title: Purification, characterization and activation of fish muscle prokallikrein.
abstract: Fish prokallikrein was isolated and characterized from skeletal muscle of the black sea bass, Centropristis striata. The prokallikrein was purified to apparent homogeneity by anion exchange perfusion chromatography and reversed phase high performance liquid chromatography. Initial identification was by its weak immunoreactivity with human tissue kallikrein antiserum. Two-dimensional gel electrophoresis and immunoblotting identified the protein as 36 kDa with a pI of 4.95-5.15. The prokallikrein was trypsin-activated to produce an approximately 36 kDa active enzyme as identified on an SDS-polyacrylamide gel overlayed with a membrane impregnated with the fluorogenic tripeptidyl substrate D-Val-Leu-Arg-7-amino-4-trifluoromethyl-coumarin. A potential dimer at 72 kDa was also enzymatically active. Bass kallikrein cleaved low molecular weight dog kininogen to release kinin peptide as determined by radioimmunoassay. The enzyme's amidolytic activity, with a pH optimum at 9.0, was inhibited by aprotinin, benzamidine, and phenylmethanesulphonyl fluoride, but not by elastatinal, soybean trypsin inhibitor, or limabean trypsin inhibitor. Polyclonal antiserum raised against the purified bass muscle prokallikrein recognized 36 kDa and 72 kDa proteins in bass heart, skeletal muscle, spleen, swimbladder, gill, and kidney by Western blot analyses. The wide distribution of immunoreactive proteins in the tissues suggests a potential physiological role for fish kallikreins in muscle contraction and/or relaxation, the regulation of local blood flow, and in osmoregulation. The detection of fish prokallikrein and its activation leads the way for an evaluation of the impact of kallikreins in fish health and disease processes and for studying the evolution of kallikreins and related serine proteinases.
authors.affiliation: U.S. Dept. of Commerce/NOAA, National Marine Fisheries Service, Charleston, SC 29412-9110, USA. gary.richards@noaa.gov
authors.forename: G P
authors.initials: GP
authors.lastname: Richards
authors.forename: Y M
authors.initials: YM
authors.lastname: Liang
authors.forename: J
authors.initials: J
authors.lastname: Chao
authors.forename: L
authors.initials: L
authors.lastname: Chao
mesh_terms: D000818:Animals; D001492:Bass / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D003429:Cross Reactions; D015180:Electrophoresis, Gel, Two-Dimensional; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007610:Kallikreins / Q000302:isolation & purification* / Q000378:metabolism; D008567:Membranes, Artificial; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D014018:Tissue Distribution
chemical_list: D004792:Enzyme Precursors; D008567:Membranes, Artificial; D011480:Protease Inhibitors; D007610:Kallikreins