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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The amino-terminal propeptide, consisting of 77 amino acid residues, is known to be required as an intramolecular chaperone to guide the folding of mature subtilisin E, a serine protease, into active mature enzyme. Many mutations within the pro-sequence have been shown to abolish the production of active subtilisin E (Kobayashi, T., and Inouye, M. (1992) J. Mol. Biol. 226, 931-933). Here we report characterization, refolding, and inhibitory abilities of six single amino acid substitution mutations (Ile-67-->Val, Ile-48-->Thr, Gly-44-->Asp, Lys-36-->Glu, Ala-30-->Thr, and Pro-15-->Leu) and a nonsense mutation (N59-mer) at the codon for Lys-18. These mutant propeptides were expressed in Escherichia coli using a T7 expression system and were purified to homogeneity. Surprisingly, Lys-36-->Glu, Ala-30-->Thr and Pro-15-->Leu were found to still function as a chaperone for in vitro refolding of denatured subtilisin BPN' with 60, 80, and 54% efficiency compared to the wild-type propeptide, respectively. The Ki values against subtilisin BPN' were 1.6 x 10(-9) M, and 2.1 x 10(-9) M, respectively. The Ki values against subtilisin BPN' were 1.6 x 10(-9) M, and 2.1 x 10(-9) M, respectively, almost identical to the Ki value exhibited by the wild-type propeptide (1.4 x 10(-9) M). In contrast, Ile-67-->Val and Gly-44-->Asp were able to refold denatured subtilisin BPN' with only 18 and13% efficiencies and had Ki values of 10 and 11 x 10(-9) M, respectively. The Ile-48-->Thr mutant propeptide was unable to refold denatured subtilisin BPN' and gave a 100-fold higher Ki (118 x 10(-9) M) than the wild-type propeptide. The N59-mer propeptide extending from Leu-19 to Met-78 was unable to function as a chaperone. Like the wild-type propeptide, none of the mutant propeptides had secondary structures as judged by their circular dichroism spectra. The present results demonstrate that the ability of the propeptide as a chaperone to refold the denatured protein is well correlated with its ability as a competitive inhibitor for the active enzyme. This supports the notion that the secondary and tertiary structures of the propeptide are identical or highly homologous between the renatured propeptide-subtilisin complex and the inhibitory complex formed between the propeptide and the active enzyme.
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PMID:Functional analysis of the propeptide of subtilisin E as an intramolecular chaperone for protein folding. Refolding and inhibitory abilities of propeptide mutants. 755 46

Granzyme B (also termed fragmentin 2) is a prototypic member of a subfamily of serine proteases expressed in the cytoplasmic granules of cytotoxic T lymphocytes and natural killer cells, and has been implicated in the destruction of targeted cells. Studies on the role of all granzymes in the cytolytic response would be greatly facilitated by the availability of specific anti-granzyme antisera. Three synthetic peptides corresponding to amino acid residues 1-17, 92-109 and 139-157 of human granzyme B were predicted to be immunogenic in the mouse, based on their hydrophilicity, accessibility to solvent, polymorphism with respect to mouse granzyme B and by comparison with X-ray crystallographic models of the rat mast cell protease II. Each peptide was conjugated to keyhole limpet hemocyanin and used to produce monoclonal antibodies in BALB/c mice. The monoclonal antibodies produced generally exhibited strong and specific reactivity with the respective immunizing peptide. However, only those antibodies detecting the peptide corresponding to residues 139-157 were able to detect native or denatured granzyme B, in direct binding studies with purified granzyme B or by immunoblotting. As an alternative approach for antiserum production, mice were immunized with whole, proteolytically active granzyme B isolated by immuno-affinity purification from NK tumour cell lysates, using one of the monoclonal antibodies generated. Despite the overall structural similarities between the various human granzymes, these mouse antisera surprisingly reacted only with granzyme B. Indeed, the reactivity of these polyclonal antisera was specifically abrogated by preincubation with the peptide corresponding to amino acid residues 139-157. This peptide stretch therefore represents an immunodominant portion of the granzyme B molecule in the mouse. Given the analogous structures of serine protease families expressed in leukocytes, these findings have implications for the production of monospecific antisera to granzymes and related proteases.
Mol Immunol 1995 Aug
PMID:The peptide loop consisting of amino acids 139-157 of human granzyme B (fragmentin 2) contains an immunodominant epitope recognized by the mouse. 756 17

