Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Primary biliary cirrhosis (PBC) is characterised by the presence of antimitochondrial antibodies. The PBC-specific, immunoreactive, trypsin-sensitive antigens on the inner mitochondrial membrane (M2) have hitherto not been identified. A major 70 kD M2 autoantigen is the E2 component (lipoate acetyltransferase) of the pyruvate dehydrogenase enzyme complex located within mitochondria. This has been confirmed by immunoblotting of PBC patients' sera against purified E2 protein: sera from 38/40 (95%) patients with established clinical, biochemical, and histological features of PBC (18 stage II/III, 22 stage IV) reacted positively with E2; whilst no sera from 39 controls (27 non-PBC chronic liver disease, 12 healthy normal women) gave a positive response. Immunoblotting showed that a second subunit of the pyruvate dehydrogenase complex, a 50 kD polypeptide of unknown function (component X), is also an M2 autoantigen. Identification of these M2 mitochondrial antigens should facilitate the development of a specific serological test for PBC and the study of autoimmunising epitopes.
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PMID:Primary biliary cirrhosis: identification of two major M2 mitochondrial autoantigens. 289 10

The pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was treated with chymotrypsin at pH 7 and 0 degrees C. Loss of the overall catalytic activity lagged behind the rapid cleavage of the lipoate acetyltransferase polypeptide chains, whose apparent Mr fell from 57 000 to 45 000 as judged by sodium dodecylsulphate/polyacrylamide gel electrophoresis. The inactive chymotrypsin-treated enzyme had lost the lipoic-acid-containing regions of the lipoate acetyltransferase chains, yet remained a highly assembled structure. Treatment of this chymotryptic core complex with trypsin at pH 7.0 and 0 degrees C caused a further shortening of the lipoate acetyltransferase polypeptide chains to an apparent Mr of 28 000 and was accompanied by disassembly of the complex. The lipoic-acid-containing regions are therefore likely to be physically exposed in the intact complex, protruding from the structural core formed by the lipoate acetyltransferase component between the subunits of the other component enzymes. Proton nuclear magnetic resonance spectroscopy demonstrated that the enzyme complex contains large regions of polypeptide chain with remarkable intramolecular mobility, most of which were retained after excision of the lipoic-acid-containing regions with chymotrypsin. It is likely that the highly mobile regions are in the lipoate acetyltransferase component and facilitate movement of the lipoic acid residues. Such polypeptide chain mobility provides the molecular basis of a novel system of active-site coupling in the 2-oxo acid dehydrogenase multienzyme complexes.
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PMID:Limited proteolysis and proton NMR spectroscopy of Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex. 628 90

A computer modeling system developed to analyze experimental data for inactivation of the Escherichia coli alpha-ketoglutarate dehydrogenase complex (KGDC) accompanying release of lipoyl moieties by lipoamidase and by trypsin [Hackert, M.L., Oliver, R.M. & Reed, L.J. (1983) Proc. Natl. Acad. Sci. USA 80, 2226-2230] was used to analyze analogous data for the E. coli pyruvate dehydrogenase complex (PDC). The model studies indicate that the activity of PDC, as found for KGDC, is influenced by redundancies and random processes, which we describe as a multiple random coupling mechanism. In both complexes more than one lipoyl moiety services each pyruvate dehydrogenase (EC 1.2.4.1) or alpha-ketoglutarate dehydrogenase (EC 1.2.4.2) (E1) subunit, and an extensive lipoyl-lipoyl interaction network for exchange of electrons and possibly acyl groups must also be present. The best fit between computed and experimental data for PDC was obtained with a model that has four lipoyl domains with four or, more probably, eight lipoyl moieties servicing each E1 subunit. The lipoyl-lipoyl interaction network for PDC has lipoyl domain interactions similar to those found for KGDC plus the additional possibility of interaction of a lipoyl moiety and its paired mate on each dihydrolipoamide acetyltransferase (EC 2.3.1.12) (E2) subunit. The two lipoyl moieties on an E2 subunit in PDC appear to be functionally indistinguishable, each servicing the acetyltransferase site of that E2 subunit and a dihydrolipoamide dehydrogenase (EC 1.6.4.3) (E3) subunit if the latter is bound to that particular E2 subunit. The observed difference between inactivation of PDC by lipoamidase and by trypsin appears to be due to dead-end competitive inhibition by lipoyl domains that have been modified by excision of lipoyl moieties by lipoamidase.
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PMID:A computer model analysis of the active-site coupling mechanism in the pyruvate dehydrogenase multienzyme complex of Escherichia coli. 634 73

