. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 190 G. C. CHIN ET AL. Table II Potencies of peplides al membrane-bound and sohibilized FMRFamide receptors Peptide IC50 (membrane) IC50 (solubilized) FMRFa FLRFa acFnLRFa YGGFMRFa FFRFa FYRFa YMRFa FVRFa FWRFa 10 FHRFa 55 FMHFa 85 100 FCRFa 120 25 HMRFa 200 50 FMKFa 7000 3000 Potencies of peptides (nM) in competing for [125I]-daYFnLRFa binding to intact and CHAPS-solubilized SQM (with and oA/ radio- ligand respectively) were determined
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 190 G. C. CHIN ET AL. Table II Potencies of peplides al membrane-bound and sohibilized FMRFamide receptors Peptide IC50 (membrane) IC50 (solubilized) FMRFa FLRFa acFnLRFa YGGFMRFa FFRFa FYRFa YMRFa FVRFa FWRFa 10 FHRFa 55 FMHFa 85 100 FCRFa 120 25 HMRFa 200 50 FMKFa 7000 3000 Potencies of peptides (nM) in competing for [125I]-daYFnLRFa binding to intact and CHAPS-solubilized SQM (with and oA/ radio- ligand respectively) were determined from displacement curves as de- scribed in the Methods. specific binding by each sample was calculated and com- pared to a standard curve of FMRFamide (IC50 = pmole). In the radioreceptor assay, duplicate ali- quots (25 n\) were combined with 20 /ug of SQM protein and 50,000 dpm ['25I]-daYFnLRFa ( n/V/) in 300 Ml of receptor binding buffer. Radioligand binding was de- termined after incubation for 1 h at 25°C; control-specific binding (5224 dpm) accounted for 82% of the total bind- ing. The inhibition of specific binding was calculated, compared to a standard curve of FMRFamide (IC50 = pmole), and the receptor-reactivity was plotted against elution time. The elution profiles of the FMRFamide immunoreactivty and receptor-reactivity were compared to the elution times of FMRFamide and FLRFa standards. Identification of a FMRFamide precursor fragment in squid genomic DNA A squid optic lobe was heated to 95°C for 10 min in M NaOH. T ru mixture was spun in an Eppendorf centrifuge at 1-4 m, and //I of the supernatant was used for PCR. ;-action mixture of 100 n\ con- tained 200 juA/dNTPs, 4 TTaq polymerase (Promega), and about 200 pmol of each primer: Sense primer: A C C GAI AAG CGI TTC TTG AGG TTC GG Anti-sense primer: A TC A T CAT GAA ICG TTT ICC GAA ICG CAT The reaction mixture was covered with mineral oil and then subjected to 40 cycles of PCR under
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Keywords: ., bookauthorlilliefrankrat, booksubjectbiology, booksubjectzoology
