. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 462 H. K. LEHMAN ET AL. merthiolate and 25 mM disodium EDTA; pH ) was used to dilute the antiserum, trace, and standards. The tubes were incubated at 4°C for about 16 hours, and dextran-coated charcoal solution was then added to each tube to separate the bound and free antigen (1 ml of g/1 charcoal and 250 mg/1 dextran in 10 mM phosphate buffer, pH ). Results When the extract of Aplysia gangUa was appUed to Sephadex G-15, ir-FMRFamide and ir-SCPfl were clearly separable (Fig. 1). Approximately nmoles of
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 462 H. K. LEHMAN ET AL. merthiolate and 25 mM disodium EDTA; pH ) was used to dilute the antiserum, trace, and standards. The tubes were incubated at 4°C for about 16 hours, and dextran-coated charcoal solution was then added to each tube to separate the bound and free antigen (1 ml of g/1 charcoal and 250 mg/1 dextran in 10 mM phosphate buffer, pH ). Results When the extract of Aplysia gangUa was appUed to Sephadex G-15, ir-FMRFamide and ir-SCPfl were clearly separable (Fig. 1). Approximately nmoles of ir- FMRFamide were recovered from fractions 20-23, whereas ir-SCPs eluted earUer; nmoles were recovered from fraction 15-18. Fraction 22 from Sephadex G-15 contained the majority of ir-FMRFamide, and this material was retained on the cation exchange column. A single peak of ir- FMRFamide ( nmoles) eluted from the ion exchanger in fractions 21, 22, and 23 (Fig. 2). Authentic FMRFamide behaves similarly on CM-Sephadex under these same experimental conditions (Price and Greenberg, 1977b; Greenberg and Price, 1979). On HPLC (^-Bondapak Cig column), purified ir-FMRFamide and synthetic peptide coelute at the same position in a methyl alcohol: trifluoroacetic acid gradient (30% methyl alcohol to 50% in 20 min); synthetic SCPb elutes later. Two amino acid analyses of the ion exchange purified peptide revealed the following compositions in nanomoles: Phe (.923; ); Arg (.461; .566); and Met (.401; .408). The ratios are as expected for authentic FMRFamide {, about 2:1:1). Other amino 6 c O CO C\J <u (J c o .a ^. o (/) <. c o u o » o e c u o 0) o c 3 E E Fraction Number Figure 1. Separation of FMRFamide and SCPb from Aplysia head ganglia by gel chromatography. An acetone extract of the head ganglia was passed through a X cm Sephadex G-15 column pre- equilibrated in A/acetic acid, and eluted with the same. Approximately 8 ml fractions (300 dro
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