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Abstract
Chorion hardening is triggered by the contents of cortical alveoli that are released upon fertilization of medaka (Oryzias latipes) eggs. We purified the chorion hardening-inducing activity as a single protein from the exudate of cortical alveoli of medaka eggs. This activity was co-purified with proteolytic activity of the chorion protein ZI-1,2. Based on the amino acid sequence of purified protein, we cloned the cDNA of this protein from a medaka ovarian cDNA library. Sequence analyses revealed typical sequence features, a zinc-binding motif and a methionine turn motif, of the astacin metalloproteinase family. We termed this protein "alveolin." Alveolin has a molecular mass of 21.5 kDa deduced by the amino acid sequence and neutral optimal pH range. Alveolin hydrolyzes ZI-1,2. Alveolin activity was strongly inhibited by metal-chelating agents but not by various proteinase inhibitors. To our knowledge, this is the first description of the isolation and identification of the chorion hardening-inducing factor from cortical alveoli exudate of teleost eggs.
Highlights
In oviparous fishes, the egg is surrounded by the chorion, a single, thick extracellular envelope [1]
Heat treatment of the chorion prevented the hardening process from further cross-linking resulting in only 61– 62-kDa intermediate proteins on the gel (Fig. 1, lane 3). The exudate contained both activities as a chorion hardening-inducing factor and a ZI-1,2-specific proteinase
This is the first description of the isolation and identification of chorion hardening-inducing activity from the exudate of cortical alveoli of teleost eggs. It is a single protein with a molecular mass of 21.5 kDa and transforms the chorion into a hard and insoluble structure in vitro. As this protein was isolated from the exudate of cortical alveoli, we named this protein “alveolin.” Based on the amino acid sequences from purified alveolin, we cloned alveolin cDNA from a medaka ovarian cDNA library
Summary
Preparations of Egg Exudate and Chorions—Medaka (Oryzias latipes, orange-red type) were maintained in laboratory aquaria under controlled conditions [29]. Cloning of Alveolin, the Chorion Hardening Inducer of Medaka incubated in 25 l of medaka saline containing exudate or chromatography fractions for 2 h at 25 °C. Exudate (3.6 ml) from 3,000 naked eggs was mixed with one-ninth the volume of buffered solution (250 mM Tris-HCl, 0.1 M NaCl, pH 8.0) and applied to a Q-Sepharose Fast Flow anion exchange column (1 ml) equilibrated with buffer A solution. Active fractions were applied to a Superdex 75 column (3.2 ϫ 300 mm; Amersham Pharmacia Biotech) equilibrated with buffer B (25 mM Tris-HCl, 2 M NaCl, pH 8.0). Determination of N-terminal and Internal Amino Acid Sequences— Purified enzyme (2 g) was separated by SDS-PAGE, and protein bands were stained with 40% methanol and 1% acetic acid containing 0.5% (w/v) Coomassie Brilliant Blue R-250. The arrowheads on the right indicate intermediate proteins with molecular masses of 61– 62 kDa
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