Proteolysis

With the aim to distinguish between local and global conformational changes induced by trifluoroethanol in RNase A, spectroscopic and activity measurements in combination with proteolysis by unspecific proteases have been exploited for probing structural transitions of RNase A as a function of trifluoroethanol concentration. At 30% (v/v) trifluoroethanol (pH 8.0; 25 °C), circular dichroism and fluorescence spectroscopy indicate a cooperative collapse of the tertiary structure of RNase A coinciding with the loss of its enzymatic activity....

7p system191 01-06-2013 48 3   Download

The 26S proteasome, a protein complex consisting of a 20S proteasome and a pair of 19S regulatory particles (RP), is involved in ATP-dependent proteolysis in eukaryotes. In yeast, theRP contains six different ATPase subunits and, at least, 11 non-ATPase subunits. In this study, we identi®ed the rice homologs of yeast RP subunit genes from the rice expressed sequence tag (EST) library.

10p research12 29-04-2013 39 3   Download

Bovineb-lactoglobulin was hydrolyzed with trypsin or chymotrypsin in the course of heat treatment at 55, 60 and 65°C at neutral pH. At these temperaturesb-lactoglobulin undergoes signi®cant but reversible structural changes. In the conditions used in the present study,b-lactoglobulinwas virtually insensitive to proteolysis by either enzyme at room temperature, but underwent extensive proteolysis when either proteasewas present during the heat treatment.

11p research12 29-04-2013 33 3   Download

Soluble methane mono-oxygenase (sMMO) ofMethylo-coccus capsulatus (Bath) catalyses the O2-dependent and NAD(P)H-dependent oxygenation of methane and numer-ous other substrates. During puri®cation, the sMMO enzyme complex, which comprises three components and has a molecular mass in excess of 300 kDa, becomes inac-tivated because of cleavage of just 12 amino acids from the N-terminus of proteinB, which is the smallest component of sMMOand theonlyonewithout prosthetic groups.

9p research12 23-04-2013 24 3   Download

Tyrosinase (monophenol,L-DOPA:oxygen oxidoreductase) was isolated from the ink of the squid,Illex argentinus. Squid tyrosinase, termed ST94, was found to occur as a covalently linked homodimeric protein with a molecular mass of 140.2 kDa containing two copper atoms per a subunit. The tyrosinase activity of ST94 was enhanced by proteolysis with trypsin to form a protein, termed ST94t, with a molecular mass of 127.6 kDa.

13p tumor12 20-04-2013 30 3   Download

Testican-1, a secreted proteoglycan enriched in brain, has a single thyropin domain that is highly homologous to domains previously shown to inhibit cysteine proteases. We demonstrate that purified recombinant human testican-1 is a strong competitive inhibitor of the lysosomal cysteine pro-tease, cathepsin L,withaKiof 0.7nM, but it does not inhibit the structurally related lysosomal cysteine protease cathep-sin B. Testican-1 inhibition of cathepsin L is independent of its chondroitin sulfate chains and is effective at both pH 5.5 and7.2.

8p tumor12 20-04-2013 33 4   Download

The catalytic activity of phenylalanine hydroxylase (PAH, phenylalanine 4-monooxygenase EC 1.14.16.1) is regulated by three main mechanisms, i.e. substrate (L-phenylalanine, L-Phe) activation, pterin cofactor inhibition and phos-phorylation of a single serine (Ser16) residue.To address the molecular basis for the inhibition by the natural cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin, its effects on the recombinant tetrameric human enzyme (wt-hPAH) was studied using three different conformational probes, i.e.

10p tumor12 20-04-2013 34 3   Download

Here, we show that recombinant bovine PDE5A1 is pro-teolysedby recombinant caspase-3 inin vitroand transfected Cos-7 cells. In addition, the treatment of PDE5A1-trans-fectedCos-7andPC12cellswithstaurosporine, anapoptotic agent that activates endogenous caspase-3, also induced proteolysis and inactivation of PDE5A1. These findings suggest that there is specificity in the interaction between caspase-3 and PDE5A1 that requires application of an apoptotic stimulus.

9p tumor12 20-04-2013 57 5   Download

Macrophages at an inflammatory site release massive amounts of proteolytic enzymes, including lysosomal cys-teine proteases, which colocalizewith their circulating, tight-binding inhibitors (cystatins, kininogens), so modifying the protease/antiprotease equilibrium in favor of enhanced proteolysis. We have explored the ability of human cath-epsins B, KandL to participate in the production of kinins, using kininogens and synthetic peptides that mimic the insertion sites of bradykinin on human kininogens....

8p tumor12 20-04-2013 44 5   Download

Cytidine 5¢-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP using either ammonia orL-glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Limited trypsin-catalysed proteolysis, Edman degradation, and site-directed muta-genesis were used to identify peptide bonds C-terminal to three basic residues (Lys187, Arg429, and Lys432) of Escherichia coliCTP synthase thatwere highly susceptible to proteolysis. ...

