Synthesis of the segment (11-23) located in the first tandem repeat of plasma kallikrein: comparative binding studies of this and another segment (328-343) to high-molecular-mass kininogen.
The synthesis of porcine plasma kallikrein (pPK) segment (11-23), of sequence Phe-Phe-Arg-Gly-Gly-Asp-Val-Ser-Ala-Met-Tyr-Thr-Pro, present in the first tandem repeat sequence of the regulatory chain of PK, has been accomplished following the peptide fragments (5 + 4 + 4) condensation strategy in solution, as well as by fluorenylmethoxycarbonyl solid-phase chemistry. This and another synthetic PK segment of residues (328-343) present in the fourth tandem repeat sequence [Cys(ACM)-Ser-Leu-Arg-Leu-Ser-Thr-Asp-Gly-Ser-Pro-Thr-Arg-Ile-Thr-Tyr] and synthesized by a solid-phase method, were fully characterized by 1H nuclear magnetic resonance, fast atom bombardment mass spectrometry, amino acid composition and reversed-phase high-performance liquid chromatography. Proteolysis of these peptides by either rat PK (rPK) or trypsin resulted in cleavages between Arg decreases Gly for pPK (11-23) and between Arg decreases Leu and Arg decreases Ile for rPK (328-343). Kinetic studies revealed that for peptide pPK (11-23), the catalytic efficiency (kcat/Km) of rPK is congruent to 9-fold higher than that of trypsin, but for the other peptide, rPK (328-343), kcat/Km of trypsin is congruent to 49-fold higher than that of rPK. The facile cleavage of pPK (11-23) by rPK confirms the Arg13 decreases Gly14 position as the site of autolytic degradation of PK and also explains its special preference for Phe-Phe-Arg sequence.
Basak
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J.A. de Sève Laboratory of Molecular Neuroendocrinology, Clinical Research Institute of Montreal, Quebec, Canada.
Yuan
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Harris
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Seidah
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Chrétien
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D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006868:Hydrolysis; D007610:Kallikreins / Q000138:chemical synthesis* / Q000378:metabolism*; D007700:Kinetics; D007704:Kininogens / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000378:metabolism*; D011485:Protein Binding; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013552:Swine
D000596:Amino Acids; D007704:Kininogens; D010446:Peptide Fragments; C082256:kallikrein (11-23); C082257:kallikrein (328-343); D007610:Kallikreins