Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
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Kinin release and its involvement in inflammatory exudation were assessed by immuno-blot analysis of the kinin-precursor protein, high molecular weight kininogen (HK). HK consists of heavy (H) chain, bradykinin and light (L) chain. After bradykinin was released by plasma kallikrein, HK remains two-chain-kinin-free form;, i.e., H-chain and L-chain link each other through a disulfide bond. By Western blot analysis using antibody recognizing the light chain of HK, a band of 110-kD mass, which corresponds to intact HK, was detected in plasma after SDS-PAGE under reducing conditions, while a 46-kD band, corresponding to the light chain of HK, but no 110-kD band, was found in the exudate of rats with carrageenin-induced pleurisy at 3 hr as well as at 16 h. This result indicates that in the exudate most all of the HK molecules had released kinin to form kinin-free-HK, whereas the HK in the plasma remained intact. On the contrary, low molecular weight kininogen (LK) in the exudate was mostly in its intact form. These results indicate that plasma kallikrein could be activated in the exudate to release kinin from HK, as it reacts exclusively with HK and not with LK, and they are also mostly consistent with the features of the kinin release from the exudate and the plasma. That is, no kinin was detected in the exudate when the latter was incubated with plasma kallikrein, whereas salivary kallikrein did release kinin, indicating that kinin had already been released from HK, but not from LK in the exudate. Immunoblot analysis of HK in the pleural exudate also demonstrated no kinin involvement in phorbol myristate acetate- or zymosan-induced pleurisy, since no light chain band, but an intact HK band, was found in the exudates from these pleurisies.
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PMID:The kinin released from high molecular weight-kininogen is responsible for inflammatory exudation in rats: detection of kinin-free-kininogen in the exudate by immunoblot analysis. 823 50

The major constituent of senile plaques (one of the hallmark lesions of Alzheimer's disease) is a 42(43)-amino-acid polypeptide, termed the A4 or beta-amyloid peptide. The beta-amyloid peptide or A4 is derived from one or more larger beta-amyloid precursor proteins. The precursor protein from whence the A4 peptide is derived is highly conserved throughout evolution, and humans, monkeys, dogs, and bears develop brain deposits of A4 peptide in amyloid fibrils. However, similar accumulations of A4 amyloid are negligible in the brains of rats and mice for reasons that remain unexplored. Notably, the A4 sequence of rodents, deduced from the cDNA clones, differs only in three amino acids from the A4 isolated from the brain of humans. Hence, these differences could account for the inability of rodents to develop Alzheimer-like A4 amyloid plaques. To test this hypothesis directly, using physical and chemical model systems, we synthesized, purified, and characterized A4 peptides corresponding to the human and rodent sequences. Circular dichroic and Fourier-transform infrared spectroscopy were used with various membrane-mimicking solvents, different peptide concentrations, and variable pH to identify those environmental conditions that promoted beta-pleated sheet formation of the human versus rodent A4. At an intermediate alkaline pH (< or = 10), the rodent peptide has more beta-pleated sheet structure than the human sequence. The beta-pleated sheets for both peptides could be eliminated at very high pH (> or = 12). The amount of the beta-structure increased in an octyl glucoside solution, compared to that found in SDS, as well as in several of the other solutions tested here. This suggests that particles originated from prior membrane damage may play a role in the stabilization of beta-pleated sheets with subsequent formation of amyloid deposits. Finally, we found that higher beta-pleated sheet content was observed for the rodent sequences in acetonitrile/water mixtures. In contrast, more beta-pleated sheets were detected with the human A4 in trifluoroethanol/water mixtures at neutral pH. Remarkably, at relatively low peptide concentrations, only the human sequences assumed an extended secondary structure. These data suggest that subtle inter-species amino-acid differences may account for the inability of the rodent peptide to form amyloid fibrils in situ.
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PMID:Human and rodent Alzheimer beta-amyloid peptides acquire distinct conformations in membrane-mimicking solvents. 842 35

