Tung‐Bin Lo

The primary structures of growth hormones of three cyprinid species: bighead carp, silver carp and grass carp

The primary structures of growth hormone (GH) of three cyprinid species, bighead carp, silver carp, and grass carp, were determined by a chemical method and/or by molecular cloning. The bighead carp GH was extracted from pituitary tissue by use of an alkaline medium, then successively purified by gel filtration, hydrophobic interaction column chromatography, and reverse-phase high-pressure liquid chromatography. The primary structure of bighead carp GH determined chemically is identical to that deduced from the cDNA nucleotide sequence. By molecular cloning, the primary structures of silver carp and grass carp GHs were also determined. The GHs of these three cyprinid species all contain 188 amino acid residues and their sequences are identical. When four of the five cysteines of cyprinid GHs were arranged to match the same positions of cysteines of other vertebrate GHs, a maximally matched alignment was achieved. Among fishes, GHs are relatively conserved within the same order (82 to 100% identity) but they are more diversified between orders (49 to 68% identity). In further comparison, fish GHs are even more different from tetrapod GHs (37 to 58% identity). Although the primary structures of vertebrate GHs are relatively variable, four homologous sequences, notably one located at the C-terminal, are found.

Purification, characterization, and molecular cloning of gonadotropin subunits of silver carp (Hypophthalmichthys molitrix).

The alpha and beta subunit of silver carp gonadotropin (scGTH-alpha and scGTH-beta) were isolated by high-performance liquid chromatography. Heterogeneity of N-terminal amino acid sequence was observed in scGTH-alpha but not in scGTH-beta. For determining the complete primary structures of scGTH-alpha and scGTH-beta, their cDNAs were cloned. Combining the data of N- and C-terminal sequences determined from proteins and the amino acid sequences deduced from cDNAs, we infer that scGTH-alpha consists of 95 and/or 93 residues and scGTH-beta consists of 115 residues. Both scGTH-alpha and scGTH-beta are glycoprotein. Their carbohydrate content is about 20 g per 100 g protein. The molecular weights of scGTH-alpha and scGTH-beta were calculated to be 12,700 and 15,700 Da, respectively. The amino acid sequences of scGTH-alpha and scGTH-beta are very similar to those of the corresponding subunit of carp GTH, different in only 2 and 4 residues, respectively. In addition, a high extent of homology (70%) was also observed between the alpha subunits of fish and mammalian GTHs. In the case of beta subunit, homology among various species of fish (75 to 98%) is much higher than that between fish and mammal (40%). These data suggest that the alpha subunit is conserved while the beta subunit is diversified during the molecular evolution of vertebrate GTH.

Organization and nucleotide sequence of carp gonadotropin α subunit genes

We have used PCR to amplify and align the sequence of two genes encoding cGTHα. Both genes comprise four exons and three introns. The organization of cGTHα genes is very similar to that of mammalian GTHα genes. However, the cGTHα genes only span a region of 1.2 kb which is much smaller than those mammalian GTHα genes.

Molecular cloning of silver carp and bighead carp prolactin

The cDNAs encoding the prolactin of silver carp (scPRL) and bighead carp (bcPRL) have been cloned. Deduced from the nucleotide sequences, both scPRL and bcPRL are composed of 187 amino acid residues. Only one residue is different between scPRL and bcPRL. Homology analysis indicates that scPRL and bcPRL are highly homologous to carp PRL (97%), relatively conserved in relation to PRLs of salmon, trout, and tilapia (64-69%), and diversified from avian and mammalian PRL (30-35%). Similar to PRLs of other species of fish, scPRL and bcPRL lack the first 12 N-terminal residues of avian and mammalian PRLs.

STUDIES ON BUNGARUS FASCIATUS VENOM

The crude venom of Bungarus fasciatus was separated into thirteen fractions in a CM-cellulose column with ammonium acetate buffer by two-stage gradient elution at 5°C. The pharmacological activities and various enzymic activities of these fractions were investigated. Two fractions (Fractions V and VI) showed cardiotoxic activity and one fraction (Fraction VIII) exhibited neurotoxic action. Three cardiotoxic principles were isolated in pure forms and their pharmacological activities were different from cobra cardiotoxin. The amino acid composition of the three cardiotoxic principles were very similar and molecular weights were about 10,500. The isoelectric points determined by electrofocusing were 9.16, 9-32 and 9.70, respectively. The CD spectra of the three cardiotoxin-like components were almost equivalent and were entirely different from that of cobra cardiotoxin. The cardiotoxin-like components showed no cross reaction to purified antibody of cobra cardiotoxin. The sequence of N-terminal nine amino acid residues are exactly the same as that of cobra phospholipase A2 but shows no phospholipase A activity.