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At least 703 records · Page 39Linked to original sources
PubMed1996

Calcium ion modulation of meizothrombin autolysis at Arg55-Asp56 and catalytic activity.

When a recombinant variant of prothrombin with the cleavage site mutations R155A, R271A, and R284A (rMZ) is exposed to either prothrombinase or ecarin, a form of meizothrombin (rMZa) is generated that is stable for weeks in the presence of Ca2+ (Côté, H. C. F., Stevens, W. K., Bajzar, L., Banfield, D. K., Nesheim, M. E., and MacGillivray, R. T. A. (1994) J. Biol. Chem. 269, 11374-11380). In the absence of Ca2+ however, rMZa is rapidly cleaved within a disulfide bonded loop in the F1 domain at Arg55 in the sequence RTPR downward arrowDKL, yielding a molecule with 3 chains joined by two disulfide bonds (rMZa*). Cleavage kinetics are first order regardless of the rMZa concentration, indicating an intramolecular cleavage. This cleavage does not occur at Ca2+ concentrations in excess of 1.0 mM. To assess the role of the F1 domain in rMZa activity, another variant lacking the R155A mutation (rMZdesF1) was expressed, which when activated yields meizothrombin lacking the F1 domain (rMZdesF1a). Rates of hydrolysis of the tripeptide substrate S2238 by rMZa or rMZa* increase from 60% to 90% that of recombinant thrombin as Ca2+, Mg2+, or Mn2+ concentrations are varied from 0 to 10 mM. Km and kcat values for rMZa in the absence and presence of 5 mM Ca2+ are 1.9 and 2.2 microM and 65 and 105 s-1. TAME esterase activity of rMZa also increases with 5 mM Ca2+. No such metal ion-dependent effects are obtained with either thrombin or rMZdesF1a. Fibrinogen clotting activities, relative to that of thrombin, increase in a manner analogous to those obtained with small substrates, for rMZa and rMZa* but not rMZdesF1a. Complexes of the active site probe dansylarginine N-(3-ethyl-1,5-pentanediyl)amide with rMZa and rMZa*, but not thrombin or rMZdesF1a exhibit large cation-dependent decreases in fluorescence intensity, suggesting that metal ion binding in the F1 domain alters the environment of the probe at the active site. These results indicate that in the absence of divalent cations, the activity of rMZa is inhibited, perhaps by obstruction of the active site by the F1 domain, and that Ca2+ binding to the F1 domain modulates the properties of not only the F1 domain but also the protease domain.

Arginine↗
PubMed2000

Inflated wings, tissue autolysis and early death in tissue inhibitor of metalloproteinases mutants of Drosophila.

In vertebrates, tissue inhibitors of metalloproteinases (TIMPs) play key roles in extracellular matrix (ECM) homeostasis and growth control. Deletion of the recently cloned Timp gene of Drosophila results in a subviable phenotype. Adult flies display inflated wings similar to integrin mutants, suffer from a bloated gut and progressive dissolution of internal tissues, and die prematurely. Our results demonstrate that the Timp gene product controls selective aspects of ECM function in Drosophila, and suggest that it is involved in cell adhesion/cell signaling pathways. Hence, Drosophila Timp mutants may prove useful as a model system for a wide variety of pathological conditions related to ECM dysregulation.

Abdomen↗
PubMed1999

A defect in cell wall recycling triggers autolysis during the stationary growth phase of Escherichia coli.

The first gene of a family of prokaryotic proteases with a specificity for L,D-configured peptide bonds has been identified in Escherichia coli. The gene named ldcA encodes a cytoplasmic L, D-carboxypeptidase, which releases the terminal D-alanine from L-alanyl-D-glutamyl-meso-diaminopimelyl-D-alanine containing turnover products of the cell wall polymer murein. This reaction turned out to be essential for survival, since disruption of the gene results in bacteriolysis during the stationary growth phase. Owing to a defect in muropeptide recycling the unusual murein precursor uridine 5'-pyrophosphoryl N-acetylmuramyl-tetrapeptide accumulates in the mutant. The dramatic decrease observed in overall cross-linkage of the murein is explained by the increased incorporation of tetrapeptide precursors. They can only function as acceptors and not as donors in the crucial cross-linking reaction. It is concluded that murein recycling is a promising target for novel antibacterial agents.