Amyloid precursor protein (APP) and cholesterol metabolism are genetically linked to Alzheimer's disease, the latter through apolipoprotein E, a lipid and cholesterol transport protein. We have examined the hypothesis that the processing of APP is disrupted by elevated cholesterol, which is known to modulate the activity of several transmembrane proteins. In the current study, cholesterol, solubilized by methyl- beta-cyclodextrin or ethanol, was added to the culture media of APP 751 stably transfected HEK 293 cells. Radiolabeled APP and APPsol (the soluble N-terminal derivative following alpha-secretase cleavage) were precipitated from lysates and conditioned media of stably transfected HEK 293 cells; the relative levels were determined by quantitative densitometry following separation by SDS-polyacrylamide gel electrophoresis. The data show that cholesterol, solubilized by methyl-beta-cyclodextrin, greatly reduced the levels of APPsol. Low doses of ethanol-solubilized cholesterol similarly caused a dramatic reduction of APPsol. By contrast, levels of APP holoprotein remained the same or increased. The large decrease seen in APPsol production was not due to nonspecific inhibition of secretion because several secreted proteins increased in level. Cholesterol, which impedes membrane fluidity, may lower APPsol production by impeding the interaction of the substrate with its protease(s). If APPsol were to function trophically, as suggested by other studies, the current conclusion suggests that changes in cellular cholesterol levels in Alzheimer's disease could contribute to neuronal degeneration by decreasing the production of APPsol.
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PMID:Cholesterol modulates alpha-secretase cleavage of amyloid precursor protein. 862 95

Active interleukin-1beta-converting enzyme (ICE) is composed of 20- and 10-kDa polypeptides (p20 and p10) derived from the processing of a cytosolic 45-kDa precursor protein (p45). The cleavage and activation of the native p45 ICE precursor have been characterized by use of specific inhibitors and antibodies recognizing various regions of ICE. The processing of p45 in vitro in THP.1 monocytic cell cytoplasmic extracts is inhibited only by protease inhibitors that inhibit ICE and not by inhibitors of other protease classes. The addition of L-742,395, a biotinylated irreversible ICE inhibitor, to these extracts labels only p45 and simultaneously inhibits p45 processing, demonstrating that the p45 has catalytic activity. Following a cleavage of p45 at a site that becomes the COOH terminus of p20, a more active intermediate is formed which migrates on SDS-polyacrylamide gel electrophoresis with an molecular mass of 35 kDa (ED50 of approximately 0.1 microM L-742,395 labeling versus 5 microM for p45). This new more active ICE form serves both as an intermediate enzyme to cleave p45 as well as a substrate for the formation of the final active ICE (ED50 of 1 nM L-742,395 labeling of p20 and for p22, an NH2-terminally extended form of p20). While initial cleavage of p45 can be found at the sites corresponding to both the NH2 termini of p22 and p20, these fragments cannot be labeled by L-742,395 and are hence inactive. p45 is not processed at the site corresponding to the NH2 terminus of the p10. Less than 50% of the p45 is cleaved down to active p20 or p22 ICE as determined by band shift on SDS-polyacrylamide gel electrophoresis of the biotinylated fragments, indicating that the in vitro activation is highly inefficient. The ICE fragmentation occurs by an intermolecular process and is highly dilution sensitive. Cleavage of p45 by exogenous p20/p10 ICE differs from that of the endogenous p45 cleavage activity in that the p20/p10 activity is more salt sensitive, and it produces a different pattern of cleavage fragments, principally 35- and 12-kDa fragments. These results indicate that the nature of the ICE activity changes as p45 is processed down to the p20/p10 form of the enzyme.
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PMID:Activation of the native 45-kDa precursor form of interleukin-1-converting enzyme. 866 43

The precursor of aqualysin I, an extracellular subtilisin-type protease produced by Thermus aquaticus, consists of four domains: an N-terminal signal peptide, an N-terminal pro-sequence, a protease domain, and a C-terminal extended sequence. In an Escherichia coli expression system for the aqualysin I gene, a 38 kDa precursor protein consisting of the protease domain and the C-terminal extended sequence is accumulated in the membrane fraction and processed to a 28 kDa mature enzyme upon heat treatment at 65 degrees C. The 38 kDa precursor protein is separated as a soluble form from denatured E. coli proteins after heat treatment. Accordingly, purification of the 38 kDa proaqualysin I was performed using chromatography. The purified precursor protein gave a single band on SDS-polyacrylamide gels. The precursor protein exhibited proteolytic activity comparable to that of the mature enzyme. The purified precursor protein was processed to the mature enzyme upon heat treatment. The processing was inhibited by diisopropyl fluorophosphate. The processing rate increased upon either the addition of mature aqualysin I or upon an increase in the concentration of the precursor, suggesting that the cleavage of the C-terminal extended sequence occurs through an intermolecular self-processing mechanism.
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PMID:A 38 kDa precursor protein of aqualysin I (a thermophilic subtilisin-type protease) with a C-terminal extended sequence: its purification and in vitro processing. 873 36

