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Query: UMLS:C0026918 (Mycobacterium)
 52,428 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Trypsin treatment of solubilized coupling factor-latent ATPase from Mycobacterium phlei alters its subunit structure and functional properties. This coupling factor exhibits ATPase activity following trypsin treatment. Concurrently, both the ability of the enzyme to rebind to membranes depleted of coupling factor and its capacity for coupled phosphorylation are lost. The native alpha (64 000 dalton) subunit undergoes limited proteolytic digestion, and the delta (14 000 dalton) subunit is partially lost. During the course of tryptic proteolysis, the coupling factor molecule may exist in one of ten unique structural state (e.g. the native, ATPase-inactive molecule exists in the alpha alpha alpha state). Rigorous analysis of the experimental data by theoretical modeling provided information concerning the intermediate structural states leading to the fully ATPase-activated alpha" alpha" alpha" state under different conditions of trypsin treatment. The theoretical models of structure-function relationships that best-represented the experimental data predicted that the native coupling factor molecule contains three copies of the alpha (64 000 dalton) form of the alpha subunit, that the alpha" (58 000 dalton) alpha subunit species contributes maximally and the alpha' (61 000 dalton) form about half-maximally to ATPase activity, that membrane rebinding ability is proportional to the number of native alpha subunits in the enzyme, and that at least one native alpha subunit/molecule is required for full expression of coupled phosphorylation. These results indicate an essential role for the alpha subunit in the regulation of ATPase activity and in the ability of the solubilized coupling factor to rebind to depleted membranes.
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PMID:Tryptic proteolysis of coupling factor-latent ATPase from mycobacterium phlei. Theoretical modeling of structure-function relationships. 15 57

This report investigates the extent of the expression of fibronectin (FN) binding properties among the mycobacteria and provides preliminary characteristics of the bacterial molecule(s) mediating attachment. Eight BCG substrains, three Mycobacterium tuberculosis strains and four other mycobacterial species all expressed FN-binding capacity. Treatment of organisms with detergent prior to the binding assay destroyed the FN-binding capacity of BCG but not that of Staphylococcus aureus. Trypsin pretreatment eliminated the FN-binding capacity of both BCG and S. aureus. [35S]Methionine-labelled material in supernatants from BCG and M. tuberculosis cultures attached to FN-coated surfaces. These culture supernatants inhibited the attachment of BCG but not S. aureus to FN-coated surfaces. This inhibitory activity of the supernatants was removed by affinity chromatography on FN-Sepharose but was not affected by similar passage over a control column (human serum albumin attached to Sepharose). These results demonstrate that the ability to bind FN is present in all mycobacterial species tested and suggest that attachment is mediated by trypsin-sensitive cell-surface component(s).
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PMID:Attachment of mycobacteria to fibronectin-coated surfaces. 314 7

Trypsin- and chymotrypsin-treated delipidated cell walls of Mycobacterium smegmatis were digested overnight with lysozyme. The water-soluble products thus obtained were filtered on a column of Sephadex G-50; the first peak, excluded from the column, has immunological adjuvant activity. The material of the excluded peak is obtained after lyophilization as a snow-white, fluffy material, soluble in water and insoluble in organic solvents. It behaves as a slightly polydisperse macromolecule in an ultracentrifuge, with an approximate molecular weight of 20,000. All the constituents of this material are typical bacterial cell-wall constituents; thus, the water-soluble adjuvant is considered to be an "oligomer" of the cell wall. When added to Freund's incomplete adjuvant with an antigen (e.g., ovalbumin) and injected into hind-foot pads of guinea pigs, this water-soluble adjuvant increases the amount of precipitating antibodies and induces hypersensitivity to ovalbumin and the biosynthesis of gamma(2)-type precipitating antibodies. The water-soluble material has a stronger adjuvant activity than equal amounts of whole bacteria, cell walls, or wax D, and seems to be the first well-defined, water-soluble, adjuvant-active fraction isolated from Mycobacteria.
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PMID:Isolation and properties of a macromolecular, water-soluble, immuno-adjuvant fraction from the cell wall of Mycobacterium smegmatis. 450 37

Results of the previous investigation in which it was found that DNA extracted from D29 mycobacteriophage was infectious for Mycobacterium smegmatis 607, have been extended. DNA extracted from mycobacteriophage D4 and D32 produced plaques when plated on their respective hosts; D28 DNA, extracted in the same manner and tested under similar conditions, failed to show infectivity. Species barriers were not crossed by mycobacteriophage DNA; bacteria resistant to intact phage were not infected with the phage DNA. The efficiency of plating of the DNA is very much lower than that of intact phage; infection of a given host was not accomplished by DNA when titration for plaque formation by the intact phage was less than 10(9) PFU. The base composition of DNA extracted from the four mycobacteriophages and the three propagating hosts was very similar. The bases were paired, adenine with thymine and guanine with cytosine. A relatively higher per cent of guanine-cytosine than of adenine-thymine, was found. The buoyant density of each DNA in CsCl was linearly related to its guanine-cytosine content whereas with the exception of D28 DNA, thermal denaturation temperatures failed to show this relationship. However, the thermal transition profiles were characteristic of double stranded DNA. Additional evidence that D29 DNA forms complexes with basic proteins was obtained. Binding between calf thymus histone and between RNAase and D29 DNA readily occurs with a resultant loss in DNA infectivity. Trypsin and D29 DNA are only weakly reactive.
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PMID:Further studies of infectious DNA extracted from mycobacteriophages. 594 24

