Abstract
We report the purification and characterization of proteoliaisin, a protein that participates in the assembly of the sea urchin fertilization envelope. Proteoliaisin was purified from egg cortical granule exudate to greater than 99% homogeneity using chromatography on DEAE-Sepharose and on phenyl-Sepharose. Native proteoliaisin is a highly asymmetric protein (f/fo = 2.0) composed of a single Mr approximately 230,000 peptide. Its asymmetry was demonstrated both by analytical ultracentrifugation and by nondenaturing polyacrylamide gel electrophoresis, a novel analysis that detects molecular asymmetry in heterogeneous protein mixtures. Proteoliaisin is enriched in six amino acids: aspartic acid/asparagine, glutamic acid/glutamine, glycine, and cysteine, which account for over 50% of its mass. Nearly all of the cysteine residues are disulfide bonded. The protein contains a small proportion of aromatic amino acids with phenylalanine greater than tyrosine greater than tryptophan. At neutral pH its absorbance maximum is at 274.5 nm, with an extinction coefficient of 0.43 ml mg-1 cm-1. Proteoliaisin forms a 1:1 Ca2+-stabilized complex with ovoperoxidase, another component of the fertilization envelope, with Kd = 1.1 X 10(-6) M. Proteoliaisin, a constituent of the specialized echinoderm extracellular matrix called the fertilization envelope, has certain structural similarities to mammalian extracellular matrix proteins.
Highlights
Proteoliaisin, a protein that participates in the assembly of the sea urchin fertilization envelope
Proteoliaisin waspurified from egg cortical granule exudate to greater than 99%homogeneity using chromatographyon DEAE-Sepharose and on phenyl-Sepharose
Proteoliaisin is enriched in six amino acids: aspartic acidlasparagine, glutamic acidlglutamine, glycine, and cysteine, which account for over 50%of its mass
Summary
Prepared cortical granule exudate was concentrated by ammonium sulfate precipitation a t 10%saturation and solubilized in buffer A (see “Experimental Procedures”) containing 250 mM NaCl. After removing insoluble material by centrifugation at 17,900X g for 30 min, the concentrated cortical granule exudate was applied to a. 1.0 liter column of DEAE-Sepharose equilibrated with 250 mM NaCl in buffer A. The column was loaded and eluted at 10 “Cwith a linear, increasing NaCl gradient (250-500 mM, 3.0 liter). Total Total Specific PurifiDrotein activitv activitv cation mg units unitslrng -fold
Sign-in/Register to view highlights & summary 
Published Version
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
