Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Gene fragments encoding serine proteases expressed in adult buffalo fly (Haematobia irritans exigua) were amplified from cDNA using generic oligonucleotide PCR primers, based on conserved residues surrounding the active-site His and Ser amino acids found in all serine proteases. The PCR product consisted of a broad band extending from about 450 bp to 520 bp, which suggested that the PCR product actually consisted of numerous DNA fragments of slightly variable sizes. Seventeen independent clones of these fragments, each with an insert of approximately 480 bp, were digested with HaeIII. Comparison of restriction fragment patterns indicated that 13 of these clones harboured different PCR products. This was confirmed by DNA sequence analysis of 9 clones. Each of the sequenced clones contained an open reading frame which included structurally conserved regions characteristic of the serine protease superfamily. This study reveals the expression of a large and highly variable repertoire of serine proteases in adult buffalo fly. Importantly, these data also demonstrate the utility of such an approach in obtaining DNA probes for use in further investigations of gene family organization and expression, as well as providing recombinant antigens in the form of fusion proteins which may be used as candidates for vaccine production.
Mol Gen Genet 1993 Jul
PMID:A family of serine protease genes expressed in adult buffalo fly (Haematobia irritans exigua). 834 Dec 58

Moulting fluid of pharate adult tobacco hornworm moths, Manduca sexta, contains a novel cuticle-degrading proteinase, designated as MFP-1. The enzyme has been purified using heparin affinity chromatography and partially characterized. Before purification MFP-1 is associated with a large complex having an apparent native molecular mass > 669 kDa. After purification MFP-1 has a molecular mass of 41 kDa. The pI of the enzyme is 5.54. MFP-1 can be classified as generally trypsin-like on the basis of its substrate specificity and inhibition by soybean trypsin inhibitor. The enzyme's preferred substrate, Tos-Gly-Pro-Arg-pNA, its inhibition by hirudin, and its affinity for heparin, all indicate that MFP-1 has some characteristics in common with the vertebrate blood-clotting enzyme thrombin. MFP-1 is probably a serine protease, since it is inhibited by both DFP and PMSF (specific inhibitors of serine proteinases). However, the enzyme was also inhibited by a number of agents that affect cysteine proteinases. Purified MFP-1 degrades Manduca cuticle in vitro. We suggest that the enzyme may act as the first step in the degradation of the cuticle during the moulting process.
Insect Biochem Mol Biol 1993 Jul
PMID:A cuticle-degrading proteinase from the moulting fluid of the tobacco hornworm, Manduca sexta. 835 21

Lipoprotein lipase (LpL) activity in Manduca sexta flight muscle tissue was measured using in vivo radiolabeled lipophorin as a substrate. LpL hydrolyses diacylglycerol in the low density lipophorin (that occurs during flight) at a higher rate than diacylglycerol in the high density lipophorin (present in the resting insect). LpL has a pH-optimum of 7.5 and is less sensitive to NaCl than mammalian LpL. LpL is inhibited by bovine albumin and chicken ovalbumin. LpL is inhibited by the serine protease inhibitors diisopropylfluorophosphate (DFP) and phenylmethanesulfonyl fluoride (PMSF), which indicates the presence of an active site serine similar to mammalian LpL. Flight muscle LpL shows affinity for immobilized copper as well as for immobilized heparin. Using radiolabeled DFP, a protein of 37 kDa was identified (after SDS-PAGE) as the DFP-binding protein in a partially purified preparation of LpL. This 37 kDa protein is proposed to be the LpL or a subunit thereof.
Insect Biochem Mol Biol 1993 Oct
PMID:Characterization and identification of a lipoprotein lipase from Manduca sexta flight muscle. 837 12

A structure-activity study has been carried out on several compounds known as inhibitors of the serine protease prolyl endopeptidase. Conformational analysis has been done using different molecular mechanics methods such as molecular dynamics, or a randomized conformational search method. The conformers obtained were classified using geometric and energetic criteria. A pattern recognition analysis was done in order to divide conformers according to families. The resulting dominant families, for all compounds investigated, showed very similar geometric features. Based on the lowest energy conformers obtained after randomized conformational analysis, a 3D-QSAR model was established using the CoMFA approach. The validity of this model was verified by predicting correctly the activity of other molecules not used in the construction of this model.
J Comput Aided Mol Des 1993 Jun
PMID:Inhibitors of prolyl endopeptidase: characterization of the pharmacophoric pattern using conformational analysis and 3D-QSAR. 837 23

