1.Fluorogenic peptide-based substrates for monitoring thrombin activity.
van Berkel SS1, van der Lee B, van Delft FL, Wagenvoord R, Hemker HC, Rutjes FP. ChemMedChem. 2012 Apr;7(4):606-17. doi: 10.1002/cmdc.201100560. Epub 2012 Jan 31.
The synthesis of a series of peptides containing C-terminal 7-amino-4-methylcoumarin (AMC) for use in the thrombin generation test (TGT) is described. The lead structure in this project was H-Gly-Gly-Arg-AMC, of which the water solubility and kinetic parameters (K(M) and k(cat)) are greatly improved over those of the substrate in current use in the TGT: Cbz-Gly-Gly-Arg-AMC. A series of N-terminally substituted Gly-Gly-Arg-AMC derivatives were synthesized, as well as implementation of structural changes at either the P(2) or P(3) position of the peptide backbone. Furthermore, two substrates were synthesized that have structural similarities to the chromogenic thrombin substrate SQ68 or that contain a 1,2,3-triazole moiety in the peptide chain, mimicking an amide bond. To determine the applicability of newly synthesized fluorogenic substrates for monitoring continuous thrombin generation, the K(M) and k(cat) values of the conversion of these fluorogenic substrates by thrombin (FIIa) and factor Xa (FXa) were quantified.
2.The control of neutrophil chemotaxis by inhibitors of cathepsin G and chymotrypsin.
Lomas DA1, Stone SR, Llewellyn-Jones C, Keogan MT, Wang ZM, Rubin H, Carrell RW, Stockley RA. J Biol Chem. 1995 Oct 6;270(40):23437-43.
Neutrophil chemotaxis plays an important role in the inflammatory response and when excessive or persistent may augment tissue damage. The effects of inhibitors indicated the involvement of one or more serine proteinases in human neutrophil migration and shape change in response to a chemoattractant. Monospecific antibodies, chloromethylketone inhibitors, and reactive-site mutants of alpha 1-antitrypsin and alpha 1-antichymotrypsin were used to probe the specificity of the proteinases involved in chemotaxis. Antibodies specific for cathepsin G inhibited chemotaxis. Moreover, rapid inhibitors of cathepsin G and alpha-chymotrypsin suppressed neutrophil chemotaxis to the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and zymosan-activated serum in multiple blind well assays and to fMLP in migration assays under agarose. The concentrations of antichymotrypsin mutants that reduced chemotaxis by 50% would inactivate free cathepsin G with a half-life of 1.
3.Proteins of the Rpf (resuscitation promoting factor) family are peptidoglycan hydrolases.
Telkov MV1, Demina GR, Voloshin SA, Salina EG, Dudik TV, Stekhanova TN, Mukamolova GV, Kazaryan KA, Goncharenko AV, Young M, Kaprelyants AS. Biochemistry (Mosc). 2006 Apr;71(4):414-22.
The secreted Micrococcus luteus protein, Rpf, is required for successful resuscitation of dormant "non-culturable" M. luteus cells and for growth stimulation in poor media. The biochemical mechanism of Rpf action remained unknown. Theoretical predictions of Rpf domain architecture and organization, together with a recent NMR analysis of the protein structure, indicate that the conserved Rpf domain has a lysozyme-like fold. In the present study, we found that both the secreted native protein and the recombinant protein lyse crude preparations of M. luteus cell walls. They also hydrolyze 4-methylumbelliferyl-beta-D-N,N',N''-triacetylchitotrioside, a synthetic substrate for peptidoglycan muramidases, with optimum activity at pH 6. The Rpf protein also has weak proteolytic activity against N-CBZ-Gly-Gly-Arg-beta-naphthylamide, a substrate for trypsin-like enzymes. Rpf activity towards 4-methylumbelliferyl-beta-D-N,N',N''-triacetylchitotrioside was reduced when the glutamate residue at position 54, invariant for all Rpf family proteins and presumably involved in catalysis, was altered.
4.A sensitive enzymatic method (SK-013) for detection and quantification of specific periodontopathogens.
Ishihara K1, Naito Y, Kato T, Takazoe I, Okuda K, Eguchi T, Nakashima K, Matsuda N, Yamasaki K, Hasegawa K, et al. J Periodontal Res. 1992 Mar;27(2):81-5.
Porphyromonas gingivalis, Bacteroides forsythus, and Treponema denticola have been found to predominate in periodontal pockets of patients with adult periodontitis. These microorganisms hydrolyze the synthetic peptide N-benzoyl-DL-arginine-2-naphthylamide (BANA). In this study, we developed an enzymatic method, designated SK-013, to detect the existence of these microorganisms in subgingival plaque bacteria. This enzymatic method was based on the observation of the hydrolysis of N-carbobenzoxy-glycyl-glycyl-arginyl-3,5-dibromo-4-hydroxyaniline (N-CBz-Gly-Gly-Arg-DBHA) and made more sensitive by adding an enhancing system. The SK-013 was specifically positive for P. gingivalis, B. forsythus, T. denticola, and some strains of Capnocytophaga species, but was not specific for any of the other bacterial strains tested. This SK-013 system may be valuable for detection and quantification of periodontal disease-associated bacteria in subgingival plaque and thus for diagnosis of periodontal infections.