The pyruvate dehydrogenase complex of Escherichia coli was treated with o-phenylene bismaleimide in the presence of the substrate pyruvate, producing almost complete cross-linking of the lipoate acetyltransferase polypeptide chains as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This took place without effect on the catalytic activities of the other two component enzymes and with little evidence of cross-links being formed with other types of protein subunit. Limited proteolysis with trypsin indicated that the cross-links were largely confined to the lipoyl domains of the lipoate acetyltransferase component of the same enzyme particle. This intramolecular cross-linking had no effect on the very sharp resonances observed in the (1)H n.m.r. spectrum of the enzyme complex, which derive from regions of highly mobile polypeptide chain in the lipoyl domains. Comparison of the spin-spin relaxation times, T(2), with the measured linewidths supported the idea that the highly mobile region is best characterized as a random coil. Intensity measurements in spin-echo spectra showed that it comprises a significant proportion (probably not less than one-third) of a lipoyl domain and is thus much more than a small hinge region, but there was insufficient intensity in the resonances to account for the whole lipoyl domain. On the other hand, no evidence was found in the (1)H n.m.r. spectrum for a substantial structured region around the lipoyl-lysine residues that was free to move on the end of this highly flexible connection. If such a structured region were bound to other parts of the enzyme complex for a major part of its time, its resonances might be broadened sufficiently to evade detection by (1)H n.m.r. spectroscopy.
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PMID:Cross-linking and 1H n.m.r. spectroscopy of the pyruvate dehydrogenase complex of Escherichia coli. 675 33

1. Bovine kidney pyruvate dehydrogenase multienzyme complex is inactivated by elastase in a similar manner as described earlier for papain. The core component, lipoate acetyltransferase, is cleaved by elastase into an active fragment (Mr 26000) and a fragment with apparent Mr of 45000 as analyzed by dodecylsulfate gel electrophoresis. Due to the fragmentation of the core, the enzyme complex is disassembled into its component enzymes which retain their complete enzymatic activities as assayed separately. 2. A different mechanism was found for the inactivation of pyruvate dehydrogenase complex with trypsin and some other proteases (chymotrypsin, clostripain). In these cases, the pyruvate dehydrogenase component is inactivated rapidly by limited proteolysis. More slowly, the enzyme complex is disassembled simultaneously with fragmentation of the lipoate acetyltransferase which again results in an active fragment of Mr 26000 and another fragment of apparent Mr 45000. Upon prolonged proteolysis, the latter fragment is cleaved further to give products of Mr 36000 or lower. 3. The enzyme-bound lipoyl residues of the pyruvate dehydrogenase complex have been labelled covalently by incubation with [2-14C]pyruvate. After treatment of this [14C]acetyl-enzyme with papain, elastase, or trypsin, radioactivity was associated exclusively with the 45000-Mr and 36000-Mr fragments but not with the active 26000-Mr fragment. 4. It is concluded that the bovine kidney lipoate acetyltransferase core is composed of 60 subunits each consisting of two dissimilar folding domains. One of these contains the intersubunit binding sites as well as the active center for transacylation whereas the other possesses the enzyme-bound lipoyl residues.
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PMID:Bovine kidney pyruvate dehydrogenase complex. Limited proteolysis and molecular structure of the lipoate acetyltransferase component. 678 Mar 50

Although a series of protein levels from several protein pathways have been shown to differ between white (WA) and brown (BA) adipocytes, proteomic work on this subject with the exception of mitochondrial protein differences is limited. It was, therefore, the aim of the study to compare WA with BA soluble protein levels. Proteins were extracted from WA and BA and the soluble fraction was run on two-dimensional gel electrophoresis. Quantification of spot volume was carried out and protein spots, statistically different between groups (P < 0.01), were in-gel digested with trypsin and peptides were identified using nano-LC-ESI-MS/MS in the CID and ETD mode. Differences between selected proteins were evaluated by immunoblotting. A network was generated using the ingenuity pathway analysis. Five proteins, protein DJ-1, dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, isocitrate dehydrogenase subunit alpha, electron transfer flavoprotein subunit alpha and immunoglobulin-binding protein 1, were increased in BA based on a gel-based proteomic method and differential expression was verified by immunoblotting. These individual proteins were represented by one spot each and sequence coverages were between 28 and 65%. A network generated based on these results indicated a link to ubiquitination. Differential protein levels between WA and BA allow interpretation of previous work on adipocyte biochemistry and form the basis for future studies with genetic or pharmacological inhibition of these proteins accompanied by work on phenotype and adipocyte function.
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PMID:Proteomic differences between white and brown adipocytes. 2439 Apr 58