Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Amino acid replacements in a homodimeric protein, Streptomyces subtilisin inhibitor, at the position of Val13, which is located at the center of the beta-sheet interface of the domain-domain interaction, changed both the overall stability and the denaturational scheme. All the mutant forms lost stability, losses in free energy at 82.21 degrees C at pH 7.0 obtained by calorimetric measurements ranging from 10.3 kcal (mol dimer)-1 for the Gly mutant, which involved a loss in enthalpy without a compensating loss in entropy, to 0.84 kcal (mol dimer)-1 for the Ile mutant, which involved comparable losses in enthalpy and entropy. A gain in enthalpy for the Ala mutant was overcome by a gain in entropy, resulting in a loss in free energy by 6.78 kcal (mol dimer)-1. It was found that decreases in enthalpy and entropy showed good correlation with increasing side-chain hydrophobicity, while no such correlation was present for free energy. Replacements with Gly, Ala, Met and Phe altered the thermal denaturational scheme from N2 reversible 2D, which was the case for the Leu and Ile mutants as well as for the wild-type, to N2 reversible 2N* reversible 2D, where the first step can be regarded as a dissociation of the native dimer followed by a major unfolding in the second step. The free energy acquired by forming the dimer was estimated to be 2.03 kcal mol-1 for the Ala mutant at 66.95 degrees C at pH 9.5 and lower for the other three mutants.
J Mol Biol 1995 Jun 09
PMID:A thermodynamic study of mutant forms of Streptomyces subtilisin inhibitor. II. Replacements at the interface of dimer formation, Val13. 778 16

To investigate the contribution of the carboxy-terminal domain in the process of tubulin folding and dimer formation, we constructed a beta 1-beta 3 tubulin chimaera and two truncated carboxy-terminal beta 3-tubulins. The capacity of these altered polypeptides to incorporate into dimers and into microtubules was tested by non-denaturing electrophoresis and co-assembly experiments. The chimaera and the truncated protein with a deletion encompassing the last 12 amino acid residues (beta 3 delta C12) were incorporated into dimers and microtubules, though the level of incorporation was diminished compared to wild-type beta 3-tubulin. However, the level of incorporation of beta 3 delta C12 into subtilisin-digested dimers was similar to the incorporation of wild-type beta 3-tubulin. Since subtilisin deletes the carboxy-terminal region, these results suggest a regulatory role of the carboxy-terminal region in the folding process itself and not in the formation of the dimer.
J Mol Biol 1995 Mar 10
PMID:Beta-tubulin folding is modulated by the isotype-specific carboxy-terminal domain. 787 81

Tubulin and microtubules were modified with the protease, subtilisin. The modification reduced the length of alpha- or beta-tubulin by cleaving a peptide fragment from the C-terminals. Generation of alpha'beta'-tubulin, which is cleaved at both the alpha- and beta-subunit terminals, and alpha beta'-tubulin, which is cleaved at the beta-subunit C-terminal, have already been reported. In this work an isotype, alpha'beta-tubulin, was produced. The three modified tubulin isotypes were compared for their ability to interact with glycolytic enzymes. Cleavage of alpha led to a poorer interaction when tested via affinity chromatography. Tubulin also inhibits the activity of aldolase and glyceraldehyde 3-phosphate dehydrogenase. When the alpha-subunit C-terminal was intact, inhibition was greatest. These results imply that the C-terminal of the tubulin alpha-subunit is responsible for interactions with glycolytic enzymes.
J Mol Recognit 1993 Dec
PMID:Glycolytic enzyme-tubulin interactions: role of tubulin carboxy terminals. 791 12

The heterotetrameric enzyme hydroxynitrile lyase (HNL) from sorghum (EC 4.1.2.11) is involved in the catabolism of the cyanogenic glycoside dhurrin. We have isolated a cDNA clone comprising about 90% of the COOH terminal sequence of a precursor which encodes both subunit of HNL from Sorghum bicolor L. (SbHNL). Hence the subunits of SbHNL must be the result of post-translational processing. The deduced amino acid sequence of HNL shares significant sequence homology with members of the serine carboxypeptidase family. In particular, HNL from sorghum shares the catalytical triad Asp. His, and Ser with these enzymes which evolved in 3 groups of enzymes (carboxypeptidase, chymotrypsin, and subtilisin) by convergent evolution. Moreover, like serine carboxypeptidases, HNL from sorghum consists of two pairs of glycosylated cysteine linked A and B chains forming a heterotetramer of a molecular weight of 105,000 (carboxypeptidases 120,000). Thus, HNL from sorghum closely resembles to serine carboxypeptidases but differs from all other HNLs described so far. Western blotting experiments revealed cross reaction between carboxypeptidase from wheat and anti SbHNL antisera. Therefore, convergent evolution of HNLs from various ancestoral enzymes is conceivable. Hybridization of SbHNL cDNA to northern blots of total RNAs isolated from various organs of young sorghum seedlings shows the same expression pattern of HNL as found by means of western blotting or enzyme assays. Using PCR and Southern blot analysis, we demonstrated that the gene of SbHNL is free of introns. Further sequence analysis of cDNA clones and genomic DNA revealed a stretch of 23 adenine residues in the 3'-untranslated part of the gene. Both, intronless organisation of the gene and a genomic stretch of oligo A suggests that SbHNL may have evolved by a reverse transcription event.
Plant Mol Biol 1994 Oct
PMID:Molecular cloning of hydroxynitrile lyase from Sorghum bicolor (L.). Homologies to serine carboxypeptidases. 794 27

