Purification, physicochemical characterization, and immunohistochemical localization of a major 11.7 S glycoprotein from the jelly coats of the anuran Lepidobatrachus laevis.
Embryos of the frog Lepidobatrachus laevis are encased by a fertilization envelope and two jelly layers, termed J1 (innermost) and J2 (outermost). From preparations of total jelly solubilized from cleavage-stage embryos by a solution of alkaline beta-mercaptoethanol we have purified one jelly coat glycoprotein to homogeneity via FPLC gel permeation chromatography on Superose 6H. The purified glycoprotein was 94% protein and 6% carbohydrate, had an s0(20),w of 11.7 S, with a molecular weight of 245,000 measured by sedimentation equilibrium and 263,000 by gel permeation chromatography. SDS-PAGE revealed that the glycoprotein is composed of a single subunit near 29,700 molecular weight; thus we propose that eight of these subunits comprise the native molecule. Amino acid analysis of the glycoprotein indicated a high content of Glx + Asx (32.4 mole%), a low content of basic amino acids (Arg + Lys = 12.2 mole%), and a single cysteine residue per subunit. The N-terminal amino acid was threonine and the sequence of the first twenty amino acids was determined. Monospecific antisera to the glycoprotein were prepared in rabbits and were used to immunohistochemically localize the glycoprotein throughout the matrix of both jelly layers. Antiserum against the glycoprotein had virtually no effect on the fertilizability of jellied eggs in vitro; thus we hypothesize that the glycoprotein fulfills a structural role in both jelly layers.
Carroll
E J
EJ
Department of Biology, University of California, Riverside 92521.
Wei
S H
SH
Nagel
G M
GM
D000595:Amino Acid Sequence; D000818:Animals; D001001:Anura / Q000196:embryology; D002462:Cell Membrane / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005306:Fertilization; D006023:Glycoproteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D008623:Mercaptoethanol / Q000494:pharmacology; D008969:Molecular Sequence Data; D010063:Ovum / Q000737:chemistry / Q000648:ultrastructure
D006023:Glycoproteins; D008623:Mercaptoethanol