Photon Factory Activity Report 2004 #22 Part B (2005)
Biological Science

15A/2004G176

Lamellar structures of ternary mixtures of GM3/SM/cholesterol.
Sinzi MATUOKA
Sapporo Medical University, S.1 W.17, Chuo-ku, Sapporo, Hokkaido 060-8556, Japan
Introduction
It has been recognized that sphingolipid enrichedmicrodomains are present in cell plasma membranes. In
these microdomains sphingomyeline(SM) and cholesterolare enriched as well as glycosphingolipid. Thesemicrodomains has been observed also in the model
membrane consisting of a fluid phosphatidylcholine, SMand cholesterol. In this report, to investigate whether
cholesterol affects the structure and biological function in
the sphingolipid enriched microdomain, x-ray diffraction
measurements of ternary mixtures of GM3/SM/
cholesterol was carried out and the results were comparedwith those in binary mixtures of GM3/SM.

By the experiment of precipitation of unilamellar
vesicles (diameter 100 nm) by anti-GM3 antibody(M2590), the threshold for the reactivity of M2590 in
GM3/SM/cholesterol system was 6 mol%. This
corresponds to the GM3 content where lamellar repeat
distance was saturated as shown in Fig.1 and is similar tothat in SM/GM3 system. Thus, the formation of thestructure recognized by the antibody was not affected by
the presence of cholesterol.

Materials and Methods
Ganglioside GM3(bovine brain) was purchased from
Alexis Corp.(San Diego, USA). Sphingomyeline(SM)and cholesterol were purchased from Sigma Chemical(St.Louis,MO,USA). To prepare multibilayer vesicles,
mixtures of GM3, SM and cholesterol (27mol%)dissolved in chloroform-methanol were dried undervacuum and then were hydrated with phosphate buffer at55oC that is above the main transition temperature.
X-ray diffraction measurements were carried out at BL15A. The diffraction patterns were detected by imaging
plates (Type BAS-III, Fuji Photo Film Co., Ltd., Japan).

Fig. 1 Lamellar repeat distance of ternary mixtures of
GM3/SM/cholesterol (•) and binary mixtures of
GM3/SM(×) as a function of GM3(bovine brain)in the L α !phase. Line represents smoothed splinewith binary mixtures of GM3/SM.

Results
Fig. 1 displays spacings due to the lamellar repeat
distance as a function of GM3 in ternary mixtures of
GM3/SM/cholesterol and in binary mixtures of GM3/SMin the Lα phase. From 2 to 6 mol% of GM3 content
lamellar repeat distance rapidly increased and above 6mol% gradually increased. The dependence of lamellar
repeat distance on GM3 content was not affected by thepresence of cholesterol.
Fig. 2 displays x-ray diffraction profiles of ternary
mixtures of GM3/SM/cholesterol in the Lα phase. Thereobserved the 1st-order, the 2nd-order and the 3rd-order(above 6 mol%GM3) diffraction peaks due to the lamellarstructure. At 4 mol% GM3, there observed a shoulder inthe x-ray diffraction profile suggesting two distinct
lamellar structures with different lamellar repeat distance.
It is possible that phase separation of collective GM3structures takes place. In GM3/SM system two distinct
lamellar structures were not observed , however, the peakwidth of x-ray diffraction profile was large at 4 - 5 mol%
GM3 content. This may suggest that the collective GM3
structures forms also in GM3/SM system.

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Users' Report

Fig. 2

X-ray diffraction profiles of multibilayer vesicles
of ternary mixtures of SM/cholesterol containing
different molar ratios of GM3(bovine brain) in
the L α phase(48oC).