2Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 119997 Moscow, Russia; fax: (495) 330-6538; E-mail: email@example.com
3Faculty of Chemistry, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (495) 939-3417; E-mail: firstname.lastname@example.org
* To whom correspondence should be addressed.
Received February 24, 2009; Revision received May 19, 2009
The relative location of proteins and lipids in particles of medicinal leech salivary gland secretion (SGS) is revealed for the first time. Their sizes and morphology are described. Using scanning electron microscopy and transmission electron microscopy, it was determined that SGS consists of particles of different sizes and form. This picture is supported by confocal laser scanning microscopy of SGS preparations treated with fluorescein isothiocyanate. After incubation with nonionic detergents (Brij 35 and Tween 20), transmission electron microscopy revealed the dissociation of fragments composing protein–lipid particles (PLP), and in this case an increase in free protein concentration determined by a modification of the Lowry method was observed. Perylene probing of lipids in SGS preparations showed that they are concentrated mainly inside PLP and are almost absent on the surface. Cholesterol was detected during SGS probing using the cholesteryl-Bodipy (hydrophobic fluorescent analog of cholesterol) on surface sections during confocal analysis of electron microphotographs of SGS. This analysis detected PLP structures in SGS resembling caveoles full of cholesterol. SGS, preliminary frozen at –70°C, transformed into a multitude of similar small particles visualized by transmission electron microscopy, whose fixed distribution resembled water crystal structure.
KEY WORDS: medicinal leech salivary gland secretion, scanning electron microscopy, transmission electron microscopy, confocal laser scanning microscopy, nonionic detergents, lipids, cholesterol