alpha-chymotrypsin and succinyl-alanyl-alanyl-prolyl-phenylalanine-4-nitroanilide

The entrapment of α-chymotrypsin (α-CT) within 70-140 nm liposomes formed from POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) leads to an unexpected and remarkable increase in the thermal stability of the enzyme. This finding is based on the observation that heating aqueous suspensions of α-CT-containing POPC liposomes to 80 °C for 30 minutes resulted in partial enzyme inactivation, whereas the same treatment of aqueous solutions of free α-CT inactivated the enzyme completely. The stabilizing effect of enzyme confinement in the attoliter volumes of the liposomes was found to increase with decreasing numbers of α-CT molecules per liposome. Single-enzyme confinement was particularly effective, as intermolecular interactions between heat-denatured α-CT molecules (causing irreversible inactivation) are not possible.

Topics: Aniline Compounds; Animals; Ascomycota; Cattle; Chymotrypsin; Endopeptidase K; Heating; Oligopeptides; Particle Size; Phosphatidylcholines; Protein Stability; Unilamellar Liposomes

The stereochemical features of 2,8,14,20-tetrakis(D-leucyl-D-valinamido)resorc[4]arenecarboxylic acid and the N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine-4-nitroanilide polypeptide substrate were investigated by nuclear magnetic resonance spectroscopy. Proton selective relaxation parameters gave the basis for the inhibitory activity of resorcin[4]arene in the hydrolysis of the polypeptide substrate by α-chymotrypsin. Results showed that an interaction between the resorcin[4]arene and α-chymotrypsin does occur, and involves the hydrophobic moiety of the macrocycle. This interaction is further reinforced by polar groups located on the side chains of the resorcin[4]arene, whereas the macrocycle-polypeptide substrate interaction is negligible. Conformational analysis and interaction studies carried out by molecular modeling are in good agreement with the NMR data, thus providing an additional support to the rationalization of the inhibitory potential of resorcin[4]arenes on the α-chymotrypsin activity.

Topics: Animals; Chymotrypsin; Hydrolysis; Hydrophobic and Hydrophilic Interactions; Kinetics; Molecular Docking Simulation; Nuclear Magnetic Resonance, Biomolecular; Oligopeptides; Phenylalanine; Protease Inhibitors; Protons; Solutions; Static Electricity; Stereoisomerism; Swine; Valine

A model system, α-chymotrypsin (Cht) (a protease) and a cleavable peptide-chromogen (pro-drug) covalently incorporated into a hydrogel, was investigated to understand the mechanisms of covalent loading and release by enzymatic cleavage in bio-responsive delivery systems. Using EDC and Sulfo-NHS, terminal carboxyl groups of N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide, a cleavable chromogen, were conjugated to primary amines of a hydrated poly(HEMA)-based hydrogel. Hydrogel disks were incubated in buffered Cht causing enzyme-mediated cleavage of the peptide and concomitant release of the chromophore for monitoring. To investigate substrate loading and the effects of hydrogel morphology on the system, the concentration of the amino groups (5, 10, 20, and 30 mol%) and the cross-linked density (1, 5, 7, 9 and 12 mol%) were independently varied. Loading-Release Efficiency of the chromogen was shown to exhibit a positive relation to increasing amino groups (AEMA). The release rates demonstrated a negative relation to increasing cross-linked density attributed to decreasing void fractions and increasing tortuosities. The diffusion coefficient of Cht, D(0,Cht), was determined to be 6.9±0.5×10(-7)cm(2)s(-1), and the range of D(eff) of Cht for 1 to 12 mol% TEGDA was determined to be 6.9×10(-8) to 0.1×10(-8)cm(2)s(-1). We show how these parameters may be optimized and used to achieve programmed release rates in engineered bio-responsive systems.

Topics: Chromogenic Compounds; Chymotrypsin; Cross-Linking Reagents; Drug Carriers; Drug Delivery Systems; Hydrogels; Methacrylates; Models, Chemical; Molecular Structure; Oligopeptides; Substrate Specificity

Certain oral treponemes express a highly proteolytic phenotype and have been associated with periodontal diseases. The periodontal pathogen Treponema denticola produces dentilisin, a serine protease of the subtilisin family. The two-gene operon prcA-prtP is required for expression of active dentilisin (PrtP), a putative lipoprotein attached to the treponeme's outer membrane or sheath. The purpose of this study was to examine the diversity and structure of treponemal subtilisin-like proteases in order to better understand their distribution and function. The complete sequences of five prcA-prtP operons were determined for Treponema lecithinolyticum, "Treponema vincentii," and two canine species. Partial operon sequences were obtained for T. socranskii subsp. 04 as well as 450- to 1,000-base fragments of prtP genes from four additional treponeme strains. Phylogenetic analysis demonstrated that the sequences fall into two paralogous families. The first family includes the sequence from T. denticola. Treponemes possessing this operon family express chymotrypsin-like protease activity and can cleave the substrate N-succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroanilide (SAAPFNA). Treponemes possessing the second paralog family do not possess chymotrypsin-like activity or cleave SAAPFNA. Despite examination of a range of protein and peptide substrates, the specificity of the second protease family remains unknown. Each of the fully sequenced prcA and prtP genes contains a 5' hydrophobic leader sequence with a treponeme lipobox. The two paralogous families of treponeme subtilisins represent a new subgroup within the subtilisin family of proteases and are the only subtilisin lipoprotein family. The present study demonstrated that the subtilisin paralogs comprising a two-gene operon are widely distributed among treponemes.