The fibronectin (Fn) monomer contains two major sites of fibrin binding affinity present within the NH2-terminal and COOH-terminal domains; they consist of five (1F1-5F1) and three (10F1-12F1) consecutive type 1 modules, respectively. Recently, we have reported that the fourth and fifth type 1 module pair (4F1.5F1) of the NH2-terminal domain of fibronectin demonstrated fibrin binding ability (Williams, M. J., Phan, I., Harvery, T. S., Rostagno, A., Gold, L. I., and Campbell, I. D. (1994) J. Mol. Biol. 235, 1303-1311). In an attempt to further localize fibrin binding activity and to characterize the nature of the interaction between different type 1 modules of Fn and fibrin, we have tested a range of recombinant proteins and subtilisin generated proteolytic fragments of Fn in an enzyme-linked immunosorbent assay (ELISA) and by fibrin affinity chromatography. Of the recombinant proteins, we found that only the 4F1.5F1 exhibited significant fibrin binding activity, while 1F1, 1F1.2F1, 7F1, and 10F1 had little to no affinity for fibrin. On a molar basis, 4-5 times more 4F1.5F1 than a proteolytic fragment, corresponding to 1F1-5F1 (25.9 kDa) was required to cause 50% inhibition (IC50) of intact biotinylated Fn binding to fibrin in a competitive ELISA. This suggests that all five type 1 modules in tandem engender higher fibrin binding activity than the 4F1.5F1 alone. Furthermore, since fibrin binding activity of the intact Fn molecule was inhibited, by 70-80%, by the 4F1.5F1, the 25.9-kDa fragment, and a MoAb mapped to an epitope on the 4F1.5F1, the fibrin-binding site within the 4F1.5F1 contributes greatly to the non-covalent interaction of intact Fn with fibrin. These results provide significant insight into the Fn/fibrin interaction, a major component of the processes of wound repair and fibrin matrix assembly.
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PMID:Further characterization of the NH2-terminal fibrin-binding site on fibronectin. 798 69

The extracellular portion of the interleukin-1 receptor (IL-1R) is sufficient for high-affinity binding to IL-1; however, the structural basis for binding of the receptor to IL-1 is not known. To produce individual domains of IL-1 receptor for structural studies, we constructed a molecular fusion of IL-1 receptor with immunoglobulin G heavy chain that contains a protease specific sequence joining the two portions of the molecule (IL-1R-G-IgG). We introduced the hexapeptide sequence AAHY:TL (where ":" denotes the scissile bond) at the junction of the IL-1R and IgG regions, for specific cleavage by an H64A variant of subtilisin BPN' (Genenase I), an endoprotease that cleaves selectively at this sequence (Carter et al., (1989) Proteins 6, 240-248). Plasmid DNA encoding the fusion protein was used to transfect human embryonic kidney 293 cells transiently, and secreted IL-1R-G-IgG was purified from cell supernatants by protein A chromatography. The IL-1 receptor's extracellular region was then generated by enzymatic cleavage with Genenase I which was immobilized on controlled-pore glass. Incubation of IL-1R-G-IgG with immobilized Genenase I resulted in specific cleavage at the target site, as confirmed by SDS-PAGE, immunoblotting and direct sequencing of the newly generated termini. The resulting soluble IL-1R was separated from the immunoglobulin Fc cleavage product by re-chromatography on protein A. The purified, soluble IL-1R retained quantitatively the ability to bind to its ligand, IL-1 beta. This approach offers a generic means by which the extracellular region of a given type I transmembrane receptor can be expressed as an immunoadhesin, released enzymatically and then easily purified for crystallographic or ligand binding studies.
Mol Immunol 1994 Dec
PMID:Generation of soluble interleukin-1 receptor from an immunoadhesin by specific cleavage. 799 45

