Activation of peptide prodrugs by hK2
a technology of peptide prodrugs and hk2, which is applied in the direction of peptides, enzymology, drug compositions, etc., can solve the problems of inability to effectively treat men with metastatic prostate cancer, and the prolonged administration of effective concentrations of standard chemotherapeutic agents is usually impossibl
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example 1
Production of Recombinant hK2
[0052]Recombinant hK2 was produced and purified as described in Lövgren et al., Biochem. Bioph. Res. Co., 238, 549-555 (1997). Semenogelin I and II were isolated from human semen as described previously in Malm et al., Eur. J. Biochem., 238, 48-53 (1996). The tripeptide aminomethylcoumarin (AMC) substrates Bos-Phe-Ser-Arg-AMC, Boc-Gln-Gly-Arg-AMC, H-Pro-Phe-Arg-AMC, boc-Val-Pro-Arg-AMC, H-D-Val-Leu-Lys-AMC, Tos-Gly-Pro-Arg-AMC-Tos-Gly-Pro-Lys-AMC, Z-Leu-Leu-Arg-AMC, Z-Val-Val-Arg-AMC, Z-Ala-Arg-Arg-AMC, and H-Arg-Gln-Arg-Arg-AMC were from Bachem (Bubendorf, Switzerland). The heptapeptide substrates Mu-Ala-Pro-Val-Leu-Ile-Leu-Ser-Arg-AMC and Mu-Val-Pro-Leu-Ile-Gln-Ser-Arg-AMC corresponding to the pro peptides of PSA hK2 were from Enzyme Systems Product (Livermore, Calif., USA). ACT was purified from human blood plasma as described in Christenssson et al., Eur. J. Biochem., 194, 755-63 (1990). PCI was provided by Prof. Johan Stenflo (Malmö University Hospi...
example 2
Determination of hK2 Cleavage Sites in Semenogelin I and II
[0053]Purified semenogelin I and II (40 μg), was incubated with hK2 (8 μg) in 50 mM Tris pH 7.5, 0.1 M NaCl, 0.15 M urea at 37° C. for 4 hours. The fragments generated were purified by reverse phase HPLC using a C-8 column. Elution was achieved with a 0-30% (0.25% / min.) linear acetonitrile gradient and fractions corresponding to individual peaks were collected. The amino terminal sequences of the individual peaks were determined by automated amino terminal sequencing with an Applied Biosystems 470 A gas-phase sequencer. Cleavage of either Sg I or Sg II with hK2 results in generation of a multitude of peptides. After partial separation of the peptides by reversed phase HPLC on a C-8 column we obtained sequences of four cleavage sites in Sg I and seven cleavage sites in Sg II. The semenogelins contain three types of internal repeats, as described in Lilja et al., J. Biol. Chem., 264, 1894-2000 (1989) and Lilja et al., PNAS USA...
example 3
pH Dependence of the Enzymatic Action of hK2
[0054]The pH dependence of hK2 was determined using a universal buffer composed of 29 mM citric acid, 29 mM citric acid, 29 mM KH2PO4, 29 mM boric acid 0.1 M NaCl and 0.2% BSA. The buffering range is Ph 2.4-11.8. The rate of the cleavage of the substrate I-1295 (100 μM) by 1.6 pmol hK2 was followed for 20 minutes at pH 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, and 10.
[0055]All experiments were done close to physiological pH, at pH 7.5, which is very close to the pH optimum of hK2.
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