Topics: Amino Acid Sequence; Animals; Bacterial Proteins; Binding Sites; Chymotrypsin; Dogs; Humans; Lipoproteins; Molecular Sequence Data; Oligopeptides; Operon; Peptide Hydrolases; Phylogeny; RNA, Bacterial; RNA, Ribosomal, 16S; Sequence Alignment; Substrate Specificity; Subtilisins; Treponema; Virulence Factors

A potentially general kinetic method for the investigation of active-site availability in preparations of macromolecular catalysts was developed. Three kinetic models were considered: (a) the conventional two-step model of enzyme catalysis, where the preparation contains only active catalyst (E(a)) and inert (i.e. non-binding, non-catalytic) material (E(i)); (b) an extension of the conventional model (a) involving only E(a) and E(i), but with non-productive binding to E(a) (in addition to productive binding); (c) a model in which the preparation contains also binding but non-catalytic material (E(b)), predicted to be present in polyclonal catalytic antibody preparations. The method involves comparing the parameters V(max) and K(m) obtained under catalytic conditions where substrate concentrations greatly exceed catalyst concentration with those (klim/obs, the limiting value of the first-order rate constant, k(obs), at saturating concentrations of catalyst; and Kapp/m) for single-turnover kinetics, in which the reverse situation obtains. The active-site contents of systems that adhere to model (a) or extensions that also lack E(b), such as the non-productive binding model (b), may be calculated using [E(a)](T)=V(max)/klim/obs. This was validated by showing that, for alpha-chymotrypsin, identical values of [E(a)](T) were obtained by the kinetic method using Suc-Ala-Ala-Pro-Phe-4-nitroanilide as substrate and the well-known 'all-or-none' spectroscopic assay using N-trans-cinnamoylimidazole as titrant. For systems that contain E(b), such as polyclonal catalytic antibody preparations, V(max)/klim/obs is more complex, but provides an upper limit to [E(a)](T). Use of the kinetic method to investigate PCA 271-22, a polyclonal catalytic antibody preparation obtained from the antiserum of sheep 271 in week 22 of the immunization protocol, established that [E(a)](T) is less than approx. 8% of [IgG], and probably less than approx. 1% of [IgG].

Topics: Animals; Antibodies, Catalytic; Binding Sites; Catalysis; Chymotrypsin; Haptens; Imidazoles; Immune Sera; Immunoglobulin G; Kinetics; Macromolecular Substances; Mathematics; Models, Chemical; Oligopeptides; Reproducibility of Results; Sheep; Thermodynamics; Titrimetry

Potato chymotrypsin inhibitor-1 (pCTI-1) was biotinylated by reaction with sulfosuccinimidyl-6-(biotinamido)hexanoate. This derivative was used as a probe on Western blots for the detection and quantitation of chymotrypsin and the detection of a chymotrypsin-like serine proteinase synthesized by ovine chondrocytes in alginate bead culture. Densitometric analysis demonstrated that there was a linear relationship between the amount of chymotrypsin electrophoresed, over the range 0.1 to 10 ng, and the intensity of the band detected on Western blots using biotinylated pCTI-1 as probe, indicating that the technique could be used for the quantification of active proteinases. The biotinylated pCTI-1 detection technique was convenient to use, reproducible, and more sensitive than zymography.

Topics: Animals; Biotin; Blotting, Western; Cartilage, Articular; Cattle; Cells, Cultured; Chromatography, Gel; Chromogenic Compounds; Chymotrypsin; Oligopeptides; Plant Proteins; Protease Inhibitors; Serine Endopeptidases; Sheep

