Rat plasma kallikrein: purification, NH2-terminal sequencing and development of a specific radioimmunoassay.
Rat plasma kallikrein (rPK) was purified to homogeneity form plasma using affinity and high-performance liquid chromatography techniques, and subjected to NH2-terminal sequencing. The data showed that the sequenced segments of the regulatory (heavy) and catalytic (light) chains of the proteinase, respectively, display 73 and 91% sequence similarity with their counterpart in human plasma kallikrein. This sequence homology in conjunction with the determined molecular structure and inhibitor sensitivity support the identity of the isolated enzyme as plasma kallikrein. A polyclonal antiserum against rPK was obtained after immunization of rabbits with the purified enzyme, and a specific radioimmunoassay was developed. Since Tyr-iodinated rPK was not recognized by the antiserum, two alternative approaches were found to be successful. These included the use of a tracer consisting of rPK modified with either the affinity reagent 125I-labeled DTyr-Glu-Phe-Lys-Arg chloromethyl ketone or with the Bolton Hunter reagent. The usable range of the assay is between 15-150 fmol per tube. The antibody was shown to bind both monomeric and dimeric forms of rPK. Denaturation of the enzyme in sodium dodecyl sulfate does not abolish immune recognition only as long as the regulatory subunit is attached to the catalytic chain. Oxidation or reduction of rPK results in complete loss of immunoreactivity. This observation suggests that perhaps the disulfide linkage of the catalytic and regulatory polypeptides somehow helps to protect the antigenic epitope from denaturation. Alternatively, the epitope(s) recognized by the antibody spans a domain which includes both Tyr and Cys residues necessary for immune recognition.
Paquin
J
J
J.A. de Sève Laboratory of Biochemical, Neuroendocrinology, Clinical Research Institute of Montreal, Quebec, Canada.
Benjannet
S
S
Sawyer
N
N
Lazure
C
C
Chrétien
M
M
Seidah
N G
NG
D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D007610:Kallikreins / Q000037:antagonists & inhibitors / Q000097:blood* / Q000302:isolation & purification; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D013379:Substrate Specificity; D013552:Swine
D046911:Macromolecular Substances; D011480:Protease Inhibitors; D007610:Kallikreins