Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A Ca2+-independent phospholipase A2 (PLA2) maximally active at pH 4 and specifically inhibited by the transition-state analogue 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33) was isolated from rat lungs. The sequence for three internal peptides (35 amino acids) was used to identify a 1653-base pair cDNA clone (HA0683) from a human myeloblast cell line. The deduced protein sequence of 224 amino acids contained a putative motif (GXSXG) for the catalytic site of a serine hydrolase, but showed no significant homology to known phospholipases. Translation of mRNA produced from this clone in both a wheat germ system and Xenopus oocytes showed expression of PLA2 activity with properties similar to the rat lung enzyme. Apparent kinetic constants for PLA2 with dipalmitoylphosphatidylcholine as substrate were Km = 0.25 mM and Vmax = 1.89 nmol/h. Activity with alkyl ether phosphatidylcholine as substrate was decreased significantly compared with diacylphosphatidylcholine. Significant lysophospholipase, phospholipase A1, or 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine acetylhydrolase activity was not observed. Enzyme activity was insensitive to p-bromophenacyl bromide, bromoenol lactone, trifluoromethylarachidonoyl ketone, mercaptoethanol, and ATP, but was inhibited by MJ33 and diethyl p-nitrophenyl phosphate, a serine protease inhibitor. SDS-polyacrylamide gel electrophoresis with autoradiography of the translated [35S]methionine-labeled protein confirmed a molecular mass of 25.8 kDa, in good agreement with the enzyme isolated from rat lung. By Northern blot analysis, mRNA corresponding to this clone was present in both rat lung and isolated rat granular pneumocytes. These results represent the first molecular cloning of a cDNA for the lysosomal type Ca2+-independent phospholipase A2 group of enzymes.
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PMID:Identification of a human cDNA clone for lysosomal type Ca2+-independent phospholipase A2 and properties of the expressed protein. 899 71

Members of the serpin (serine protease inhibitor) family share a similar backbone structure but expose a variable reactive-site loop, which binds to the catalytic groove of the target protease. Specificity originates in part from the sequence of this loop and also from secondary binding sites that contribute to the inhibitor function. To clarify the intrinsic contribution of the reactive-site loop, alpha1-antichymotrypsin has been utilized as a scaffold to construct chimeras carrying the loop of antithrombin III, protease nexin 1, or alpha1-antitrypsin. Reactive-site loops not only vary in sequence but also in length; therefore, the length of the reactive-site loop was also varied in the chimeras. The efficacy of the specificity transfer was evaluated by measuring the stoichiometry of the reaction, the ability to form an SDS-stable complex, and the association rate constant with a number of potential targets (chymotrypsin, neutrophil elastase, trypsin, thrombin, factor Xa, activated protein C, and urokinase). Overall, substitution of a reactive-site loop was not sufficient to transfer the specificity of a given serpin to alpha1-antichymotrypsin. Specificity of the chimera partly matched that of the loop donor and partly that of the acceptor, whereas the behavior as an inhibitor or a substrate depended upon the targeted protease. Results suggest that, aside from the contributions of the loop sequence and the framework-specific secondary binding sites, an intramolecular control may be essential for productive interaction.
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PMID:Intrinsic specificity of the reactive site loop of alpha1-antitrypsin, alpha1-antichymotrypsin, antithrombin III, and protease nexin I. 919 29

Neuroserpin is a serine protease inhibitor of the serpin family that has been identified as an axonally secreted glycoprotein in neuronal cultures of chicken dorsal root ganglia. To obtain an indication for possible functions of neuroserpin, we analyzed its expression in the developing and the adult CNS of the mouse. In the adult CNS, neuroserpin was most strongly expressed in the neocortex, the hippocampal formation, the olfactory bulb, and the amygdala. In contrast, most thalamic nuclei, the caudate putamen, and the cerebellar granule cells were devoid of neuroserpin mRNA. During embryonic development, neuroserpin mRNA was not detectable in neuroepithelia, but it was expressed in the differentiating fields of most CNS regions concurrent with their appearance. In the cerebellum, the granule cells and a subgroup of Purkinje cells were neuroserpin-positive during postnatal development. As a further step toward the elucidation of neuroserpin function, we performed a study to identify potential target proteases. In vitro, neuroserpin formed SDS-stable complexes and inhibited the amidolytic activity of tissue plasminogen activator, urokinase, and plasmin. In contrast, no complex formation with or inhibition of thrombin was found. Expression pattern and inhibitory specificity implicate neuroserpin as a candidate regulator of plasminogen activators, which have been suggested to participate in the modulation or reorganization of synaptic connections in the adult. During development, neuroserpin may attenuate extracellular proteolysis related to processes such as neuronal migration, axogenesis, or the formation of mature synaptic connections.
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PMID:Expression of neuroserpin, an inhibitor of tissue plasminogen activator, in the developing and adult nervous system of the mouse. 936 46

