Isolation and characterization of a protease inhibitor from Acacia karroo with a common combining loop and overlapping binding sites for chymotrypsin and trypsin.

By using affinity and reversed-phase HPLC (RP-HPLC) chromatographies two chymotrypsin-trypsin inhibitors were isolated from seeds of Acacia karroo, a legume of the subfamily Mimosoideae. The primary structure of one of these inhibitors, named AkCI/1, was determined. The inhibitor consists of two polypeptide chains, 139 and 44 residues respectively, which are linked by a single disulfide bridge.

The catalytic aspartate is protonated in the Michaelis complex formed between trypsin and an in vitro evolved substrate-like inhibitor: a refined mechanism of serine protease action.

The mechanism of serine proteases prominently illustrates how charged amino acid residues and proton transfer events facilitate enzyme catalysis. Here we present an ultrahigh resolution (0.93 Å) x-ray structure of a complex formed between trypsin and a canonical inhibitor acting through a substrate-like mechanism.

Probing conformational plasticity of the activation domain of trypsin: the role of glycine hinges.

Trypsin-like serine proteases play essential roles in diverse physiological processes such as hemostasis, apoptosis, signal transduction, reproduction, immune response, matrix remodeling, development, and differentiation. All of these proteases share an intriguing activation mechanism that involves the transition of an unfolded domain (activation domain) of the zymogen to a folded one in the active enzyme. During this conformational change, activation domain segments move around highly conserved glycine hinges.

When the surface tells what lies beneath: combinatorial phage-display mutagenesis reveals complex networks of surface-core interactions in the pacifastin protease inhibitor family.

Pacifastin protease inhibitors are small cysteine-rich motifs of approximately 35 residues that were discovered in arthropods. The family is divided into two related groups on the basis of the composition of their minimalist inner core. In group I, the core is governed by a Lys10-Trp26 interaction, while in group II it is organized around Phe10.

Unconventional translation initiation of human trypsinogen 4 at a CUG codon with an N-terminal leucine. A possible means to regulate gene expression.

Chromosomal rearrangements apparently account for the presence of a primate-specific gene (protease serine 3) in chromosome 9. This gene encodes, as the result of alternative splicing, both mesotrypsinogen and trypsinogen 4. Whereas mesotrypsinogen is known to be a pancreatic protease, neither the chemical nature nor biological function of trypsinogen 4 has been explored previously.

Local binding with globally distributed changes in a small protease inhibitor upon enzyme binding.

Complexation of the small serine protease inhibitor Schistocerca gregaria chymotrypsin inhibitor (SGCI), a member of the pacifastin inhibitor family, with bovine chymotrypsin was followed by NMR spectroscopy. (1)H-(15)N correlation (HSQC) spectra of the inhibitor with increasing amounts of the enzyme reveal tight and specific binding in agreement with biochemical data. Unexpectedly, and unparalleled among canonical serine protease inhibitors, not only residues in the protease-binding loop of the inhibitor, but also some segments of it located spatially far from the substrate-binding cleft of the enzyme were affected by complexation.

Myelin basic protein, an autoantigen in multiple sclerosis, is selectively processed by human trypsin 4.

Demyelination, the proteolytic degradation of the major membrane protein in central nervous system, myelin, is involved in many neurodegenerative diseases. In the present in vitro study the proteolytic actions of calpain, human trypsin 1 and human trypsin 4 were compared on lipid bound and free human myelin basic proteins as substrates. The fragments formed were identified by using N-terminal amino acid sequencing and mass spectrometry.

Inhibition of Colorado potato beetle larvae by a locust proteinase inhibitor peptide expressed in potato.

The cDNA for a 73-mer peptide containing two locust serine proteinase inhibitors was cloned, fused to the constitutive CaMV35S promoter and introduced into potato by Agrobacterium-mediated transformation. From 23 independent transgenic lines, three with high mRNA level and proteinase inhibitory activity were propagated in vitro and transferred to pots. The peptide from the leaves was identified by its N-terminal sequence and by K(i) values against chymotrypsin and trypsin.

Extended intermolecular interactions in a serine protease-canonical inhibitor complex account for strong and highly specific inhibition.

We have previously shown that a trypsin inhibitor from desert locust Schistocerca gregaria (SGTI) is a taxon-specific inhibitor that inhibits arthropod trypsins, such as crayfish trypsin, five orders of magnitude more effectively than mammalian trypsins. Thermal denaturation experiments, presented here, confirm the inhibition kinetics studies; upon addition of SGTI the melting temperatures of crayfish and bovine trypsins increased 27 degrees C and 4.5 degrees C, respectively.