Heparin accelerates the inhibition of neutrophil elastase by mucus proteinase inhibitor (MPI), the physiological antielastase of airways as a result of its binding with the inhibitor [Faller, B., Mély, Y., Gérard, D., & Bieth, J. G. (1992) Biochemistry 31, 8285-8290]. To explore the heparin-binding site of the inhibitor, we have modified the lysine and arginine residues of MPI and its isolated C-terminal domain by using 4-N,N-(dimethylamino)azobenzene-4'-isothiocyano-2'-sulfonic acid (S-DABITC) [Chang, J. Y. (1989) J. Biol. Chem. 264, 3111-3115] and (p-hydroxyphenyl)glyoxal (HPG) (Yamasaki, R. B., Vega, A., & Feeney, R. E. (1980) Anal. Biochem. 109, 32-40], respectively. The derivatizations were done in the absence and presence of a 4.5 kDa heparin fraction with a low degree of polydispersity. The effect of chemical modification of the inhibitors on their affinity for heparin was tested using two complementary procedures, one based on the ability of heparin to accelerate the inhibition of chymotrypsin by the inhibitors and the other exploiting the affinity of the inhibitors for immobilized heparin. Modification of a limited number of lysine and arginine residues in full-length MPI led to a 6-fold decrease in affinity for heparin. The presence of the polymer during the modification reactions significantly prevented this effect. Amino acid sequencing unambiguously identified the heparin-protected lysines as Lys 13 and Lys 87, located on the N-terminal and C-terminal domains of MPI, respectively. Heparin apparently protects mainly two arginine residues from modification by HPG.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Binding Sites; Chymotrypsin; Heparin; Humans; In Vitro Techniques; Isothiocyanates; Kinetics; Models, Molecular; Molecular Sequence Data; Oligopeptides; p-Dimethylaminoazobenzene; Peptide Mapping; Proteinase Inhibitory Proteins, Secretory; Proteins; Recombinant Proteins; Serine Proteinase Inhibitors; Substrate Specificity

Topics: Adult; Animals; Bacteria; Benzoylarginine Nitroanilide; Case-Control Studies; Chymotrypsin; Culture Media; Dental Plaque; Dipeptides; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Endopeptidases; Female; Humans; Male; Middle Aged; Milk; Oligopeptides; Pancreatic Elastase; Periodontal Index; Periodontitis; Porphyromonas gingivalis; Prevotella intermedia; Substrate Specificity; Trypsin

The alpha-chymotrypsin-catalyzed hydrolysis of succinyl-L-alanyl-L-alanyl- L-prolyl-L-phenylalanyl p-nitroanilide has been studied in reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane. It has been found that alpha-chymotrypsin is strongly inhibited competitively by the acidic peptide product which is formed during the course of the reaction. It has also been shown that the application of the integrated form of the Michaelis-Menten equation can be useful to detect possible inhibition effects and abnormal kinetic behavior of enzymes in reverse micelles. Furthermore, it has been shown that the turnover number (kcat) at low water content is lower than in water and increases as the water content in the system is lower than in water and increases as the water content in the system (wo = [H2O]/[AOT]) increases, kcat reaching the value in water at high wo. If however, initial velocity data, as obtained under conditions where the enzyme is not saturated with substrate, are plotted against wo, the curves are bell-shaped, with a maximum around wo = 15.

Topics: Amino Acid Sequence; Catalysis; Chromatography, High Pressure Liquid; Chymotrypsin; Dioctyl Sulfosuccinic Acid; Hydrolysis; Micelles; Molecular Sequence Data; Oligopeptides; Surface-Active Agents

1. We have investigated the collagenolytic activity of the following serine proteases: proteinase K, subtilisin Novo, Staphylococcal endoproteinase Glu-C, Streptomyces pronases, the trypsins and chymotrypsins from shrimp midgut and bovine pancreas. 2. By assays on both the insoluble 3H-collagen fibrils and the soluble type I collagen, it was demonstrated that the shrimp midgut serine proteases, and less efficiently, the pronases from Streptomyces griseus, could hydrolyze collagen while the other serine proteases tested could not. 3. Our data indicate that the trypsins and chymotrypsins of shrimp (Penaeus monodon) directly and indirectly digest native collagen, and that the indirect pathway probably involves activation of procollagenase in the native collagen by these serine proteases.

Topics: Amino Acid Sequence; Animals; Bacteria; Cattle; Chymotrypsin; Collagen; Decapoda; Digestive System; Hydrolysis; Kinetics; Molecular Sequence Data; Oligopeptides; Serine Endopeptidases; Trypsin

In this new photometric assay fecal samples are pretreated with detergent and high concentrations of salts. The subsequent kinetic enzyme determination step involves the chromogenic substrate succinyl-Ala-Ala-Pro-Phe-4-nitroanilide. The sample pretreatment assures a nearly complete solubilization of the formerly particle-bound enzyme, thus permitting determination of the enzyme activity in either the suspension or the supernate after centrifugation. Furthermore this pretreatment enhances the enzyme activity and decreases the Km value. Results by this assay correlate well with those by classical titrimetry and the method is easily adapted to automated systems.

Topics: Autoanalysis; Buffers; Calcium Chloride; Chromogenic Compounds; Chronic Disease; Chymotrypsin; Feces; Humans; Hydrogen-Ion Concentration; Oligopeptides; Pancreatitis; Quaternary Ammonium Compounds; Reference Values; Sodium Chloride; Solubility; Spectrophotometry