Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C1389183 (autodigestion)
 317 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Prosomes are ribonucleoprotein particles constituted by a variable set of about 20 proteins found associated with untranslated mRNA. In addition, they contain a small RNA, the presence of which has been an issue of controversy for a long time. The intact particles have a multicatalytic proteinase (MCP) activity and are very stable; we have never observed autodigestion of the particle by its intrinsic proteinase activity. Surprisingly it was found that Zn2+ and Cu2+ ions at concentrations of 0.1-1 mM disrupt the prosome particles isolated from HeLa cells and duck erythroblasts and abolish instantaneously its MCP activity, without altering the two-dimensional electrophoretic pattern of the constituent proteins. Fe2+, however, seems to induce autodegradation rather than dissociation of the prosome constituents. Most interestingly, protein or oligopeptide substrates protect the particle and its proteinase activity from disruption by Zn2+ or Cu2+. Nuclease-digestion assays reveal that the prosomal RNA, which is largely resistant in the intact particle, becomes digestible after dissociation of prosomes by Zn2+. These data give, for the first time, unambiguous proof of the presence of an RNA in the particle. Furthermore, they demonstrate a structure-function relationship between the complex and its enzyme activity, which seems to be based on the particle as an entity and not on the single constituent proteins.
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PMID:Disruption of prosomes by some bivalent metal ions results in the loss of their multicatalytic proteinase activity and cancels the nuclease resistance of prosomal RNA. 144 37

Poliovirus protease 3C, type 1 Mahoney strain, was expressed in Escherichia coli under phage T7 promoter control and purified to homogeneity from resolubilized inclusion bodies. The renatured protein was as enzymatically active as the protease found in the soluble portion of the bacterial lysate. Proteolytic activity was assayed using as substrate either [35S]methionine-labeled recombinant poliovirus proteins 2C3AB or a truncated version of 3ABC, or synthetic peptide 16-mers corresponding to the cleavage sites at 2C/3A and 3A/3B. Poliovirus protein 3CD (protease-polymerase) was also expressed in bacteria. About 25% of this protein apparently autodigested in vivo, releasing immunoprecipitable protein 3D (polymerase). No further autodigestion of 3CD could be detected in vitro, nor could addition of purified protein 3C effect digestion in trans. Both the serine protease inhibitors PMSF, TPCK, and 3,4-dichloroisocoumarin, and the cysteine protease inhibitors cystatin and zinc, were effective inhibitors of the 3C protease. Six new mutants of the protease, with altered or no enzymatic activity, were identified based on the observation that low level expression of wild type enzyme severely retards growth of bacterial colonies harboring the expression plasmid.
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PMID:Purification, properties, and mutagenesis of poliovirus 3C protease. 165 83

Incubation of alpha-chymotrypsin and alpha-lytic protease with chloro(2,2':6',2''-terpyridine)platinum(II), [Pt(trpy)Cl]+, results in attachment of Pt(trpy)2+ tags at both His 57 and His 40 in the former and His 57 in the latter. The [Pt(trpy)His]2+ chromophores are readily detected and quantitated owing to their characteristic, strong UV absorption. Although the tagging of His 57 modifies the catalytic triad (Ser 195, His 57, and Asp 102) and disrupts the charge relay, the platinated enzymes retain significant esterase and amidase activity for both specific and nonspecific substrates. Unlike suicide inhibitors, which inactivate the enzymes by filling the active site and imitating the tetrahedral intermediate, [Pt(trpy)Cl]+ reacts with a particular amino acid and permits binding of substrates. The kinetic constants for the following are reported: two esters and two amides with alpha-chymotrypsin and an amide with alpha-lytic protease. The kcat values are between 1 and 25% of, and the Km values are a little higher than, the corresponding values for the native enzymes. The catalytic activity is not due to the native enzymes, trypsin, or some zinc-containing protease. Activities of the native and of the platinated alpha-chymotrypsin depend similarly on pH although the pKa of His 57 is raised to 9.7 upon platination. The platinated enzymes undergo autodigestion slower than do the native enzymes. Because the Pt(trpy)2+ tags are noninvasive, stable, and yet easily removable by thiourea, [Pt(trpy)Cl]+ may be used to retard autodigestion of stored proteolytic enzymes.
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PMID:Catalytic activity of the serine proteases alpha-chymotrypsin and alpha-lytic protease tagged at the active site with a (terpyridine)platinum(II) chromophore. 222 78