A protein site is a region of a three-dimensional protein structure with a distinguishing functional or structural role. Certain sites recur in different protein structures (for example catalytic sites, calcium binding sites, and some types of turns), but maintain critical shared features. To facilitate the analysis of such protein sites, we have developed a computer system for analyzing the spatial distributions of biochemical properties around a site. The system takes a set of similar sites and a set of control nonsites, and finds differences between them. Specifically, it compares distributions of the properties surrounding the sites with those surrounding the nonsites, and reports statistically significant differences. In this paper, we use our method to analyze the features in the active site of the serine protease enzymes. We compare the use of radial distributions (shells) with 3-D grids (blocks) in the analysis of the active site. We demonstrate three different strategies for focusing attention on significant findings, based on properties of interest, spatial volumes of interest, and on the level of statistical significance. Finally, we show that the program automatically identifies conserved sequential, secondary structural and biophysical features of the serine protease active site, using noncatalytic histidine residues as a control environment.
Proc Int Conf Intell Syst Mol Biol 1995
PMID:Characterizing oriented protein structural sites using biochemical properties. 758 27

Signaling activity of bacterial chemotaxis transmembrane receptors is modulated by reversible covalent modification of specific receptor glutamate residues. The level of receptor methylation results from the activities of a specific S-adenosylmethionine-dependent methyltransferase, CheR, and the CheB methylesterase, which catalyzes hydrolysis of receptor glutamine or methylglutamate side-chains to glutamic acid. The CheB methylesterase belongs to a large family of response regulator proteins in which N-terminal regulatory domains control the activities of C-terminal effector domains. The crystal structure of the catalytic domain of the Salmonella typhimurium CheB methylesterase has been determined at 1.75 A resolution. The domain has a modified, doubly wound alpha/beta fold in which one of the helices is replaced by an anti-parallel beta-hairpin. Previous biochemical and mutagenesis data, suggest that the methylester hydrolysis catalyzed by CheB proceeds through a mechanism involving a serine nucleophile. The methylesterase active site is tentatively identified as a cleft at the C-terminal edge of the beta-sheet containing residues Ser164, His190 and Asp286. The three-dimensional fold, and the arrangement of residues within the catalytic triad distinguishes the CheB methylesterase from any previously described serine protease or serine hydrolase.
J Mol Biol 1995 Jul 07
PMID:Crystal structure of the catalytic domain of the chemotaxis receptor methylesterase, CheB. 760 74

Previous structural and kinetic characterization of mutations within the active site of alpha-lytic protease have demonstrated that amino acid residues in direct contact with the substrate are major substrate specificity determinants. The experiments described here identify residues 216-226 of alpha-lytic protease as a region of structure peripheral to the active site that also plays an important role in establishing the substrate specificity of the enzyme. Alanine substitution mutations within this surface loop of 19 amino acid residues significantly perturb the enzyme's specificity profile, despite being as far as 21 A from the hydroxyl group of Ser195. The kinetic consequences of the mutations are remarkably independent of position within the loop and suggest that active site plasticity is affected more than static structure. Kinetic characterization of double mutants with the Met190-->Ala broad-specificity active site mutation reveals varying degrees of non-additivity and indicates that active site plasticity can be influenced through multiple sets of interactions. Although these results clearly demonstrate that tuning of serine protease activity is possible through remodelling of structure surrounding the active site, practical issues such as retaining compatibility with the folding mechanism and stability of the mature enzyme present significant obstacles to general application of the technique.
J Mol Biol 1995 Aug 04
PMID:Functional linkage between the active site of alpha-lytic protease and distant regions of structure: scanning alanine mutagenesis of a surface loop affects activity and substrate specificity. 764 81

Dichelobacter nodosus, a Gram negative obligate anaerobe and causative organism of ovine footrot, secretes a family of extracellular acidic serine proteases with pI's in the range of 5.2 to 5.6, and a basic serine protease with a pI of approximately 9.5. Four acidic proteases (V1, V2, V3 and V5) from virulent and five acidic proteases (B1 to B5) from benign strains of D. nodosus were purified by chromatography on Sephadex G-100 and DEAE-Sepharose CL-6B. Proteases V2, V5 and B5 were found to yield two forms (a and b) on purification which probably arise from limited autolysis of the parent molecule. Amino acid compositions, peptide profiles produced on autolysis and apparent Mr on SDS-PAGE of proteases V1-V3 showed that they were similar to each other and to proteases B1 to B4, and that these proteases were clearly distinct from proteases V5 and B5, which were found to be identical.
Biochem Mol Biol Int 1994 Dec
PMID:Purification of the extracellular acidic proteases of Dichelobacter nodosus. 769 88