Subtilisin BPN' is an extracellular serine protease from Bacillus amyloliquefaciens that requires an N-terminal 77 amino acid pro-sequence for correct folding of the catalytic domain. We have expressed an inactive, stable pro-subtilisin variant in Escherichia coli and show that it has structural properties similar to native subtilisin in terms of its near- and far-UV circular dichroism spectra, its compactness, and its capacity to bind calcium ions stoichiometrically. Unlike subtilisin, the pro-subtilisin variant unfolds reversibly with guanidinium chloride, and unfolding occurs via a folding intermediate. This intermediate is similar to the metastable intermediate state recently found for folding of subtilisin in the absence of the pro-sequence. The intermediate state has native-like secondary but little tertiary structure, and has a compactness between that of the native and unfolded state. Pro-subtilisin folds from the intermediate to the folded state in a single co-operative transition mediated by the pro-sequence. The isolated pro-sequence does not appear from its circular dichroism and 1H-NMR spectrum to have enough intrinsic stabilizing interactions to fold autonomously. However, the difference circular dichroism spectra of the pro-subtilisin variant and native subtilisin suggest that it is folded in the context of the pro-subtilisin molecule. The inability of the pro-subtilisin variant to bind a polypeptide inhibitor supports further the hypothesis that the pro-sequence interacts with subtilisin in the region where the active site is exposed. Our results suggest that the interactions provided by the pro-sequence are important only late on the folding pathway of pro-subtilisin and stabilize the transition state for folding. Kinetic analysis of the refolding reaction in the presence and absence of the pro-sequence reveal this stabilization to be in excess of 7.5 kcal/mol; folding is accelerated more than five orders of magnitude.
J Mol Biol 1993 Sep 20
PMID:Folding of subtilisin BPN': role of the pro-sequence. 837 4

Membrane cofactor protein (MCP) is a complement regulatory protein that acts as a cofactor for the cleavage of C3b and C4b by the serine protease factor I. We have previously reported the characterization of a functional MCP molecule on the acrosomal membrane. This protein migrated as a single band with a molecular weight of 40,000 Da, which is 10,000-20,000 Da smaller than the known MCP molecules, and is devoid of N- and O-linked sugars. We have proposed that the difference in molecular weight resulted from the lack of sugars. To investigate if this is due to the absence of glycosylation sites, we have characterized a cDNA clone from a human testis cDNA library. This cDNA corresponds to a peculiar MCP form previously described, which is characterized by the presence of the serine/threonine/proline-rich exon C (STPC) and the cytoplasmic tail known as CYT2, and we conclude that the absence of mature oligosaccharide of the sperm MCP cannot be totally attributed to a defect of N- and O-glycosylation sequences but rather reflects an alteration of the mechanisms of glycosylation in spermatozoa. The presence of functional MCP on the acrosomal membrane, as well as the other complement regulatory protein, decay-accelerating factor, strongly suggests that these proteins may act concomitantly to protect the acrosome-reacted spermatozoa from the attack of the complement present in the female genital tract.
Mol Reprod Dev 1993 Jan
PMID:Characterization of a cDNA clone coding for human testis membrane cofactor protein (MCP, CD46). 841 11

The problem of why serine protease inhibitors having substrate-like structure around the reactive site are not degraded by the cognate protease has prompted us to investigate the structural requirements in Streptomyces subtilisin inhibitor (SSI) for its inhibitory action. We removed the disulfide bridge between Cys71 and Cys101 near the reactive site by oligonucleotide-directed mutagenesis, replacing both Cys residues with Ser residues. Inhibitory activity of the mutated SSI toward subtilisin BPN' was initially potent, but decreased remarkably with increasing incubation time after mixing (temporary inhibition), due to degradation of the mutated SSI by subtilisin via a specific intermediate with a nick at the reactive site (Met73-Val74). Binding affinity of subtilisin for the mutated SSI was reduced by more than 10(3)-fold, and the mutated SSI showed a 20 deg.C decrease in melting temperature, which probably mainly reflects the destruction of the region of alpha-helix containing Cys101. These results imply that the susceptibility of the mutated SSI to protease, and the irreversibility of the peptide bond cleavage at the reactive site, result from increased flexibility around the reactive site in the complex of the disulfide-bond-removed SSI with the protease, demonstrating the requirement of conformational rigidity around the reactive site of the inhibitor for its native inhibitory action.
J Mol Biol 1993 Mar 20
PMID:Requirement for a disulfide bridge near the reactive site of protease inhibitor SSI (Streptomyces subtilisin inhibitor) for its inhibitory action. 846 55