Ustilago maydis, a fungal pathogen of corn, can alternate between yeast-like and filamentous growth. This dimorphic switch is governed by the mating-type loci. We have identified an abundant class of small SDS-insoluble cell wall proteins, designated repellents, specifically present in the filamentous form. Genetic analysis revealed that these peptides are processed from a single precursor protein, Rep1. Rep1 comprises 652 amino acids with a leader sequence for secretion. A characteristic feature of Rep1 is 12 repeats of a 37 amino acid consensus sequence; 10 of these repeats are separated by Kex2 protease cleavage sites. In (delta)rep1 mutants formation of aerial hyphae and surface hydrophobicity were reduced dramatically. This and the fact that expression of rep1 is regulated by the mating-type loci indicates that repellents play a structural role in the formation of aerial hyphae.
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PMID:A novel class of small amphipathic peptides affect aerial hyphal growth and surface hydrophobicity in Ustilago maydis. 886 56

The primary structure of beta 2-microglobulin (beta 2m), the major constituent protein of beta 2-microglobulin amyloidosis (A beta 2m) or dialysis-amyloidosis, was initially shown to be identical to serum beta 2m, thereby strongly suggesting the polymerization of intact beta 2m in tissues. Recent biochemical data have been controversial, showing beta 2m acidic isoforms, fragmentation and amino acid sequence alteration of deposited beta 2m. The aim of this study was to reinvestigate beta 2m amyloid deposits for the presence of beta 2m fragments and/or amino acid sequence alteration. Four amyloid-laden tissues (3 femoral bone amyloid cysts and 1 heart tissue) from dialysis patients were used to isolate amyloidogenic beta 2m. Amyloid fibrils were isolated using the classic water extraction method, and purified in 6 M guanidine on a gel-filtration column. The protein was further purified on 17% SDS-PAGE gel, and transferred to a nitrocellulose membrane for immunostaining with antihuman beta 2m. beta 2m samples were microsequenced using the standard 03RPTH program on a 470A gas-phase sequencer, and HPLC was performed after digestion with trypsin. Two peaks were obtained with the gel filtration column, the second corresponding by molecular weight to beta 2m. SDS-PAGE analysis of this peak under reducing conditions, demonstrated one major band at 12,000 Da and a minor band at 25,000 Da (monomer and dimer), and no lower molecular weight bands were observed. The 12 kDa band was micro-sequenced and the amino acid sequence corresponded to that of normal beta 2m through the 40th residue. Amino acid sequence analysis showed no difference from normal beta 2m in any of the beta 2m proteins contained in the amyloid deposits isolated from the four studied tissues. Also, the HPLC profile of the four protein samples were strictly normal and identical to a commercial preparation of beta 2m. The present study demonstrates that beta 2m molecules polymerized in amyloid fibrils and deposits are intact and have a normal amino acid sequence, and produced by a specific and unique fibrillogenetic mechanism, which does not require proteolytic processing from the precursor protein to the amyloid fibrils.
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PMID:Polymerization of normal and intact beta 2-microglobulin as the amyloidogenic protein in dialysis-amyloidosis. 888 86

