Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using partial proteolytic cleavage, the nerve growth factor (NGF) binding site and the epitopes for two anti-NGF receptor (NGFR) monoclonal antibodies were localized on the recombinant extracellular domain (RED) of the NGFR. The RED was prepared in the baculovirus-insect cell system and was purified by immunoaffinity and ion-exchange chromatography. The four cysteine-rich repeat domains and some additional C-terminal sequences were resistant to proteolysis with papain or proteinase K. The Mr 32,000 papain-resistant fragment (P32) and the Mr 30,000 proteinase K-resistant fragment (K30) share the same N terminus as the intact RED and have C termini in the vicinity of residue 170. Even though P32 and K30 have the same N terminus and probably differ by only a small number of amino acids at the C terminus, P32, but not K30, binds 125I-NGF. As judged by Western blot analysis, two anti-NGFR antibodies (ME20.4 and NGFR5) bind to P32 but have a lesser affinity for K30. Since antibody ME20.4 inhibits NGF binding but antibody NGFR5 does not, these antibodies bind to distinct epitopes. However, these epitopes apparently are closely spaced since these antibodies compete with each other for binding to biotinylated RED. NGF, but not the control protein cytochrome c, protects RED from papain digestion. Therefore, the P32 C terminus is important for the expression of the NGF binding site and the antibody-defined epitopes, even though the NGF binding site and antibody-defined epitopes probably are not encoded by the P32 C terminus. These data suggest that complex interactions occur between different regions of the RED, and that optimum NGF binding requires the integrity of multiple RED domains, including a short sequence to the C terminus of residue 170.
 ...
PMID:Structural domains of the extracellular domain of human nerve growth factor receptor detected by partial proteolysis. 137 92

The topographical localization of the N-terminus of cytochrome b in the inner mitochondrial membrane was determined by mild proteolysis of the yeast mitochondrial cytochrome bc1 complex and identification of the proteolytic fragments derived from subunits of the complex with an established orientation in the inner membrane. The cytochrome bc1 complex was incorporated into proteoliposomes which were separated by cytochrome c affinity chromatography into two populations in either the mitochondrial or the submitochondrial orientation. Core protein I which protrudes from the matrix side of the inner membrane was digested by proteinase K only in proteoliposomes with the submitochondrial orientation and not in those with the mitochondrial orientation. By contrast, cytochrome c1 with protrudes from the cytoplasmic side of the inner membrane was digested by proteinase K only in proteoliposomes with the mitochondrial orientation and not in those with the submitochondrial orientation. Cytochrome b was digested by SV8 protease only in proteoliposomes with the mitochondrial orientation to yield two aggregating fragments of 25.6 and 24.5 kDa. These peptides were isolated by preparative gel chromatography and sequenced to establish that the cleavage of cytochrome b by SV8 protease occurred at glutamate residues 59 and 66. These residues are localized in the extramembranous loop between the two hydrophobic membrane-spanning helices A and B and thus face the cytoplasmic side of the inner mitochondrial membrane. These results indicate that the N-terminus of yeast cytochrome b protrudes from the matrix side of the inner membrane consistent with the eight-helix model for the orientation of cytochrome b in the membrane.
 ...
PMID:Biochemical evidence for the orientation of cytochrome b in the yeast mitochondrial membrane in the eight-helix model. 803 Nov 40

Mice infected with Listeria monocytogenes (LM) generate H2-M3wt-restricted CD8 effectors which recognize a heat-killed LM-associated antigen (HAA) presented by macrophages. To characterize HAA, we extracted a bioactive component from LM using SDS or NaOH. Extracted HAA aggregated in hydrophilic solvents but dissociated in the presence of SDS into a smaller subunit which migrated in Sephadex G-200 between chymotrypsinogen (25 kDa) and cytochrome c (12.5 kDa). HAA bioactivity and size was unaffected by proteinase K under conditions which degraded virtually all detectable protein. HAA was also unaffected by other proteases, RNase and DNase, but HAA bioactivity was destroyed by periodate, an agent that degrades carbohydrates. These studies demonstrate that H2-M3wt can present a hydrophobic, non-peptide, microbial antigen, probably glycolipid in origin, to CD8 T cells.
 ...
PMID:H2-M3wt-restricted, Listeria monocytogenes-specific CD8 T cells recognize a novel, hydrophobic, protease-resistant, periodate-sensitive antigen. 867 23