A high molecular mass alkaline proteinase was purified by DEAE-Sepharose and Mono Q chromatography. The mol. wt was estimated to be about 600,000. Under denaturing conditions, the enzyme dissociated into a cluster of subunits with mol. wt ranging from 25,000 to 30,000. The isoelectric point of the enzyme was about pH 7.3. The proteinase was able to hydrolyse N-terminal-blocked 4-methyl-7-coumarylamide substrates for either trypsin- or chymotrypsin-like activity. It was also able to hydrolyse haemoglobin and myosin at temperatures of about 60 degrees C. The activities responded to pH and some chemicals in different ways. The trypsin-like activity was clearly inhibited by several serine protease inhibitors. These results suggest that the enzyme is multicatalytic, having at least two different active sites.
Comp Biochem Physiol B Biochem Mol Biol 1995 Aug
PMID:Purification and characterization of a multicatalytic proteinase from Atlantic salmon (Salmo salar) muscle. 857 23

Serine proteases play an important role in a diverse array of biological processes, including embryogenesis, metastasis, angiogenesis, thrombolysis and tissue invasion by certain parasites. The latter observation prompted us to explore the possibility that the tissue-invasive ocular parasite Acanthamoeba castellanii elaborates one or more serine proteases. Acanthamoeba sp. are pathogenic free-living amoebae that can produce an invasive, blinding inflammatory disease of the cornea, termed Acanthamoeba keratitis. The present study reports the preliminary purification and characterization of a novel plasminogen activator from an ocular isolate of A. castellanii. The parasite-derived enzyme has a molecular mass of approx. 40 kDa and produces a single band of lysis on fibrinogen-agarose zymographs. Activity of the enzyme is completely inhibited by treatment with diisopropylfluorophosphate, indicating that it is a serine protease. The parasite-derived serine protease is not inhibited by amiloride which is a strong inhibitor of urokinase-type plasminogen activator. Additionally, the enzyme is not inhibited by plasminogen activator inhibitor-1 which is the primary physiological inhibitor of both urokinase and tissue-type plasminogen activator. It does not cross-react with antibodies specific for human urokinase or tissue-type plasminogen activator. The parasite-derived enzyme activates plasminogen from several mammalian species, including human, cow and pig. Thus, it is possible that this parasite-derived serine protease contributes to the pathogenesis of Acanthamoeba keratitis.
Mol Biochem Parasitol 1995 Jul
PMID:Characterization of a plasminogen activator produced by Acanthamoeba castellanii. 857 23

Factor IX is an essential vitamin K-dependent serine protease that participates in the intrinsic pathway of coagulation. The protein is expressed exclusively in the liver. The rare Leyden form of hemophilia B (inherited factor IX deficiency) results from point mutations in three proximal promoter elements that decrease factor IX expression. Recovery of expression occurs following puberty, with factor IX protein levels rising into the normal range. We have previously implicated the PAR domain D-site-binding protein (DBP) as well as an upstream element, site 5, as playing important roles in the phenotypic recovery of hemophilia B Leyden. Here we demonstrate that site 5 binds both the CCAAT/enhancer-binding protein (C/EBPalpha) and the ubiquitous Ets factor GA-binding protein (GABPalpha/beta). Transactivation of the factor IX promoter by the PAR proteins DBP and hepatic leukemia factor (HLF) is dependent on the binding of GABPalpha/beta to site 5, and coexpression of these two factors is required for optimal activation of this promoter. The binding of C/EBPalpha to site 5 also augments the activity of GABPalpha/beta. Analysis of the developmental regulation of site 5-binding proteins in rat liver has shown that C/EBPalpha and the GABPbeta subunit increase markedly in the 2 weeks after birth. These observations establish a functional association between the Ets factor GABPalpha/beta and C/EBPalpha and indicate that the two PAR proteins, DBP and HLF, may play complementary roles in factor IX activation. Given the developmental changes exhibited by these proteins, it is likely that they play a role in regulation of the normal factor IX promoter as well as promoters carrying hemophilia B Leyden mutations.
Mol Cell Biol 1996 May
PMID:Binding of the Ets factor GA-binding protein to an upstream site in the factor IX promoter is a critical event in transactivation. 862 59


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