During the past decade, strains of Bacteroides fragilis that produce an enterotoxin have been implicated in diarrheal disease in animals and humans. The extracellular enterotoxin has been purified and characterized as a single polypeptide (M(r), approximately 20,000). Single specific primer-PCR was used to clone a portion of the B. fragilis enterotoxin gene. The recombinant protein expressed by the cloned gene fragment reacted with monospecific antibodies to B. fragilis enterotoxin by enzyme-linked immunosorbent assay and immunoblot analysis. The deduced amino acid sequence revealed a signature zinc-binding consensus motif (HEXXHXXGXXH/Met-turn) characteristic of metalloproteases termed metzincins. Sequence comparisons showed close identity to matrix metalloproteases (e.g., human fibroblast collagenase) within the zinc-binding and Met-turn region. Purified enterotoxin contained 1 g-atom of Zn2+ per molecule and hydrolyzed gelatin, azocoll, actin, tropomyosin, and fibrinogen. The enterotoxin also underwent autodigestion. The N-terminal amino acid sequences of two autodigestion products were identical to the deduced amino acid sequence of the recombinant enterotoxin and revealed cleavage at Cys-Leu and Ser-Leu peptide bonds. Gelatinase (type IV collagenase) activity comigrated with the toxin when analyzed by gel fractionation and zymography, indicating that protease activity is due to the enterotoxin and not to a contaminating protease(s). Optimal proteolytic activity occurred at 37 degrees C and pH 6.5. Primary proteolytic cleavage sites in actin were identified, revealing cleavage at Gly-Met and Thr-Leu peptide bonds. Enzymatic activity was inhibited by metal chelators but not by inhibitors of other classes of proteases. Additionally, cytotoxic activity of the enterotoxin on human carcinoma HT-29 cells was inhibited by acetoxymethyl ester EDTA. The metalloprotease activity of the enterotoxin suggests a possible mechanism for enterotoxicity and may have additional implications in the study of disease caused by B. fragilis.
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PMID:The enterotoxin of Bacteroides fragilis is a metalloprotease. 780 55

Ginger proteases in ginger rhizome (Zingiber officinale roscoe) were extracted from the ginger acetone powder and purified on DEAE-Sepharose and Sephadex G-75 columns. Before the purification, excess p-chloromercuribenzoate was added to the enzymes to prevent their autodigestion. The mercuribenzoate-proteases were further purified and fractionated by isoelectric focusing in Ampholine of pH 3-10 or pH 4-6. The proteases were fractionated into three components by the isoelectric focusing, having pI value of 4.5, 4.6 and 4.8 respectively. All these proteases had a molecular mass of 29,000 as measured by SDS-polyacrylamide gel electrophoresis and by TSK G2000SW XL gel chromatography. The Ampholine in the purified enzymes can quickly be removed by the gel chromatography of TSK G2000SW. Some divalent metal ions, such as Hg2+, Cu2+, Cd2+, and Zn2+, strongly inhibited these purified enzymes.
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PMID:Purification of ginger proteases by DEAE-Sepharose and isoelectric focusing. 787 61

Using an anti-(glutathione S-transferase-UVS.2 cDNA) Ig and uterine egg vitelline envelope (UEVE) protein of Xenopus laevis as probes, the hatching enzyme (HE) from Xenopus was solubilized in hatching medium and purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its molecular mass and enzymatic properties. The hatching medium solubilized the UEVE and contained molecules reactive to the anti-(GST UVS.2) Ig against Xenopus HE. It was found that the HE had a molecular mass of 60 kDa, and often preparations also contained a 40-kDa form. The 60-kDa HE had a high hydrolytic and UEVE-solubilizing activity, and its activities against Boc-Leu-Gly-Arg-7-amino-4-methylcoumarin (-NH-Mec) and UEVE were inhibited by anti-(GST UVS.2) Ig in a dose-dependent manner. The 60-kDa form was easily autodigested into a 40-kDa form. The 40-kDa molecule alone had no detectable UEVE-solubilizing activity, even it still had high hydrolytic activity. It probably represents the main protease domain of the 60-kDa form after loss of two CUB repeats during autodigestion or digestion. The autodigestion of the 60-kDa molecule into 40-kDa molecule is probably a congenital behavior for successfully dissolving the embryo envelope during the hatching process. The two molecules may play different roles at different stages of the hatching process, during which they co-ordinate with each other to achieve complete solubilization of the embryo envelope, similar to the high and low choriolytic enzymes in medaka (Oryzias latipes). Their hydrolytic activity against Boc-Leu-Gly-Arg-NH-Mec was optimal at pH of 7.4, and with an apparent Km value of 200 micromol.L-1 at 30 degrees C. The HE is very sensitive to trypsin-specific inhibitors such as leupeptin, (4-amidino-phenyl)methane sulfonyl fluoride, diisopropyl fluorophosphate (DFP) and N-alpha-tosyl-L-lysylchloromethane (Tos-Lys-CH2Cl), indicates that it is a trypsin-type protease. The results on EDTA and some metal ions, combined with the occurrence of a astacin family metalloprotease-specific 'HExHxxGFxHE' sequence in the deduced HE amino-acid sequence, indicates that this HE is a Zn2+ metalloprotease.
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PMID:Properties of the hatching enzyme from Xenopus laevis. 1155 58