To improve the sequence ions of a protein in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), proteinase K was used to digest the protein followed by MALDI-MS characterization of the peptide fragments. The primary structures of three proteins, insulin B chain, cytochrome c and lysozyme, were determined by this method. A series of peptide fragments including those differentiated by one residue can be produced from the protein by using proteinase K digestion, thus providing support to the protein sequence. The peptide fragments liberated from proteinase K proteolysis of the insulin B chain allow the protein to be partially sequenced. Furthermore, some of the residues are double or triple checked by generating a variety of fragments. The same method was used to investigate cytochrome c and lysozyme denaturated in 3 M guanidine hydrochloride. The success of the method relies on the intrinsic properties of proteinase K and accurate determination of the peptide fragments by MALDI-MS.
 ...
PMID:Primary structures of proteins characterized by proteinase K digestion and matrix-assisted laser desorption/ionization mass spectrometry. 940 26

It is shown that simultaneously to the unfolding of hen egg white lysozyme and horse heart cytochrome c the sequential conformational changes and molten globule states can be detected by the combination of proteolysis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). This is demonstrated by the differences among the products and the time courses of native lysozyme as well as those unfolded in 1 and 3 M guanidine hydrochloride (GuHCl) when they were proteolyzed by proteinase K and analyzed by MALDI-MS. Due to the absence of disulfide bonds in the cytochrome c molecule, it is more sensitive to the disturbance of the denaturant. The partially unfolded state as detected at low concentrations of guanidine hydrochloride in our experiment resemble the molten globule state. One of the unique properties of the method described herein is to measure directly the peptide fragment liberated from proteolysis of the protein. It allows the identification of the sensitive sites susceptible to denaturation, which are subsequently cleaved by proteinase K proteolysis.
 ...
PMID:Protein conformational changes determined by matrix-assisted laser desorption mass spectrometry. 952 57

Recent hydrogen exchange experiments on native cytochrome c implicate a sequential unfolding pathway in contrast to a simple two-state process. We have studied the heat-induced unfolding of this protein by using spectroscopic measurements to detect changes in conformation and proteolytic enzyme digestion to identify regions of the protein that are labile. Several spectroscopic profiles were monitored: CD at 222 nm, a measurement of secondary structure change in the protein, the absorbance at 280 nm, involving the local environment of Trp 59, and absorbance at 420 nm, the Soret band of the heme. The apparent Tm values for these probes differ, consistent with an unfolding pathway containing intermediates. The limited digestion by proteinase K is consistent with population of an intermediate state in unfolding. We find a single strong region of cleavage at low temperature with retention of structure in each fragment.
 ...
PMID:Proteolysis as a probe of thermal unfolding of cytochrome c. 953 27

Limited cleavage of oxidized and reduced horse heart cytochrome c (Cyt c) and the azide complex of Cyt c by proteinase K at room temperature yields a single cut within the central loop (36-60 in the sequence). Using an assay that allows spectroscopic evaluation of the fraction of intact protein as a function of time, together with a simple kinetic model for proteolysis, fluctuation opening of the loop can be related to the free energy of the corresponding protein. This allows us to estimate quantitatively the free energy difference between the oxidized form of Cyt c and other states using proteolysis as a probe. The results we obtain indicate that oxidized Cyt c is 2.0 kcal mol(-1) less stable than the reduced form, and 0.07 kcal mol(-1) is more stable than the Cyt c: azide complex at 25 degrees C. These values agree in magnitude with results from hydrogen exchange and unfolding studies, suggesting that the stability of a protein can be directly related to its structural dynamics.
 ...
PMID:Proteolysis as a measure of the free energy difference between cytochrome c and its derivatives. 982 13

Proteolysis experiments have been used to monitor the conformational transitions from an unfolded to a folded state occurring when the apo form of horse cytochrome c (cyt c) binds the heme moiety or when two fragments of cyt c form a native-like 1:1 complex. Proteinase K was used as a proteolytic probe, in view of the fact that the broad substrate specificity of this protease allows digestion at many sites along a polypeptide chain. The rather unfolded apo form of cyt c binds heme with a concomitant conformational transition to a folded species characterized by an enhanced content of helical secondary structure. While the holoprotein is fully resistant to proteolytic digestion and the apoprotein is digested to small peptides, the noncovalent complex of the apoprotein and heme exhibits an intermediate resistance to proteolysis, in agreement with the fact that the more folded structure of the complex makes the protein substrate more resistant to proteolysis. The noncovalent native-like complex of the two fragments 1-56 and 57-104 of cyt c, covering the entire polypeptide chain of 104 residues of the protein, is rather resistant to proteolysis, while the individual fragments are easily digested. Fragment 57-104 is fast degraded to several peptides, while fragment 1-56 is slowly degraded stepwise from its C-terminal end, leading initially mostly to fragments 1-48 and 1-40 and, at later stages of proteolysis, fragments 1-38, 1-35, 1-33, and 1-31. Thus, proteolysis data indicate that the heme containing fragment 1-56 has a rather compact core and a C-terminal flexible tail. Upon prolonged incubation of the complex of fragments 1-56 and 57-104 (nicked cyt c) with proteinase K, a chain segment is removed from the nicked protein, leading to a gapped protein complex of fragments of 1-48 and 57-104 and, on further digestion, fragments 1-40 and 57-104. Of interest, the chain segment being removed by proteolysis of the complex matches the omega-loop which is evolutionarily removed in cyt c of microbial origin. Overall, rates and/or resistance to proteolysis correlates well with the extent of folding of the protein substrates, as deduced from circular dichroism measurements. Thus, our results underscore the utility of proteolytic probes for analyzing conformational and dynamic features of proteins. Finally, a specific interest of the cyt c fragment system herewith investigated resides in the fact that the fragments are exactly the exon products of the cyt c gene.
 ...
PMID:Protein interactions leading to conformational changes monitored by limited proteolysis: apo form and fragments of horse cytochrome c. 1158 45

