RESUMEN
Cholesterol is known to affect the activity of membrane-bound enzymes, including Na(+)/K(+)-ATPase. To gain insight into the mechanism of cholesterol's effect, we have used various hydrophobic fluorescent probes which insert into different regions of the membrane bilayer and report on the degree of hydration of their environment. Specifially, we have measured the generalized polarization of Laurdan and the lifetime of DPH and derivatives of DPH inserted into membranes from pig kidneys enriched in Na(+)/K(+)-ATPase. Spectral measurements were also carried out on these membranes after modification of their cholesterol content. The generalized polarization of Laurdan increased with increasing cholesterol, showing an abrupt modification at the native cholesterol content. The fluorescence lifetimes of DPH and the DPH derivatives were analyzed using a distribution model. The center value of these lifetime distributions and their widths also changed with increasing cholesterol. One DPH derivative, DPH-PC, showed a minimum value for the lifetime center at the native cholesterol concentration, whereas the other derivatives showed a maximum value for the lifetime center at that cholesterol concentration. DPH-PC is known to sense the protein-lipid interface, whereas the other derivatives sense the bulk lipid phase. These data suggest that hydration at the protein-lipid interface is maximal at the native cholesterol concentration as is the enzymatic activity. Hydration at the protein-lipid interface is therefore proposed to be required for activity. These results are in agreement with current models of membrane dynamics and thermodynamics of protein function.
Asunto(s)
Colesterol/química , Riñón/enzimología , ATPasa Intercambiadora de Sodio-Potasio/química , Animales , Membrana Celular/enzimología , Colesterol/metabolismo , Difenilhexatrieno/química , Activación Enzimática , Polarización de Fluorescencia , Membrana Dobles de Lípidos/química , Liposomas/química , Lípidos de la Membrana/química , Modelos Químicos , Fosfatidilcolinas/química , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Espectrometría de Fluorescencia , Porcinos , Agua/químicaRESUMEN
Most interferogram demodulation techniques give the detected phase wrapped owing to the arctangent function involved in the final step of the demodulation process. To obtain a continuous detected phase, an unwrapping process must be performed. Here we propose a phase-unwrapping technique based on a regularized phase-tracking (RPT) system. Phase unwrapping is achieved in two steps. First, we obtain two phase-shifted fringe patterns from the demodulated wrapped phase (the sine and the cosine), then demodulate them by using the RPT technique. In the RPT technique the unwrapping process is achieved simultaneously with the demodulation process so that the final goal of unwrapping is therefore achieved. The RPT method for unwrapping the phase is compared with the technique of least-squares integration of wrapped phase differences to outline the substantial noise robustness of the RPT technique.
RESUMEN
The Hartmann test is a well-known technique for testing large telescope mirrors. The Hartmann technique samples the wave front under analysis by use of a screen of uniformly spaced array of holes located at the pupil plane. The traditional technique used to gather quantitative data requires the measurement of the centroid of these holes as imaged near the paraxial focus. The deviation from its unaberrated uniform position is proportional to the slope of the wave-front asphericity. The centroid estimation is normally done manually with the aid of a microscope or a densitometer; however, newer automatic fringe-processing techniques that use the synchronous detection technique or the Fourier phase-estimation method may also be used. Here we propose a new technique based on a regularized phase-tracking (RPT) system to detect the transverse aberration in Hartmanngrams in a direct way. That is, it takes the dotted pattern of the Hartmanngram as input, and as output the RPT system gives the unwrapped transverse ray aberration in just one step. Our RPT is compared with the synchronous and the Fourier methods, which may be regarded as its closest competitors.