Flash-induced changes of light-absorption and of light-scattering of vertebrate rod outer segments (ROS) from frog and cattle in suspension were measured at 380 and 800 nm. The photometer used allows the observation of light intensity changes under well defined angles. We studied the successive decrease of the signal amplitude in series of flashes. One flash bleaches about 1% rhodopsin. The following results are discussed: 1. The signal at 380 nm is a superposition of the absorption change caused by formation of metarhodopsin II and of a biphasic additional signal. The latter exists only for the initial range of bleaching (15 to 25% rhodopsin). 2. At 800 nm three scattering signals are observed which are characterized by their successive amplitude decrease and time course: N: A small signal with time course and successive amplitude decrease comparable to the metarhodopsin II absorption change, probably arising from a structural change within the disc membrane. Ni: A slow signal, disappearing with the first flash, which may be understood as an outer membrane effect. P: A biphasic signal with a successive decrease rate, by a factor of 10 to 20 higher than that of the metarhodopsin II signal. The two kinetically different components are separated by variation of the observation angle. Two regions of different extension appear to change structurally with different time course. "P" may reflect an influence of the light-induced transmitter release on disc shape and/or mass.

Fluorescence probe data using 1,6-diphenyl-I ,3,5-hexatriene for various concentrations of intrinsic molecules (cholesterol, gramicidin A and cytochrome oxidase) within fluid lipid bilayers have been examined. The polarization value increases with increasing concentration of intrinsic molecule and then approaches a limiting value. Empirical curve-fitting of the experimental data, change of polarization with concentration, shows that each system can be fitted approximately by an exponential curve.A theory has been constructed based upon the assumption that only one intrinsic molecule need be adjacent to a fluorescent probe molecule to affect its motion drastically. The change in probe motion then depends upon the probability p of all positions next to a lipid chain being free of intrinsic molecules. The value of the probability p has been calculated and it is shown that p = (1 -x ) " where x = c or x = __ ~ depending on whether the intrinsic molecule spans the lipid bilayer or not. The approximation p = ePMx gives a good fit to the data for all x, thereby explaining the observed phenomenological fit.The fluorescent probe data is interpreted to show that protein-protein contacts increase as the intrinsic protein concentration increases within the lipid bilayer.An apparent dichotomy between the results from the fluorescence probe and from the deuterium magnetic resonance is explained in terms of a dominant affect on the probe being its hindrance to motion by interaction with the intrinsic molecule (protein) whilst individual C2Hz groups of the chain may exhibit greater disorder.The studies of protein-lipid interactions and cholesterollipid interactions are important for achieving a good basic understanding of biomembrane structure and function. The way in which the fluidity of the lipid bilayer matrix can be modulated by intrinsic molecules has been studied by variety of physical techniques including calorimetry and spectroscopic methods [l]. Some of these techniques involve the use of probe molecules and fluorescent probes have been developed for this purpose. Essentially, the probe molecule is considered to mimic a lipid molecular or lipid chain and then deductions are made concerning, for example, the change of lipid fluidity as the intrinsic protein (or cholesterol) concentration is increased. Some workers have interpreted probe data, particularly spin-probe studies of protein-lipid systems in terms of long-lived protein-lipid complexes where the lipid in the boundary region is rigid or ordered [2,3]. Recent deuterium magnetic resonance studies indicate that the intrinsic proteins (as distinct from cholesterol) disorder the lipid chains rather than increase their order and that rapid exchange of all lipids occurs [4,5]. There appears to be a dichotomy between the results obtained by some workers using spin-label data and the studies of deuterium magnetic resonance.In the present paper we discuss our studies of intrinsic molecules (cholesterol, gramicidin A and proteins) in fluid bilayers using the fluorescent p...

The rotational motion of the sarcoplasmic reticulum Ca2 -activated ATPase (ATP phosphohydrolase, EC 3.6.1.3) has been investigated by measuring the decay of laser flash-induced dichroism with the covalently attached triplet probe eosin isothiocyanate. The Arrhenius plot for rotational mobility indicates two discontinuities at 415'C and -350C. The experimental data are rationalized in terms of a sudden conformeric change in the ATPase at 15'C and a temperaturedependent equilibrium existing between the conformationally altered ATPase and oligomeric forms of it in the temperature range 15-350C. The enzymatic activity, as indicated by a discontinuity in the Arrhenius plot for the rate of ATP hydrolysis, appears to be sensitive only to the change at 15'C. There is a strong correlation between the activation energy below 15'C for rotational motion (33.6 + 2.2 kcal/mol) and enzymatic activity (34 ± 4 kcal/mol). The concept of biomembrane fluidity put forward by Chapman and coworkers (1) has been a fruitful one for emphasizing the

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