The complex of the type-1 plasminogen activator inhibitor (PAI-1) and its target proteinases, the urokinase and tissue-type plasminogen activators (uPA and tPA), but not the free components, bind with high affinity to the endocytosis receptors alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein (alpha2MR/LRP) and very-low-density lipoprotein receptor (VLDLR). To characterize the molecular interaction between the complexes and the receptors, alanine codons were introduced into the human PAI-1 cDNA to replace the four basic residues, Arg-78, Lys-82, Arg-120 and Lys-124, as double mutations. The purified recombinant mutant proteins, rPAI-1/R78A-K124A and rPAI-1/K82A-R120A, produced by the yeast Pichia pastoris, were indistinghuisable from wild-type recombinant and natural human PAI-1 with respect to inhibitory activity against uPA, stability of SDS-resistant complexes with uPA, and vitronectin binding. Radiolabelled mutant uPA.PAI-1 complexes bound with a 10- to 20-fold, and 3- to 7-fold reduced affinity to purified alpha2MR/LRP and VLDLR respectively. alpha2MR/LRP-mediated endocytosis of the mutant complexes by COS-1 cells was reduced to 48 and 38% of the level of endocytosis of wild-type PAI-1. Binding of the mutant complexes to the uPA receptor was not affected. These findings suggest that the binding mode of the uPA.PAI-1 complex to both alpha2MR/LRP and VLDLR is similar. The four residues are surface exposed in the region defined by alpha-helix D and beta-strand 1A in the serine protease inhibitor (serpin) structure. Our study represents the first identification of residues in a surface region implicated in molecular recognition of protease.serpin complexes by endocytosis receptors of the low-density lipoprotein receptor family.
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PMID:Binding of urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex to the endocytosis receptors alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein and very-low-density lipoprotein receptor involves basic residues in the inhibitor. 940 75

Diisopropylfluorophosphate (DFP), a general serine protease inhibitor, inhibited the DNA fragmentation and cell death in MDCK cells treated with ricin, modeccin, Pseudomonas toxin, or diphtheria toxin. A trypsin-like serine protease inhibitor, N-tosyl-L-lysine chloromethyl ketone (TLCK) also prevented ricin-induced DNA fragmentation and cell death, albeit less effectively than DFP. Microscopic observation showed that the morphological changes of MDCK cells induced by ricin were prevented by DFP. DFP did not affect the binding, internalization, or subsequent excretion of ricin, but reduced the degradation of ricin in MDCK cells, suggesting that DFP inhibits at least the cellular protease that may be involved in the degradation of internalized ricin. In addition, SDS-PAGE analysis of cytosolic proteins suggested that DFP-sensitive endogenous proteases are activated in the ricin-treated cells. In the cells treated with DFP, the protein synthesis inhibitory activity of ricin was increased rather than inhibited. The activities of modeccin and Pseudomonas toxin were also slightly increased by DFP, but no effect of DFP on the activity of diphtheria toxin was observed. Therefore, these results suggest that protein toxins have a DFP-sensitive common pathway leading to apoptosis that is distinct from the pathway leading to the inhibition of cellular protein synthesis.
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PMID:Diisopropylfluorophosphate (DFP) inhibits ricin-induced apoptosis of MDCK cells. 953 90

An important biochemical hallmark of apoptosis is the cleavage of chromatin into oligonucleosomal fragments. Here, we purified a Mg2+-dependent endonuclease from etoposide-treated HL-60 cells undergoing apoptosis. High levels of Mg2+-dependent endonuclease activity were detected in etoposide-treated HL-60 cells, and this activity increased in a time-dependent manner following etoposide treatment. Such an activity could not be detected in untreated cells or in cells treated with etoposide in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethyl ketone (zVAD-fmk) or the serine protease inhibitor tosyl-L-phenylalanine chloromethyl ketone (TPCK). This Mg2+-dependent endonuclease was purified by a series of chromatographic procedures. The enzyme preparation showed a single major protein band with Mr 34,000, determined by SDS-PAGE. The presence of the Mr 34,000 Mg2+-dependent endonuclease was also confirmed by activity gel analysis. The enzyme required only Mg2+ for full activity. pH optimum was in the range of 6.5-7.5. This enzyme introduced single- and double-strand breaks into SV40 DNA and produced internucleosomal DNA cleavage in isolated nuclei from untreated cells. The DNA breaks were terminated with 3'-OH, consistent with characteristic products of apoptotic chromatin fragmentation. We propose to designate this Mr 34,000 Mg2+-dependent endonuclease AN34 (apoptotic nuclease Mr 34,000).
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PMID:Purification and characterization of a Mg2+-dependent endonuclease (AN34) from etoposide-treated human leukemia HL-60 cells undergoing apoptosis. 963 81