The physical properties and activities of the purified catalase-peroxidase hydroperoxidase I (HPI) of Escherichia coli (EcHPI) and HPI with a carboxyl-terminal extension of Mycobacterium tuberculosis (MtHPI-e) are compared to those of commercial preparations of horseradish peroxidase (HRP). The catalase-peroxidase proteins had similar absorption spectra and differed primarily in that MtHPI-e has a higher peroxidatic to catalatic activity ratio than EcHPI. Trypsin cleavage of MtHPI-e resulted in the formation of an active catalase-peroxidase lacking the carboxyl-terminal extension. The three enzymes, HRP, MtHPI-e, and EcHPI, mediated the isoniazid- and H2O2-dependent production of radical species, as detected by nitroblue tetrazolium reduction. A constant flux of H2O2, generated in situ from glucose oxidase and glucose was used. MtHPI-e was more effective at isoniazid-dependent radical production than EcHPI and HRP. Similar qualitative results were obtained by staining nondenaturing polyacrylamide gels for activity with nitroblue tetrazolium in the presence of isoniazid and H2O2. The absorbance spectrum of HRP exhibited changes during incubation with isoniazid and H2O2 consistent with the formation of several typical reaction intermediates, whereas the catalase-peroxidases exhibited no distinct spectral changes. The results suggest that the sensitivity of M. tuberculosis to isoniazid may be the result of isoniazid-dependent radical formation by the catalase-peroxidase in the absence of other catalase activities to remove substrate H2O2.
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PMID:Comparison of isoniazid oxidation catalyzed by bacterial catalase-peroxidases and horseradish peroxidase. 748 9

Peptide mass fingerprinting is a powerful tool for the identification of proteins. Trypsin is the most widely used enzyme for this purpose. Therefore, 104 protein digests from human Jurkat T cells and Mycobacterium were analyzed considering missed cleavage sites, tryptophan oxidation and N-terminal pyroglutamylation. About 90% of the matched peptides with missed cleavage sites could be classified into three groups: (i) lysine and arginine with a neighbouring proline on the carboxy-terminal side, (ii) neighboring lysines/arginines, and (iii) lysines and arginines with an aspartic acid or glutamic acid residue on either the amino- or carboxy-terminal side. The first group is already accounted for by search programs. The number of missed cleavage sites can be increased without reducing the precision of the database search by taking the other two groups into consideration. Peptides with tryptophan were observed in non, singly (+16 Da) and doubly (+32 Da) oxidized forms. The higher oxidized form was only observed with lower intensity in the presence of the lower oxidized form. Peptides with N-terminal glutamine were found always as pyroglutamate (-17 Da), and in the majority of cases in pairs with unmodified glutamine. These data can be used for the refinement of protein searches by peptide mass fingerprinting.
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PMID:Analysis of missed cleavage sites, tryptophan oxidation and N-terminal pyroglutamylation after in-gel tryptic digestion. 1071 61

Phagemid-based expression of cloned genes fused to the gIIIP coding sequence and rescue using helper phages, such as VCSM13, has been used extensively for constructing large antibody phage display libraries. However, for randomly primed cDNA and gene fragment libraries, this system encounters reading frame problems wherein only one of 18 phages display the translated foreign peptide/protein fused to phagemid-encoded gIIIP. The elimination of phages carrying out-of-frame inserts is vital in order to improve the quality of phage display libraries. In this study, we designed a novel helper phage, AGM13, which carries trypsin-sensitive sites within the linker regions of gIIIP. This renders the phage highly sensitive to trypsin digestion, which abolishes its infectivity. For open reading frame (ORF) selection, the phagemid-borne phages are rescued using AGM13, so that clones with in-frame inserts express fusion proteins with phagemid-encoded trypsin-resistant gIIIP, which becomes incorporated into the phages along with a few copies of AGM13-encoded trypsin-sensitive gIIIP. In contrast, clones with out-of-frame inserts produce phages carrying only AGM13-encoded trypsin-sensitive gIIIP. Trypsin treatment of the phage population renders the phages with out-of-frame inserts non-infectious, whereas phages carrying in-frame inserts remain fully infectious and can hence be enriched by infection. This strategy was applied efficiently at a genome scale to generate an ORF-enriched whole genome fragment library from Mycobacterium tuberculosis, in which nearly 100% of the clones carried in-frame inserts after selection. The ORF-enriched libraries were successfully used for identification of linear and conformational epitopes for monoclonal antibodies specific to mycobacterial proteins.
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PMID:A novel helper phage enabling construction of genome-scale ORF-enriched phage display libraries. 2408 69