Membranotropic properties of latrotoxin-like protein: Studies on liposomes

Abstract

Fusion and changes in permeability ot' artificial pho~pholipid vesicles (liposomes) caused by the influence of a-latrotoxin-like protein (L protein) from the gray matter of the bovine brain were studied using a hydrophobic fluorescent probe, RI8. It was shown that L protein stimulates fusion of negatively charged lilx)somes. This effect becomes stronger In acidified media. The influence of L protein on the permeability of phtmphatidylcholine Ilpo~me membrane is also a pH-dependent process. INTRODUCTIONFusion of the membrane structures is one of the key steps in the process of neurosecretion. During recent years, many proteins and protein complexes involved in membrane fusion in the course of neurotransmitter release were identified [1 ]. However, the molecular mechanism of this phenomenon is still not clear.It is known [2 ] that a-latrotoxin, a natural toxin from Kara-kurt black widow spider venom, being joined to a high specific receptor on the cell membrane, causes massive efflux of a neurotransmitter from the cell. In vitro investigations [3] showed that a-latrotoxin is capable of causing fusion of synaptic vesicles with synaptosomal membranes.In the cytoplasm obtained from the cells of the bovine brain, a soluble protein (molecular mass 90 kdalton), possessing antigenic determinants similar to those of a-latrotoxin, was found earlier [4]. It was supposed that this isolated protein (henceforth called ct-latrotoxin-like protein, or L protein), is an intracellular agent involved in the process of neurosecretion, and a-latrotoxin can be considired its functional analog. In particular, some similar properties of L protein and t Bogomolets Institute of Physiology, National Academy of Sciences of Ukraine, Kyiv, Ukraine. 2 Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv, Ukraine.a-latrotoxin, which were studied in experiments with artificial phospholipid membranes, supported this supposition [5]. It turned out [6] that similarly to a-latrotoxin, L protein is spontaneously incorporated into the membrane and stimulates fusion of negatively charged liposomes. In addition, the degree of liposome fusion in the presence of L protein considerably depends on pH of the media, and changes with protein molecule modifications [7 ]. The results of model experiments can provide us with substantial information about the properties and mechanisms of L protein effects.Our paper presents the results of studies of L protein interaction with artificial phospholipid vesicles (liposomes). With the use of this model system, we showed that L protein possesses pronounced membranotropic properties, which are displayed in the changes of liposome permeability and fusion in the presence of the protein. METHODSL protein was isolated from the cytoplasm obtained from the gray matter of the bovine brain. The tissue was homogenized in the solution containing (mM): sucrose, 250; EDTA, 0.5; tris-HCl, 20 (pH 7.4); phenytmethylsulfonyi fluoride, 0.1. The homogenate was centrifuged for 60 min at 100,000g. NaCl was added t...