Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The present study was conducted to explore the extent of heterogeneity of tissue kallikrein in saliva using immunological analysis and to demonstrate that such heterogeneity resulted from secretory phenomena and not degradation secondary to secretion. Human mixed saliva was collected by paraffin-stimulation and centrifuged at 10,000 rpm for 10 minutes. Special collectors were used to obtain parotid and submandibular/sublingual saliva using citric acid stimulation. Western blot analysis of human mixed saliva demonstrated major immunoreactive species with molecular masses of < 20 KD, 45 KD, 60 KD, 90 KD and > 200 KD. The polyclonal antibody used for these blotting studies was monofunctional with respect to reaction with purified salivary tissue kallikrein. Only the < 29 KD and 45 KD species were active using an enzyme overlay technique. While a similar distribution of molecular weight material was observed in both parotid and submandibular saliva, the amount of immunoreactive material was markedly less in parotid secretion. In addition, the < 20 KD material was essentially absent in parotid saliva. Treatment of the saliva with thermolysin eliminated the immunoreactive band at 60 KD while treatment with various glycosidases also eliminated some heterogeneity. These results demonstrate considerable variation in tissue kallikrein expression in salivary gland secretions.
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PMID:Molecular diversity of tissue kallikrein in human saliva. 146 63

This report describes bacterial expression, isolation, and characterization of human tissue kallikrein recombinantly produced in Escherichia coli. Successful production of enzymatically active recombinant human kallikrein requires the following processes: expression, solubilization and refolding of prokallikrein, thermolysin activation, and chromatographic separation. All experimental data confirmed that bacterially derived human kallikrein is properly folded and exhibits expected biochemical functions. As confirmed by SDS-PAGE and reverse-phase HPLC, recombinant kallikrein is apparently pure and is devoid of reduced or other partially folded kallikrein forms. Recombinant kallikrein behaves as a monomeric molecule in solution and exhibits full enzymatic activity in hydrolyzing peptide substrates. The molecule can bind to aprotinin to form kallikrein-inhibitor complex at a 1:1 molar ratio. Peptide mapping analysis derived from pepsin digestion of recombinant kallikrein assigned five disulfide bonds which match those of porcine kallikrein predicted from X-ray structure. Peptides containing unpaired cysteines or mispaired disulfide bonds were not detected. Both properly folded prokallikrein and methionyl kallikrein, containing a propeptide and an initiator methionine at their N-termini, respectively, were also produced and isolated. These two molecules are structurally similar to recombinant kallikrein, but are not enzymatically active.
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PMID:Isolation and characterization of human tissue kallikrein produced in Escherichia coli: biochemical comparison to the enzymatically inactive prokallikrein and methionyl kallikrein. 881 65