Dichelobacter nodosus, a Gram negative obligate anaerobe and causative organism of ovine footrot, secretes a family of extracellular acidic serine proteases with pI's in the range of 5.2 to 5.6, and a basic serine protease with a pI of approximately 9.5. The acidic proteases show optimum activity at pH 8 and require a divalent metal ion (eg. Ca) to maintain structural integrity. In the presence of EDTA or conditions that cause protein unfolding, the proteases undergo rapid and complete autolysis. The proteases were stable to heating to about 50 degrees C for 30 min but at higher temperatures, activity was rapidly lost; virulent proteases V1 and V2 were slightly more stable (by about 5 degrees C) than benign proteases B2 and B3. The effect of various protease inhibitors on the D. nodosus acidic proteases was the same except that the inhibitor, chymostatin, markedly inhibited protease V5 but not proteases V1-3 or B1-B4. Cleavage of the oxidized insulin B-chain showed that the specificity of proteases V1-V3 and B1-B4 was identical but that it was distinct from that of proteases V5/B5.
Biochem Mol Biol Int 1994 Dec
PMID:Properties of the extracellular acidic proteases of Dichelobacter nodosus. Stability and specificity of peptide bond cleavage. 769 89

Accessory gland peptide 76A, (Acp76A), belongs to the serpin superfamily of proteins (serine protease inhibitors). Acp76A is a secreted protein synthesized only in the Drosophila melanogaster adult male accessory gland. Accumulation of the protein in males is first detected with polyclonal antibodies at 1 day after eclosion. The level of the protein in virgin males reaches a peak 5-8 days post-eclosion, and remains constant for at least 20 days. Upon mating the amount of Acp76A in males drops dramatically, but recovers by 24 h after mating. Immediately after mating the Acp76A is found in the female uterus. By 6 h after mating the amount of Acp76A detected in females is drastically reduced.
Insect Biochem Mol Biol 1995 Feb
PMID:A Drosophila male accessory gland protein that is a member of the serpin superfamily of proteinase inhibitors is transferred to females during mating. 771 50

After exposure of cells to tumor necrosis factor (TNF), I kappa B alpha is rapidly degraded by a proteolytic activity that is required for nuclear localization and activation of transcription factor NF-kappa B. To investigate this problem, we have developed a cell-free system to study the degradation of I kappa B alpha initiated in vivo. In this in vitro system, characteristics of endogenous I kappa B alpha degradation were comparable to those observed in vivo. Recombinant I kappa B alpha, when added to lysates from cells exposed to TNF, was specifically degraded by a cellular proteolytic activity; however, it was stable in extracts from unstimulated cells. Inhibition characteristics of the proteolytic activity responsible for I kappa B alpha degradation suggest the involvement of a serine protease. Analysis of mutated forms of I kappa B alpha in the in vitro system demonstrated that an I kappa B alpha species which was unable to interact with NF-kappa B was still efficiently degraded. In contrast, deletion of the C-terminal 61 amino acids from I kappa B alpha rendered the protein resistant to proteolytic degradation. Expression of I kappa B alpha mutated forms in COS-7 cells confirmed the importance of the C-terminal domain for the degradation of the protein in vivo following cell activation. Thus, it is likely that the acidic, negatively charged region represented by the C-terminal 61 amino acids of the protein contains residues critical for TNF-inducible degradation of I kappa B alpha.
Mol Cell Biol 1995 May
PMID:Inducible degradation of I kappa B alpha in vitro and in vivo requires the acidic C-terminal domain of the protein. 773 25

A widely accepted model for the association of extrinsically bound proteins with acidic lipid-containing membranes has been that approach of the protein to the membrane induces a domain of acidic lipids that serves as the protein binding site. This model has been applied to a variety of membrane proteins including those that participate in the proteolytic complex that converts prothrombin to thrombin during the final stages of the blood coagulation cascade. The 'prothrombinase complex' consists of a serine protease (factor Xa), its protein co-factor (factor Va) and the substrate itself (prothrombin), all bound to phosphatidylserine (PS)-containing membranes derived from stimulated platelets. We have used three approaches to test the domain model as it applies to the proteins of this complex. First, phase diagrams describing the mixing of acidic and neutral lipids have failed to provide evidence for extensive acidic lipid domains (on the order of 50 or more lipid molecules) induced by protein biding. Second, pyrene-containing neutral and acidic phospholipids have been used to test for the occurrence of domains of as few as 20-30 lipids associated with binding of the membrane-binding fragment 1 region of prothrombin. Again, no evidence for domains was obtained. Finally, we have shown that binding of these proteins can be described in terms of a generalized model that presumes an acidic-lipid-independent surface adsorption combined with specific binding of acidic lipids to 'm' sites on a protein. Our results suggest that the concept of a protein-induced domain should not be applied indistriminately to explain binding of extrinsic membrane proteins such as the protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Membr Biol
PMID:Are acidic lipid domains induced by extrinsic protein binding to membranes? 776 85


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