1H, 13C and 15N NMR assignments of the backbone atoms of subtilisin 309, secreted by Bacillus lentus, have been made using heteronuclear 3D NMR techniques. With 269 amino acids, this protein is one of the largest proteins to be sequentially assigned by NMR methods to date. Because of the size of the protein, some useful 3D correlation experiments were too insensitive to be used in the procedure. The HNCO, HN(CO)-CA, HNCA and HCACO experiments are robust enough to provide most of the expected correlations for a protein of this size. It was necessary to use several experiments to unambiguously determine a majority of the alpha-protons. Combined use of HCACO, HN(COCA)HA, HN(CA)HA, 15N TOCSY-HMQC and 15N NOESY-HMQC experiments provided the H alpha chemical shifts. Correlations for glycine protons were absent from most of the spectra. A combination of automated and interactive steps was used in the process, similar to that outlined by Ikura et al. [(1990) J. Am. Chem. Soc., 112, 9020-9022] in the seminal paper on heteronuclear backbone assignment. A major impediment to the linking process was the amount of overlap in the C alpha and H alpha frequencies. Ambiguities resulting from this redundancy were solved primarily by assignment of amino acid type, using C alpha chemical shifts and 'TOCSY ladders'. Ninety-four percent of the backbone resonances are reported for this subtilisin. The secondary structure was analyzed using 3D 15N NOESY-HMQC data and C alpha secondary chemical shifts. Comparison with the X-ray structure [Betzel et al. (1992) J. Mol. Biol., 223, 427-445] shows no major differences.
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PMID:1H, 13C and 15N NMR backbone assignments and secondary structure of the 269-residue protease subtilisin 309 from Bacillus lentus. 801 37

The subtilisin molecule possesses several internal water molecules, which may be characterised as an integral part of the protein structure. We have introduced specific mutations (T71I, T71S, T71V, T71A and T71G) at position 71 in the subtilisin variant Savinase from Bacillus lentus. This position is involved in a hydrogen bonded network with several internal water molecules, forming a water channel. The water channel and most of the other internal water molecules are positioned in the interface between two half-domains of the subtilisin molecule. The data presented here indicate that the internal water molecules are structural, and may be the result of trapping during the folding process.
J Mol Biol 1994 Sep 23
PMID:Cavity mutants of Savinase. Crystal structures and differential scanning calorimetry experiments give hints of the function of the buried water molecules in subtilisins. 808 41

In prsA (protein secretion) mutants of Bacillus subtilis, decreased levels of exoproteins, including alpha-amylase and subtilisins, are found extracellularly. The effect of prsA on subtilisin secretion is elaborated here. Extracytoplasmic folding and secretion of active subtilisin is assisted by the N-terminal pro-sequence of its precursor. In this paper we present evidence that the product of the prsA gene is additionally required for these processes in vivo. We examined inducible expression of different subtilisin-alkaline phosphatase fusion genes in the prsA3 mutant. We found massive degradation of the fusion proteins, and a lack of enzymatic activity in the protein secreted. We suggest that PrsA is a novel chaperone with a predicted extracytoplasmic location, and is important in vivo for the proper conformation of various exoproteins, including those with pro-sequence (like subtilisin) and those without (like alpha-amylase).
Mol Microbiol 1993 May
PMID:Bacillus subtilis PrsA is required in vivo as an extracytoplasmic chaperone for secretion of active enzymes synthesized either with or without pro-sequences. 810 73

The complex between a member of the barley malt alpha-amylase isozyme 2 family (AMY2-2), and the endogenous bifunctional alpha-amylase/subtilisin inhibitor, BASI, has been crystallized by the hanging drop vapour diffusion technique at a AMY2-2: BASI molar ratio of 1:1. Crystals have been grown within 4 days from solutions containing polyethylene glycol and calcium chloride. Analysis of single crystals by gel electrophoresis showed the presence of both proteins in the crystal lattice. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 74.5 A, b = 96.9 A, c = 171.3 A and they diffract to 2.0 A resolution. The presence of two molecules of the 1:1 complex in the asymmetric unit gives a solvent content of 45% by volume. The 1:1 stoichiometry of the complex was confirmed by the molecular replacement method, using as a search model the recently determined three-dimensional structure of the barley alpha-amylase.
J Mol Biol 1994 Feb 11
PMID:Characterization, crystallization and preliminary X-ray crystallographic analysis of the complex between barley alpha-amylase and the bifunctional alpha-amylase/subtilisin inhibitor from barley seeds. 810 17


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