12p fptmusic 16-04-2013 39 4   Download

Serpins are the largest family of protease inhibitors and are fundamental for the control of proteolysis in multicellular eukaryotes. Most eukaryote serpins inhibit serine or cysteine proteases, however, noninhibitory mem-bers have been identified that perform diverse functions in processes such as hormone delivery and tumour metastasis. More recently inhibitory ser-pins have been identified in prokaryotes and unicellular eukaryotes, never-theless, the precise molecular targets of these molecules remains to be identified....

6p fptmusic 12-04-2013 37 2   Download

We have defined thein vivo andin vitro metabolic fate of internalized chol-era toxin (CT) in the endosomal apparatus of rat liver. In vivo, CT was internalized and accumulated in endosomes where it underwent degrada-tion in a pH-dependent manner. In vitro proteolysis of CT using an endo-somal lysate required an acidic pH and was sensitive to pepstatin A, an inhibitor of aspartic acid proteases.

13p fptmusic 11-04-2013 35 3   Download

Botulinum neurotoxin type A (BoNT⁄A), the most toxic substance known to mankind, is produced by Clostridium botulinumtype A as a complex with a group of neurotoxin-associated proteins (NAPs) through polycis-tronic expression of a clustered group of genes. NAPs are known to protect BoNT against adverse environmental conditions and proteolytic digestion. Hemagglutinin-33 (Hn-33) is a 33 kDa subcomponent of NAPs that is resistant to protease digestion, a feature likely to be involved in the protec-tion of the botulinum neurotoxin from proteolysis. ...

10p awards 06-04-2013 32 3   Download

Proteolysis of single polypeptide mammalian purple acid phosphatases (PAPs) results in the loss of an interaction between the loop residue Asp146 and the active site residues Asn91 and⁄or His92. While Asn91 is a ligand to the divalent metal of the mixed-valent di-iron center, the role of His92 in the catalytic mechanism is unknown. Site-directed mutagenesis of His92 was performed to examine the role of this residue in single polypep-tide PAP.

10p awards 06-04-2013 43 3   Download

Limited proteolysis in combination with liquid chromatography-ion trap mass spectrometry (LC-MS) was used to analyze engineered or natural proteins derived from a type I modular polyketide synthase (PKS), the 6-deoxyerythronolide B synthase (DEBS), and comprising either the first two extension modules linked to the chain-terminating thioesterase (TE) (DEBS1-TE); or the last two extension modules (DEBS3) or the first exten-sion module linked to TE (diketide synthase, DKS).

15p awards 06-04-2013 38 1   Download

The major 2S albumin allergen from Brazil nuts, Ber e 1, was subjected to gastrointestinal digestion using a physiologically relevantin vitro model sys-tem either before or after heating (100
C for 20 min). Whilst the albumin was cleaved into peptides, these were held together in a much larger struc-ture even when digested by using a simulated phase 1 (gastric) followed by a phase 2 (duodenal) digestion system. Neither prior heating of Ber e 1 nor the presence of the physiological surfactant phosphatidylcholine affected the pattern of proteolysis.

0p awards 05-04-2013 41 2   Download

accharomyces cerevisiaeis a widely used host in the pro-ductionof therapeutic peptides andproteins.Herewe report the identification of a novel endoprotease in S. cerevisiae. It is encoded by the CYM1gene and is specific for the C-terminus of basic residues of heterologously expressed peptides. Gene disruption ofCYM1not only reduced the intracellular proteolysis, but also enhanced the secretion of heterologously expressed peptides such as growth hormone, pro-B-type natriuretic peptide and pro-cholecystokinin....

10p awards 05-04-2013 39 3   Download

Limited proteolysis is a highly specific irreversible process, which can serve to initiate physiological function by con-verting a precursor protein into a biologically active form. When the activating enzyme and the activated enzyme coincide, the process is an autocatalytic zymogen activation (i.e. reactions inwhich the zymogens serves as a substrate for the corresponding active enzyme). The activity of proteases is frequently regulated by the binding of specific protease inhibitors.

0p awards 05-04-2013 34 2   Download

Approximately 400 million allergic patients are sensitized against group 1 grass pollen allergens, a family of highly cross-reactive allergens present in all grass species. We report the eukaryotic expression of the group 1 aller-gen from Timothy grass, Phl p 1, in baculovirus-infected insect cells. Domain elucidation by limited proteolysis and mass spectrometry of the purified recombinant glycoprotein indicates that the C-terminal 40% of Phl p 1, a major IgE-reactive segment, represents a stable domain....

0p awards 05-04-2013 27 2   Download

To facilitate the process of proteindesign and learn the basic rules that control the structure and stability of proteins, combinatorial methods have been developed to select or screen proteins with desired properties from libraries of mutants. One such method uses phage-display and proteo-lysis to select stably folded proteins. This method does not rely on specific properties of proteins for selection. There-fore, in principle it can be applied to any protein.

6p dell39 03-04-2013 31 4   Download