Amino Acid Sequence↗
PubMed1990

The vir locus affects the response of Bordetella pertussis to antibiotics: phenotypic tolerance and control of autolysis.

Eradication of Bordetella pertussis from the respiratory tract occurs slowly even when bactericidal antibiotics are used. The rate of killing of B. pertussis was found to be proportional to growth rate: virulent, slowly growing strains were killed over days, while rapidly growing strains (either avirulent or virulent modulated by growth conditions to avirulent) were killed over hours. Minimum inhibitory concentrations and binding of antibiotics to membrane targets were equivalent in virulent and avirulent cells, suggesting differences in antibiotic response might reflect differences in activities of the autolytic cascade. This was supported by the finding that cell wall degradation was less than 40% per day in virulent strains and greater than 70% per day in avirulent strains. Penem antibiotics, known to rapidly kill even slowly growing bacteria, demonstrated a more-than-twofold greater rate of killing of slowly growing virulent strains compared with ampicillin or erythromycin. This suggests the potential for improved therapeutic efficacy in vivo. Thus, the vir locus, which determines phase transition in B. pertussis, is apparently the first example of a sensor-transducer system controlling phenotypic tolerance and antibiotic-induced autolytic activity.

Anti-Bacterial Agents↗
PubMed1979

Characterization of active derivatives produced by acetamidination and selective autolysis of bovine trypsin.

A commercial preparation of bovine trypsin was treated with methyl acetimidate-HCl, and most of the lysine residues were converted to trypsin-resistant residues retaining their cationic charges. The modified preparation was then fractionated by ion-exchange chromatography on SE-Sephadex C-50 into two active components, amidinated alpha- and amidinated beta-trypsins. The latter component (Am-beta-trypsin), which consisted of a single polypeptide chain, was allowed to autolyze at pH 7.8, 25 degrees C for 3.5 h and a new active component named Am-delta-trypsin was isolated from the autolysate. Several lines of experimental evidence indicated that Am-delta-trypsin was derived by primary cleavage of the bond Arg105-Val106. Cleavage at Arg55-Leu56, on the other hand, appeared to lead to inactivation of Am-beta-trypsin. The kinetic properties of the catalytic hydrolyses of synthetic substrates and the affinity to Gly-Gly-Arg immobilized on Sepharose were compared among Am-delta-, Am-beta-, and Am-alpha-trypsins. Am-delta-trypsin resembled Am-beta-trypsin in these properties, but did not resemble Am-alpha-trypsin which had a cleavage at Lys131-Ser132.

Amino Acids↗
PubMed2000

Autolysis during in vitro tracheary element differentiation: formation and location of the perforation.

Tracheary elements differentiated from isolated Zinnia: mesophyll cells were observed at various times of culture under a scanning electron microscope. Perforation occurred on the primary wall at one of the longitudinal ends in single tracheary elements. In double tracheary elements, which both of two cells derived from a single cell differentiated into, the pore opened on the primary walls both at the junction of the two tracheary elements and at a longitudinal end of one of the two tracheary elements. These results suggest not only that a single tracheary element has its own program to form a perforation at one end without being affected by neighboring cells, but also that isolated cells indeed hold some traces of polarity and cell-cell communication.

Apoptosis↗
PubMed2001

Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency.

