Protease activities in serum and urine of 116 children with glomerulonephritis and 16 healthy children were tested using chromogenic peptide substrates Z-Dala_Leu-Arg-pNA and Glp-Ala-ALa-Leu-pNa. We found the dependence between activity of serine proteinases and clinical, morphological forms of primary glomerulonephritis and hypertension.
Latex of dandelion roots contains a serine proteinase that hydrolyzes a chromogenic peptide substrate Glp‐Ala‐Ala‐Leu‐pNA optimally at pH 8.0. Maximal activity of the proteinase in the roots is attained in April, at the beginning of plant development after the winter period. The protease was isolated by ammonium sulfate precipitation of the root extract followed by affinity chromatography on a Sepharose‐Ala‐Ala‐Leu‐mrp and gel filtration on Superose 6R performed in FPLC regime. Pure serine proteinase named taraxalisin was inactivated by specific inhibitors of serine proteinases, diisopropylfluorophosphate (DFP) and phenylmethylsulfonylfluoride (PMSF). Its molecular mass is 67 kDa and pI 4.5. pH stability range is 6–9 in the presence of 2 mM Ca2+, temperature optimum is at 40°C; K m=0.37±0.06 mM. The substrate specificity of taraxalisin towards synthetic peptides and insulin B‐chain is comparable with that of two other subtilisin‐like serine proteinases, cucumisin and macluralisin. The taraxalisin N‐terminal sequence traced for 15 residues revealed 40% coinciding residues when aligned with that of subtilisin Carlsberg.
New affine sorbents were synthesized involving tripeptide morpholides H-Ala-Ala-Leu-Mrp and H-D-Ala-Leu-Arg-Mrp as ligands that mimic substrates of subtilisin-like proteases and kallikrein, respectively. These were used for the isolation and purification of several proteases: trypsin, pepsin, alpha-chymotrypsin, thrombin, kallikrein, and termitase and were also efficient in the isolation of proteolytic enzymes from complex mixtures, such as the urine of children suffering from glomerulonephritis, hepatopancreas of Kamchatka crab, and dandelion roots. The ligands are competitive inhibitors of a number of proteases, and therefore, they were supposed to interact with the substrate binding sites in these enzymes.
A synthetic peptide derivative,—alanyl-alanyl-leucylmorpholide, (Ala-Ala-Leu-N(CH2CH2)2O), is suggested as a specific ligand for affinity chromatography of subtilisin-like serine proteases that prefer hydrophobic amino acid residue in P1 position of their substrates. Z-Ala-Ala-Leu-N(CH2CH2), a weak and reversible inhibitor of serine proteases, was synthesized by the carbodiimide method. Affinity sorbents were prepared by coupling the synthesized pepitide derivates to CH or AH Sepharose. Serine proteases from different sources were purified up to 17 fold on these sorbents with yields varying from 25% to 100%. Three enzymes (serine protease X, kallikrein and leucine aminopeptidase) were isolated from urine of children with glomerulonephritis with yields of 57, 22 and 55%. Proteolytic enzymes from the dandelion root, Kamchatka crab and culture filtrates of different microorganisms were also purified on the affinity sorbents.
New affine sorbents were synthesized involving tripeptide morpholides H-Ala-Ala-Leu-Mrp and H-D-Ala-Leu-Arg-Mrp as ligands that mimic substrates of subtilisin-like proteases and kallikrein, respectively. These were used for the isolation and purification of several proteases: trypsin, pepsin, alpha-chymotrypsin, thrombin, kallikrein, and termitase and were also efficient in the isolation of proteolytic enzymes from complex mixtures, such as the urine of children suffering from glomerulonephritis, hepatopancreas of Kamchatka crab, and dandelion roots. The ligands are competitive inhibitors of a number of proteases, and therefore, they were supposed to interact with the substrate binding sites in these enzymes.
