Isolation, characterization, and cDNA sequencing of alpha-1-antiproteinase-like protein from rainbow trout seminal plasma.
Seminal plasma of teleost fish contains serine proteinase inhibitors related to those present in blood. These inhibitors can be bound to Q-Sepharose and sequentially eluted with a NaCl gradient. In the present study, using a two-step procedure, we purified (73-fold to homogeneity) and characterized the inhibitor eluted as the second fraction of antitrypsin activity (inhibitor II) from Q-Sepharose. The molecular weight of this inhibitor was estimated to be 56 kDa with an isoelectric point of 5.4. It effectively inhibited trypsin and chymotrypsin but was less effective against elastase. It formed SDS-stable complexes with cod and bovine trypsin. Inhibitor II appeared to be a glycoprotein. Carbohydrate content was determined to be 16%. N-terminal Edman sequencing allowed identification of the first 30 N-terminal amino acids HDGDHAGHTEDHHHHLHHIAGEAHPQHSHG and 25 amino acids within the reactive loop IMPMSLPDTIMLNRPFLLFILEDST. The N-terminal sequence did not match any known sequence, however, the sequence within the reactive loop was significantly similar to carp and mammalian alpha1-antiproteinases. Both sequences were used to construct primers and obtain a cDNA sequence from liver. The mRNA coding the protein is 1675 nt in length including a single open reading frame of 1281 nt that encodes 426 amino acid residues. Analysis of this sequence indicated the presence of putative conserved serpin domains and confirmed the similarity to carp alpha1-antiproteinase and mammalian alpha1-antiproteinase. Our results indicate that inhibitor II belongs to the serpin superfamily and is similar to alpha1-antiproteinase.
Mak
Monika
M
Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-747 Olsztyn, Poland.
Mak
Paweł
P
Olczak
Mariusz
M
Szalewicz
Agata
A
Glogowski
Jan
J
Dubin
Adam
A
Watorek
Wiesław
W
Ciereszko
Andrzej
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D018076:DNA, Complementary / Q000032:analysis; D006031:Glycosylation; D008969:Molecular Sequence Data; D017686:Oncorhynchus mykiss / Q000378:metabolism*; D012661:Semen / Q000201:enzymology*; D016415:Sequence Alignment; D017421:Sequence Analysis; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D018076:DNA, Complementary; D014361:Trypsin Inhibitors; D000515:alpha 1-Antitrypsin