Because of its stringent sequence specificity, the catalytic domain of the nuclear inclusion protease from tobacco etch virus (TEV) is a useful reagent for cleaving genetically engineered fusion proteins. However, a serious drawback of TEV protease is that it readily cleaves itself at a specific site to generate a truncated enzyme with greatly diminished activity. The rate of autoinactivation is proportional to the concentration of TEV protease, implying a bimolecular reaction mechanism. Yet, a catalytically active protease was unable to convert a catalytically inactive protease into the truncated form. Adding increasing concentrations of the catalytically inactive protease to a fixed amount of the wild-type enzyme accelerated its rate of autoinactivation. Taken together, these results suggest that autoinactivation of TEV protease may be an intramolecular reaction that is facilitated by an allosteric interaction between protease molecules. In an effort to create a more stable protease, we made amino acid substitutions in the P2 and P1' positions of the internal cleavage site and assessed their impact on the enzyme's stability and catalytic activity. One of the P1' mutants, S219V, was not only far more stable than the wild-type protease (approximately 100-fold), but also a more efficient catalyst.

Amino Acid Sequence↗
PubMed1993

Temperature-sensitive mutation in lytF, a new gene involved in autolysis of Escherichia coli.

A temperature-sensitive mutation in a new Escherichia coli gene, located at 62.5 min on the linkage map and designated lytF, resulted in bacteriolysis at the restrictive temperature. Temperature sensitivity and lytF-mediated lysis were simultaneously suppressed by either of two previously described unlinked mutations designated smhA1 and smhB1. The smhA1 and smhB1 alleles were originally isolated as specific extragenic suppressors of temperature-sensitive mutations in three other genes known as murH (99 min), lytD (13 min) and lytE (25 min) which conferred lysis phenotypes indistinguishable from that of the lytF mutation. The murH, lytD and lytE genes have been proposed to be related on the bases of phenotypic similarities and the specificities of their extragenic suppressors. It is now further proposed that lytF belongs to this group. The isolation of new alleles of smhA and smhB as extragenic suppressors of lytF further supports this proposal.

Alleles↗
PubMed1999

Salt-Sensitive growth of Staphylococcus aureus: stimulation of salt-induced autolysis by multiple environmental factors.

The growth of Staphylococcus aureus 209P became extremely sensitive to a high NaCl concentration following lowered temperature, reduced air-supply, and decreased Ca2+ concentration in the medium. Cells in high-NaCl and low-Ca2+ concentration media either autolyzed or transformed into protoplast-like forms during growth when grown standing below 37 degrees C. The abnormal growth, however, was invariably avoided by preliminary supplementation with polyanetholesulfonate (autolysin inhibitor) in the growth media. These results suggested that the autolytic activity of this organism was precisely controlled by multiple environmental factors such as ionic strength, temperature, air supply, and the concentration of Ca2+.

Bacteriolysis↗
PubMed1986

Tunicamycin-resistant mutants of Bacillus amyloliquefaciens are deficient in amylase, protease and penicillinase synthesis and have altered sensitivity to antibiotics and autolysis.

Mutants of Bacillus amyloliquefaciens resistant to at least 10 micrograms/ml of tunicamycin were isolated and shown to be pleiotropic. The mutants were more resistant to streptomycin, chloramphenicol, kanamycin and neomycin than was the parent strain but less resistant to penicillin G and tetracycline. They were more autolytic, presumably due to an altered cell wall. The mutants produced reduced levels of amylase, penicillinase and both metal and serine protease besides having an enhanced sporulation frequency and being more motile.

Amylases↗
PubMed1990

Induction of autolysis in Enterococcus faecalis S-47 by peptide AS-48.

In addition to its bactericidal mode of action, the peptide antibiotic AS-48 exhibits a bacteriolytic effect on Enterococcus faecalis S-47 that is associated with autolysin activation. Bacteriolysis induced by the antibiotic can be modulated by addition of EDTA, divalent cations and autolysin activators (trypsin) or inhibitors (cardiolipin), suggesting that topologic regulation of the autolysins is involved in the process. In addition, inhibitors of protein and RNA synthesis interfere markedly with bacteriolysis, as do ionophores and the ATPase inhibitor DCCD, suggesting the participation of an internal messenger in autolysin activation in the presence of AS-48.

Anti-Bacterial Agents↗