Rumen fluid was taken from fistulated sheep and a cow receiving various diets based on grass hay or grass cubes with and without cereal-based concentrates. Proteinases in the extracellular fluid, and extracted from particulate material by Triton X-100, were visualized using SDS-PAGE in which gelatin was co-polymerized with the gels. Each animal sampled had a different pattern of proteinase activity. No single proteinase band predominated, although a few appeared in several samples, indicating that some microbial species were commonly involved in proteolysis but none was dominant. The banding patterns in samples taken from the same animals two weeks apart were fairly similar, indicating some stability within animals. Patterns obtained with extracellular fluid and Triton X100 extracts of small and large particulate material from the same sample were similar for the most part, although the relative intensity of the bands differed. The serine protease inhibitor, phenyl methyl sulphonyl fluoride, had little influence on the banding pattern. Concentrate in the diet appeared to increase inter-animal variation, and sheep in adjacent pens and consuming the same grass hay:concentrate diet had different banding patterns. Thus, no microbial proteolytic enzyme was predominant in protein digestion in the rumen.
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PMID:Variation in proteinase activities in the rumen. 972 42

Beta-amyloid precursor proteins (APPs) in the subcellular fractions of the homogenate of rat brain were detected immunologically. They were found to be localized in both the cytosol and microsome fractions in generally equal amounts. APPs were purified from the cytosol fraction of rat brain by column chromatography in a DEAE-anion-exchanger, Blue-Sepharose, Ni-charged chelating Sepharose, and Sephacryl S-300 columns. They migrated at about 400 kDa or above in a final gel filtration column with trypsin inhibitor activity. They gave two broad protein bands of 80 and 100 kDa and several other protein bands in sodium dodecyl sulfate-polyacryl amide gel electrophoresis (SDS-PAGE). The 80 and 100 kDa bands were highly concentrated during purification. They gave the same amino terminal sequence and were identified as rat APPs without an amino terminal signal sequence. These results suggest that rat brain APPs form a complex with themselves or with other proteins and contain APP isoforms including a serine protease inhibitor domain, APP770 or APP751, or both. An antibody produced by a rabbit immunized with the final preparation of APPs reacted with a 95 kDa protein band which migrated between the 80 and 100 kDa bands of APPs in SDS-PAGE, but it did not react with the bands of APPs. The 80 and 100 kDa APP bands were coprecipitated with a 95 kDa antigen protein band by reacting this antibody with the partially purified APPs. We conclude that APPs in the rat brain are associated directly or indirectly with another protein to yield the 95 kDa band demonstrated by SDS-PAGE.
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PMID:Purification by column chromatographies of beta-amyloid precursor proteins and their association with other 95 kDa protein in rat brain. 982 23

Human kallikrein (hK) 2 is an arginine-selective serine protease expressed predominantly in the prostate that has an 80% sequence identity with prostate-specific antigen. Expression of hK2 is elevated in the tumor epithelium compared to benign prostate tissue. We have purified, sequenced, and identified a novel hK2 complex in prostate tissue consisting of hK2 and a serine protease inhibitor known as protease inhibitor-6 (PI-6). This 64-kDa SDS-PAGE stable complex is elevated in the tumor and is approximately 10% of total hK2. No comparable complex of prostate-specific antigen was detected. PI-6, also known as cytoplasmic antiprotease, has been characterized as an intracellular inhibitor of trypsin and chymotrypsin-like proteases, which has high homology to plasminogen activator inhibitor 1 and 2. The physiological role of PI-6 in the prostate and its relationship to hK2 and prostate cancer are under investigation.
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PMID:Identification of a novel complex between human kallikrein 2 and protease inhibitor-6 in prostate cancer tissue. 1046 85

Activins are pluripotent growth factors that have recently been shown to be present in placental and fetal membrane preparations. Our previous studies have identified and purified activin A from ovine amniotic and allantoic fluids. In this study, ligand blots of side fractions from the isolation of activin A from allantoic fluid suggested the presence of activin-binding proteins other than follistatin. Further purification of one of these fractions involved two sequential reverse phase HPLC steps and a Superose 12HR fractionation. SDS-PAGE revealed a single protein band of 55 kDa, which was identified by NH2-terminal sequencing as ovine uterine milk protein (UTMP), a member of the serine protease inhibitor (serpin) superfamily of proteins. Further binding studies, using ligand blot techniques and Superose 12HR fractionation in the presence of [125I]activin, demonstrated UTMP to be an activin-binding protein with a lower affinity for activin than that of follistatin. A study of the specific binding behavior of UTMP to activin, using surface plasmon resonance, revealed an apparent equilibrium dissociation constant (Kd) of 49 +/- 25 nM, compared with the follistatin-activin Kd of 379 +/- 51 pM. Similar to another activin-binding protein, alpha2-macroglobulin, UTMP was unable to neutralize the bioactivity of activin in a bioassay based on the capacity of activin to inhibit the proliferation of an MPC-11 plasmacytoma cell line. The high concentrations of this protein in uterine fluid during pregnancy and its ability to bind activin suggest that UTMP may act as a low affinity, high capacity binding protein to sequester activin in the local uterine environment.
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PMID:Uterine milk protein, a novel activin-binding protein, is present in ovine allantoic fluid. 1049 34


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