Structure-function relations of biotin derivatives of apamin.
Apamin was biotinylated at various residues so as to produce an apamin derivative that was suitable for labelling apamin sensitive K+ channels. We labelled the sole histidine residue (His-18) of apamin with diazobenzoyl biocytin (DBB) and Lys-4 with NHS-biotin and NHS-XX-biotin. We found that at least two labelled species were produced by DBB. Proton NMR spectroscopy revealed that in addition to labelling the His-18, DBB labelled the Gln-16 and Gln-17 of apamin. Both NHS-biotin and NHS-XX-biotin appeared to specifically label Lys-4. To test the potency of these apamin derivatives, we developed an assay using apamin reversal of the adrenaline induced relaxation of the mouse ascending colon. The biological activity of the His-18 derivative was 46-fold less than that of native apamin. Biotinylation of Lys-4 with NHS-biotin reduced the activity by only 6-fold. The inclusion of a 14-carbon spacer between the Lys-4 and the biotin resulted in a derivative with only a 4-fold reduction in potency.
Shahidi
S
S
Department of Physiology, University of Sydney, NSW, Australia.
Poronnik
P
P
Barden
J A
JA
Cook
D I
DI
D000818:Animals; D001030:Apamin / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D001710:Biotin / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D003106:Colon / Q000187:drug effects / Q000502:physiology; D006639:Histidine / Q000737:chemistry; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D009119:Muscle Contraction / Q000187:drug effects; D015221:Potassium Channels; D017981:Receptors, Neurotransmitter / Q000378:metabolism; D013329:Structure-Activity Relationship
D015221:Potassium Channels; D017981:Receptors, Neurotransmitter; C033799:apamin receptor; D001030:Apamin; D006639:Histidine; D001710:Biotin