A Putative Cytosolic Saxitoxin Binding Protein in Frog Myocardium
Doyle, Donald David
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https://hdl.handle.net/2142/67597
Description
Title
A Putative Cytosolic Saxitoxin Binding Protein in Frog Myocardium
Author(s)
Doyle, Donald David
Issue Date
1981
Department of Study
Physiology and Biophysics
Discipline
Biophysics
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biophysics, General
Language
eng
Abstract
Approximately 20% of the protein in sarcolemma vesicles prepared from the hearts of the North American frog Rana pipiens pipiens is solubilized by a 96 hour dialysis of the vesicles against a low ionic strength solution of ethylene-diaminetetraacetic acid (EDTA) at pH 8.5. All of the polypeptides of the vesicle proteins which are resolvable by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 7.5% acrylamide disc gels are found in the aqueous supernatant (F1 fraction) when the dialysate is centrifuged at 100,000 xg for 1.5 hours. The yield of the individual proteins in F1 as revealed by the stained polypeptide bands on the gels varies. The total enzymatic activity of alkaline phosphatase and (Na('+) + K('+))-ATPase (ouabain sensitive) measured in the vesicles is retained throughout the EDTA treatment. 15% and 7% of their vesicular activities are found in F1 respectively. Total binding of Saxitoxin (STX), a Na('+)-channel specific neurotoxin, is only 25% retained, 6% in F1. Total 5'-nucleotidase activity is only 5% retained, less than 1% in F1. Sugar containing proteins detectable by periodic acid-Schiff base (PAS) staining of non-detergent (buffer) gels are found in F1. Proteins in F1 are associated with an appreciable amount of phospholipid. The protein-phospholipid complexes range in size from about 50,000 to several million daltons when measured by non-detergent gel electrophoresis.
85-95% of the STX binding measured in an homogenate of frog heart is found not as expected in the membrane fraction but rather in the soluble fraction. In both the soluble and membrane fractions of the homogenate, STX binding is not competed for by tetrodotoxin (TTX). STX binding in the soluble fraction is associated with a protein-containing complex of Stokes radius 52 (ANGSTROM) as determined by gel filtration chromatography. STX binding in the soluble fraction is associated with protein which focuses isoelectrically in at least 3 separate bands in the pH range 6.5-7.5.
The presence of the STX binding component in the soluble fraction was not affected by inclusion of phenylmethylsulfonyl fluoride (PMSF), EDTA, dithiothreitol (DTT), iodoacetate, leupeptin, or pepstatin in isosmotic salt homogenate solutions as inhibitors of protease activity, nor by inclusion of EDTA as an inhibitor of phospholipase activity. The soluble STX binding component also appeared when a buffered isosmotic sucrose homogenizing solution containing EDTA was used.
In contrast to STX binding, 90% of the hydrolysis of 5'-AMP by 5'-nucleotidase and alkaline phosphatase measured in the homogenate is found in the membrane fraction as expected. The activity of 5'-nucleotidase upon 5'-AMP is distinguished from that of alkaline phosphatase by the inhibition of the former and not the latter by concanavalin A.
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