Author/Editor     Vučemilo, Nataša
Title     Identifikacija in karakterizacija visoko afinitetnih vezavnih proteinov za nevrotoksične PLA2 v prašičjem možganskem korteksu
Type     monografija
Place     Ljubljana
Publisher     Medicinska fakulteta
Publication year     1999
Volume     str. 70
Language     slo
Abstract     Ammodytoxins (Atx) are neurotoxic phospholipases A2 (PLA2) from the venom of the long-nosed viper ( vipera ammodytes ammodytes) which block the release of neurotransmitter into the synaptic cleft. Using radiolabeled ammodytoxin C (125/I-AtxC) specific binding sites were found in demielinized P2 fraction of porcine cerebral cortex. Their pH and temperature stability was assessed and the influence of mono and divalent ions on specific binding of 125/I-AtxC was determined. For binding of 125/I-AtxC to porcine brain membranes the presence of Ca2+, Sr2+ or Ba2+ as well as Na+ or K+ was essential. Under pH 7.4 the specific binding of 125/I-AtxC diminished rapidly and at pH 5 it was no longer detectable. The interaction between the 125/I-AtxC and its receptors reached maximum after 30-minute incubation at room temperature and remained constant for at least another 30 minutes. In cross-linking experiments on porcine membrane preparation using 125/I-AtxC the molecular weight of one of the receptors was determined to be 25 kDa (R25). After solubilization of the membranes another AtxC-receptor was detected at 180 kDa (RI 80). The binding parameters for R25 and R180 were: Kd25 =15 nM, Kd180 =145 nM and Bmax25 =1.5 pmol/mg m.p., Bmax180 = 0.4 pmol/mg m.p., respectively. The interaction of 125/I-AtxC with both receptors was prevented only in the presence of AtxA, AtxB or mannosilated bovine serum albumin (man-BSA). 0S2, taipoxin, a myotoxic PLA2 homologue ammodytin L and two non-toxic PLA2, porcine pancreatic PLA2 and ammodytin I2 bound only to R 180. The binding of man-BSA and enzymatically inactive AtnL suggests that PLA2 activity of the ligand is not necessary for the interaction with R180. (Abstract truncated at 2000 characters)
Descriptors     CEREBRAL CORTEX
PHOSPHOLIPASES A
VIPER VENOMS
BINDING SITES
HYDROGEN-ION CONCENTRATION
VIPERIDAE
TEMPERATURE
SYNAPTIC MEMBRANES
CALCIUM
BARIUM
POTASSIUM
SODIUM
MEMBRANE PROTEINS
AUTORADIOGRAPHY
ELECTROPHORESIS, POLYACRYLAMIDE GEL