Expression of the MUC2 mucin has been demonstrated in normal gastrointestinal and respiratory epithelium and in carcinomas of the gastrointestinal and respiratory tracts, breast, ovary, and bladder using RNA probes and (or) monoclonal antibodies reactive with peptide epitopes on the 23 amino acid tandem repeat. Mouse monoclonal antibodies 4F1 and 3A2 were previously obtained by immunization with mucin derived from the LS174T colon cancer cell line and a KLH conjugate of a synthetic MUC2 VNTR peptide. These antibodies react with distinct epitopes on synthetic VNTR peptides and with normal and malignant epithelial tissues. In the present study, we examined the biosynthesis of MUC2 in LS174T colon cancer cells, using these antibodies to immunoprecipitate labelled mucin. A very high molecular mass protein was immunoprecipitated following 1 min pulse labelling with [3H]threonine and [3H]proline. A slight increase in molecular mass was observed over the next 16 min; however, unlike the MUC1 mucin, there was no large difference in apparent molecular mass between the MUC2 protein precursor and fully processed mucin using separation by SDS-PAGE. O-Glycosylation began within 1 h of synthesis of the protein core. Mucin secretion into the culture medium was detected in the 2nd hour following synthesis and was largely completed within 4 h of synthesis. Secreted mucin was far less reactive with these monoclonal antibodies than the precursor protein.
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PMID:Early steps in the biosynthesis of MUC2 epithelial mucin in colon cancer cells. 903 93

Beta-amyloid peptides (A beta peptides) form the main protein component of the amyloid deposits found in the brains of Alzheimer's disease (AD) patients. Soluble A beta peptides, which are proteolytic fragments of the amyloid-precursor protein (APP) are constitutively secreted by cells expressing APP during normal metabolism [1] and are also present in human plasma and cerebrospinal fluid [2]. Missense mutations in Codon 717 of the APP gene are responsible for a small percentage of inherited AD cases (FAD) and increase the amount of A beta peptides containing additional carboxy terminal amino acids (A beta 1-42, A beta 1-43) [3, 4]. Recent findings indicate that FAD mutations in the presenilin 1 and 2 genes also increase the amount of these longer A beta peptides [5]. A beta 1-42 polymerizes more rapidly in vitro [6] than A beta 1-40 and has been identified as the major component of the brain amyloid deposits [7-9]. We recently developed a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system [10] for the separation of these two peptides. Here we describe a modified version of the original SDS-PAGE procedure, which allows the separation of A beta 1-40, A beta 1-42, and A beta 1-43 for the first time. Detection of the three A beta peptides in the lower ng and pg range is realized by optimized silver staining or immunoblot procedures. These nonradioactive methods may validate results obtained by ELISA procedures used to study the metabolic fate of APP. They may help to define the neurotoxic potential of the longer A beta peptides in relation to their aggregation state.
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PMID:Improved electrophoretic separation and immunoblotting of beta-amyloid (A beta) peptides 1-40, 1-42, and 1-43. 915 Sep 36

Deoxyhypusine synthase catalyzes the NAD+-dependent formation of deoxyhypusine in the eIF-5A precursor protein by transferring the 4-aminobutyl moiety of spermidine. This enzyme has recently been shown to be essential for cell viability and growth of yeast [Sasaki, K., Abid, M.R., and Miyazaki, M. (1996) FEBS Lett. 384, 151 154]. We have purified and characterized the enzyme from the yeast Saccharomyces carlsbergensis. The yeast and recombinant enzymes had a specific activity of 1.21 to 1.26 pmol per min per pmol of protein, and recognized both the eIF-5A precursor proteins almost equally as judged from their similar K(m) and V(max) values. Size exclusion chromatography and SDS-PAGE indicated that the active form of the enzyme is a homotetramer consisting of 43-kDa subunits. The enzyme showed a strict specificity for its substrates, NAD+, spermidine and eIF-5A precursor protein. Among all the substrates tested, only NAD+ showed a protective effect against heat inactivation of the enzyme suggesting that NAD+ initiates some conformational change in the enzyme. NADH exhibited a strong non-competitive inhibition (product inhibition). Unexpectedly, FAD, FMN, and riboflavin showed a moderate competitive inhibition. The competitive inhibition by diamines was maximal with compounds resembling spermidine in carbon chain length. 1,3-Diaminopropane inhibited the enzyme strongly in a competitive manner (product inhibition). On the other hand, putrescine did not inhibit the enzyme or act as a substrate. A polyclonal antibody raised against the yeast recombinant enzyme specifically inhibited deoxyhypusine synthase activity. The cross-reactivity (by Western blotting) of this antibody with the crude extracts varied depending on the source, indicating species specificity.
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PMID:Biochemical and immunological characterization of deoxyhypusine synthase purified from the yeast Saccharomyces carlsbergensis. 916 30


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