Cloning and expression of ostrich trypsinogen: an avian trypsin with a highly sensitive autolysis site.

One of ostrich (Struthio camelus) trypsinogen genes was cloned from pancreatic cDNA. Its amino acid sequence compared to known trypsin sequences from other species shows high identity and suggests that it is a member of the phylogenetically anionic trypsinogen I subfamily. After cytoplasmic over expression in Escherichia coli and renaturation, the activation properties of ostrich trypsinogen were studied and compared to those of human trypsinogen 1 (also called as human cationic trypsinogen).

Isolation and functional characterization of a cyst(e)amine-sensitive protein protecting methyl-dithio groups.

We have previously shown that S-methylthio cysteine mixed disulfide and cystamine potently stimulate thiol production and glutathione synthesis of a human T-cell line in SH-poor medium. Here, we describe a simple photometric method for the determination of methylthio-mixed disulfides (MT-groups) and show that liver contains relatively large amount of MT-groups mainly associated with the globulin fractions. At least a part of methylthio (MT-) globulins is in a complex with a heat-stable protein protecting methylthio-groups against reduction and was designated as anti-methylthio factor (AMTF).

Comparison of proteolytic activities produced by entomopathogenic Photorhabdus bacteria: strain- and phase-dependent heterogeneity in composition and activity of four enzymes.

Twenty strains (including eight phase variant pairs) of nematode-symbiotic and insect-pathogenic Photorhabdus bacteria were examined for the production of proteolytic enzymes by using a combination of several methods, including gelatin liquefaction, zymography coupled to native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and activity measurement with two chromogen substrate types. Four protease activities (approximately 74, approximately 55, approximately 54, and approximately 37 kDa) could be separated. The N-terminal sequences of three of the proteases were determined, and a comparison with sequences in databases allowed identification of these proteases as HEXXH metallopeptidases.

Mutant rat trypsin selectively cleaves tyrosyl peptide bonds.

A double mutant of rat trypsinogen (Asp189Ser, DeltaAsp223) was constructed by site-directed mutagenesis. The recombinant protein was produced in Escherichia coli under the control of a periplasmic expression vector. The purified and enterokinase-activated enzyme was characterized by synthetic fluorogenic tetrapeptide and natural polypeptide substrates and by a recently developed method.

Structural characterization of the second TSP1-module of human thrombospondin.

The TSP1-module has been first identified as the type 1 repeat of thrombospondin-1. Members of this extracellular module-family have since been shown to be present in several hundred metazoan proteins as well as in proteins of some protists. Despite the widespread occurrence and biological importance of this module-type, relatively little is known about their three-dimensional structure.

Binding sites of different geometries for the 16-3 phage repressor.

Prokaryotic repressor-operator systems provide exemplars for the sequence-specific interactions between DNA and protein. The crucial atomic contacts of the two macromolecules are attained in a compact, geometrically defined structure of the DNA-protein complex. The pitch of the DNA interface seems an especially sensitive part of this architecture because changes in its length introduce new spacing and rotational relations in one step.

Comparative structure analysis of proteinase inhibitors from the desert locust, Schistocerca gregaria.

The solution structure of three small serine proteinase inhibitors, two natural and one engineered protein, SGCI (Schistocerca gregaria chymotrypsin inhibitor), SGCI[L30R, K31M] and SGTI (Schistocerca gregaria trypsin inhibitor), were determined by homonuclear NMR-spectroscopy. The molecules exhibit different specificities towards target proteinases, where SGCI is a good chymotrypsin inhibitor, its mutant is a potent trypsin inhibitor, and SGTI inhibits both proteinases weakly. Interestingly, SGTI is a much better inhibitor of insect proteinases than of the mammalian ones used in common assays.

Remarkable phylum selectivity of a Schistocerca gregaria trypsin inhibitor: the possible role of enzyme-inhibitor flexibility.

A 35-mer polypeptide isolated from the hemolymph of desert locust Schistocerca gregaria (SG) proved to be a canonical inhibitor of bovine trypsin (K(i) = 0.2 microM). Despite having a trypsin-specific arginine at the primary specificity P(1) site, it inhibits bovine chymotrypsin almost as well (K(i) = 2 microM).