Here we present a comparison between protein fragments produced by limited proteolysis and those identified by computational cutting based on the building block folding model. The principles upon which the two methods are based are different. Limited proteolysis of natively folded proteins occurs at flexible sites and never at the level of chain segments of regular secondary structure such as alpha-helices. Therefore, the targets for limited proteolysis are locally unfolded regions. In contrast, the computational cutting algorithm considers the compactness of the fragments, their nonpolar buried surface area, and their isolatedness, that is, the surface area which was buried prior to the cutting and becomes exposed subsequently. Despite the different criteria, there is an overall correspondence between sites or regions of limited proteolysis with those identified by computational cutting. The computational cutting method has been applied to several model proteins for which detailed limited proteolysis data are available, namely apomyoglobin, cytochrome c, ribonuclease A, alpha-lactalbumin, and thermolysin. As expected, more cuts are obtained computationally than experimentally and the agreement is better when a number of proteolytic enzymes are used. For example, cytochrome c is cleaved by thermolysin at 56-57, 45-46, and at 80-81, and by proteinase K at 48-49 and 50-51. Incubation of the noncovalent and native-like complex of cytochrome c fragments 1-56 and 57-104 with proteinase K yielded the gapped protein species 1-48/57-104 and finally 1-40/57-104. Computational cutting of cytochrome c reproduced the major experimental observations, with cuts at 47, 64-65 or 65-66 and 80-81 and an unstable 32-47 region not assigned to any building block. The next step, not addressed in this work, is to probe the ability of the generated fragments to fold independently. Since both the computational algorithm and limited proteolysis attempt to dissect the protein folding problem, the general agreement between the two procedures is gratifying. This consistency allows us to propose the use of limited proteolysis to produce protein fragments that can adopt an independent folding and, therefore, to study folding intermediates. The results of the present study appear to validate the building block folding model and are in line with the proposal that protein folding is a hierarchical process, where parts constituting local minima of energy fold first, with their subsequent association and mutual stabilization to finally yield the global fold.
 ...
PMID:Comparison of protein fragments identified by limited proteolysis and by computational cutting of proteins. 1207 Mar 28

The cytoplasmic membrane protein CcmC is, together with other Ccm proteins, a component for the maturation of c-type cytochromes in Gram-negative bacteria. A Pseudomonas fluorescens ATCC 17400 ccmC mutant is cytochrome c-deficient and shows considerably reduced production of the two siderophores pyoverdine and quinolobactin, paralleled by a general inability to utilize various iron sources, with the exception of haem. The ccmC mutant accumulates in a 5-aminolevulinic acid-dependent synthesis a reddish, fluorescent pigment identified as protoporphyrin IX. As a consequence a visA phenotype similar to that of a ferrochelatase-deficient hemH mutant characterized by drastically reduced growth upon light exposure was observed for the ccmC mutant. The defect of iron-protoporphyrin formation was further demonstrated by the failure of ccmC cell-free proteinase K-treated extracts to stimulate the growth of a haem auxotrophic hemH indicator strain, compared to similarly prepared wild-type extracts. In addition, the ccmC mutant did not sustain hemH growth in cross-feeding experiments while the wild-type did. Significantly reduced resistance to oxidative stress mediated by haem-containing catalases was observed for the ccmC mutant. A double hemH ccmC mutant could not be obtained in the presence of external haem without the hemH gene in trans, indicating that the combination of the two mutations is lethal. It was concluded that CcmC, apart from its known function in cytochrome c biogenesis, plays a role in haem biosynthesis. A function in the regulatory co-ordination of iron acquisition via siderophores, iron insertion into porphyrin via ferrochelatase and iron-protoporphyrin export for cytochrome c formation is predicted.
 ...
PMID:Co-ordination of iron acquisition, iron porphyrin chelation and iron-protoporphyrin export via the cytochrome c biogenesis protein CcmC in Pseudomonas fluorescens. 1466 86


1 2 Next >>