Purified sarcoplasmic reticulum (SR) vesicles from dog heart were used as an antigen to produce monoclonal antibodies (mAbs) to the Ca2+-ATPase. Nine of twelve clones of hybridoma cells produce mAbs which cross-react with seven SR preparation isolated from cardiac and skeletal muscles of various species. Three mAbs of IgM type interact with the 45-kDa tryptic fragment of rabbit skeletal muscle Ca2+-ATPase and markedly inhibit Ca2+ uptake (by 95%) and ATPase activity (by 80%) and decrease (by 30-50%) the steady-state level of the Ca2+-ATPase phosphoenzyme. The ATPase activity could be completely blocked by one of these mAbs if the incubation medium was supplemented with 2 microM orthovanadate. On the other hand, when SR vesicles were treated with increasing concentrations of a nonionic detergent C12E8, the inhibiting effect of mAb 4B4 is diminished. It is concluded that the mAbs inhibit the Ca2+-ATPase only if the enzyme exists in an oligomeric form. The inhibition of the SR activities is due to an effect of the mAbs on the whole active center of the enzyme, rather than on a single partial reaction.
Asunto(s)
Anticuerpos Monoclonales , ATPasas Transportadoras de Calcio/inmunología , Miocardio/inmunología , Retículo Sarcoplasmático/inmunología , Animales , Calcio/metabolismo , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Reacciones Cruzadas , Perros , Canales Iónicos/inmunología , Canales Iónicos/metabolismo , Microsomas/inmunología , Microsomas/metabolismo , Miocardio/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMEN
One of monoclonal antibodies (mAbs) raised against purified dog heart sarcoplasmic reticulum (SR) efficiently decreases Ca2+-pump and Ca2+-ATPase activities of various SR preparations. The ATPase activity that is insensitive to the mAb (10-20% of the initial value) is present both in light and heavy fractions of rabbit skeletal muscle SR. The residual activity is completely blocked by 2 microM vanadate. The inhibition of the ATPase by the mAb is prevented in the presence of a nonionic detergent C12E8. It is concluded that the inhibiting effect of the mAb takes place when the Ca2+-ATPase exists in an oligomeric form. Another mAb does not affect SR functions and is specific only for Ca2+-ATPase from cardiac and slow muscle cells. Decrease in the Ca2+-pump activity of SR fractions from ischemic myocardium is accompanied by a diminished binding of both mAbs with the antigen. The mAbs described could be employed for differentiating endoplasmic reticulum and plasma-lemmal calcium pump systems, visualization of SR in the cells and estimating its amount in membrane preparations and tissue homogenates.