RESUMEN
The reconstitution of influenza virus haemagglutinin into liposomes from lipid/protein/detergent mixtures by detergent removal provides vesicles that are similar in structure to viral particles. The dissociation properties of haemagglutinin aggregates and the molar ratio of lipid to protein in the starting mixture are the key factors for the individual and total yield of protein incorporation into liposomes. Structural properties of the detergent used as well as special reconstitution conditions are of minor importance for the formation of haemagglutinin liposomes. As determined by radial immunodiffusion-, haemolysis- and fusion experiments, specific properties of haemagglutinin were maintained to a large extent on liposomal incorporation, but its immunogenicity is increased, if the antigen is incorporated into the lipid bilayer of liposomes.
Asunto(s)
Hemaglutininas Virales/química , Liposomas/química , Animales , Detergentes , Hemaglutininas Virales/inmunología , Lípidos/química , Ratones , Ratones Endogámicos ICR , Orthomyxoviridae , Proteínas/químicaRESUMEN
In the haemagglutinin (HA) detergent mixtures coexist various protein complexes. Using 125I-HA tracer, gel chromatography, and centrifugation techniques we found protein aggregates comprising up to eight HA trimers. Formation of these structures seemed to be a function of the protein storage medium only. By contrast, special properties of the detergent as well as physicochemical conditions during the protein/detergent interaction had nearly no influence.
Asunto(s)
Detergentes/química , Hemaglutininas Virales/aislamiento & purificación , Orthomyxoviridae/química , Animales , Centrifugación por Gradiente de Densidad , Pollos , Cromatografía , Glicoproteínas Hemaglutininas del Virus de la Influenza , Hemaglutininas Virales/química , Hemaglutininas Virales/ultraestructura , Conformación ProteicaRESUMEN
The controlling effect of ATP, K+ and Na+ on the rate of (Na+ + K+)-ATPase inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1) is used for the mathematical modelling of the interaction of the effectors with the enzyme under equilibrium conditions. 1. Of a series of conceivable interaction models, designed without conceptual restrictions to describe the effector control of inactivation kinetics, only one fits the experimental data described in a preceding paper. 2. The model is characterized by the coexistence of two binding sites for ATP and the coexistence of two separate binding sites for K+ and Na+ on the enzyme-ATP complex. On the basis of this model, the effector parameters fitting the experimental data most closely are estimated by means of nonlinear least-squares fits. 3. The apparent dissociation constants for ATP fo the enzyme-ATP complex or of the enzyme-(ATP)2 complex are computed to lie near 0.0024 mM and 0.34 mM, respectively, irrespective of whether K+ and Na+ were absent or K+ and K+ plus Na+, respectively, were present in the experiments. 4. The origin of the high and the low affinity site for binding of ATP to the (Na+ + K+)-ATPase molecule is traced back to the coexistence of two catalytic centres which, although primarily equivalent as to the reactivity of their thiol groups with NBD-C1, are induced into anticooperative communication by ATP binding and thus show an induced geometric asymmetry. 5. On the basis of the interaction model outlined under item 2 the apparent dissociation constant for K+ or Na+ in the (K+ + Na+)-liganded enzyme-ATP complex are computed to be 1.7 mM and 3.5 mM, respectively. 6. The conclusions concerning the coexistence of two primarily equivalent but anticooperatively interacting catalytic centres and the coexistence of two separate ionophoric centres for Na+ and K+ correspond to the appropriate basic postulates of the flip-flop concept of (Na+ + K+)-ATPase mechanism.
Asunto(s)
Adenosina Trifosfato/farmacología , Potasio/farmacología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sodio/farmacología , 4-Cloro-7-nitrobenzofurazano/farmacología , Sitios de Unión , Cinética , Matemática , Modelos Químicos , Unión ProteicaRESUMEN
The paper describes the interaction of ATP, Na+ and K+ with (NaK)-ATPase exploiting the inactivation by reaction with NBD-chloride as an analytical tool for the evaluation of enzyme ligandation with the various effectors. 1. The inactivation of (NaK)-ATPase by reaction with NBD-chloride showing under all conditions studied a pseudo first-order rate rests on the alkylation of thiol groups in or near catalytic centre. ATP bound to catalytic centre prevents from enzyme inactivation by NDD-chloride through protection of these thiol groups from alkylation. Na+ and K+ affect the reactivity of the thiol groups towards NBD-chloride either indirectly via influencing ATP binding or more directly via changing the conformation of catalytic centre. Proceeding from these interrelations, the interaction of the various effectors with the enzyme was analyzed. 2. The K'D-values of various nucleotides determined by our approach correspond to the values obtained by independent methods. As shown for the first time, two catalytic centres per enzyme molecule exist. They exhibit high or low affinity to both ATP and ADP apparently caused by anticooperative interaction of the half-units of the enzyme through intersubunit communication ("half-of-the-sites reactivity"). 3. In the absence of ATP, Na+ or K+ ligandation of (NaK)-ATPase produce opposite effects on the reactivity of the thiol groups of catalytic centres reflecting different changes of their conformation. This corresponds to the well-known antagonistic effect of Na+ and K+ on some partial reactions of (NaK)-ATPase. The Na+ and K+ concentrations required to change thiol reactivity are rather high, i.e. the ionophoric centres for both Na+ and K+ are not readily accessible for cation complexation in the absence of enzyme complexation with ATP. 4. Na+ being without effect on ATP binding to the enzyme also does not influence the inactivating reaction with NBD-chloride while K+ by decreasing ATP binding dramatically decreases the protective effect of ATP. The K+ affinity of the enzyme-ATP complex is by more than two orders of magnitude higher than that of free enzyme. Na+ ligandation of the K+-liganded enzyme-ATP complex reverses the effect of K+ ligandation and produces a protective effect which distinctly surpasses that of the complexation of free enzyme with ATP. Hence, the enzyme molecule carries simultaneously ionophoric centres for both Na+ and K+. 5. The findings that per enzyme molecule ionophoric centres for Na+ and K+, and two catalytic centres with anticooperative interaction coexist corroborate the corresponding basic predictions of the flip-flop concept of (NaK)-ATPase pump mechanism, and explain some peculiar kinetic features of transport and enzyme activities of (NaK)-ATPase.
Asunto(s)
4-Cloro-7-nitrobenzofurazano/farmacología , Adenosina Trifosfato/farmacología , Médula Renal/enzimología , Oxadiazoles/farmacología , Potasio/farmacología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sodio/farmacología , Animales , Cinética , Magnesio/farmacología , Ribonucleótidos , Espectrofotometría , PorcinosRESUMEN
The [3H]-ouabain-(NaK)-ATPase complex when treated in the cold with sodium dodecyl sulfate (SDS) dissociates into a larger and smaller peptide called alpha and beta, resp.. Analysis of the released peptides by SDS-polyacrylamide gel electrophoresis reveals that [3H]-ouabain co-migrates with the alpha-peptide only, being apparently identic with the ouabain receptor molecule. The percentage occupancy of the receptor peptide with [3H]-ouabain can be increased up to 90% evidencing the stabilization of the [3H]-ouabain-alpha-peptide complex by SDS-exposure and release from the oligomeric enzyme. A hypothetic explanation for the seemingly paradoxical stabilising effect of SDS on the complex is offered.