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At least 19 recordsLinked to original sources
PubMed1990

Two bactericidal targets for penicillin in pneumococci: autolysis-dependent and autolysis-independent killing mechanisms.

It has been assumed that penicillin (and also other cell wall inhibitors) kill pneumococci predominantly by triggering their major autolytic enzyme (an N-acetylmuramoyl-L-alanine amidase; referred to as amidase), resulting in massive cell wall degradation. Three types of experiments suggest that only part of this killing is due to cell lysis by amidase. (i) Suppression of penicillin-induced lysis by specific inhibitors of amidase protected pneumococci only marginally from killing in spite of prolonged exposure to concentrations of penicillin that were 10x, 20x, or 100x greater than the MIC. (ii) Mutants from which the amidase was completely eliminated by plasmid insertion or deletion (Lyt-) were still killed, albeit at a slower rate than the wild-type Lyt+ strains (3 to 4 log units instead of 4 to 5 log units per 6 h, i.e., about 1 log unit slower than the wild type; P less than 0.001). (iii) A new mutation (cid), which was not related to the amidase gene, further reduced killing of mutants lacking amidase to 1 log unit per 6 h (Lyt- Cid- phenotype). Reintroduction of the amidase gene into Lyt- Cid- cells partially restored penicillin-induced lysis but increased only slightly the rate of killing (from 1 log unit per 6 h in Lyt- Cid- cells to 2 log units per 6 h in Lyt+ Cid- cells). We conclude that penicillin kills pneumococci by two distinct mechanisms: one that involves the triggering of the amidase (about 1 log unit of killing per 6 h) and another, amidase-independent mechanism that is responsible for 3 to 4 log units of killing per 6 h. Triggering of the amidase activity in situ in growing bacteria was significantly reduced in Lyt+ Cid- cells, indicating that there is some regulatory interaction between the cid gene product and the amidase.

Amidohydrolases↗
PubMed1995

Studies on the autolysis of m-calpain from the skeletal muscle of the amphibian Rana ridibunda.

The autolytic mechanisms responsible for the regulation of m-calpain purified from the skeletal muscle of the amphibian Rana ridibunda were examined. Both subunits of the calpain molecule were found to undergo autolysis in the presence of Ca2+. Various divalent cations were examined for their ability to induce calpain autolysis. The concentrations of these cations required for the complete calpain autolysis were: 500 microM Ca2+, 800 microM Mn2+, 2 mM Sr2+, 10 mM Ba2+, whereas Mg2+, even at 10 mM did not induce any autolysis. Calpain autolysis induced by the above divalent cations is a temperature dependent process. Presence of Mn2+ or Sr2+ reduces the Ca2+ requirement of calpain for autolysis. The rate of autolysis depends on the protease concentration; protease inhibitors such as E-64, leupeptin, antipain, and iodoacetic acid inhibit the autolysis of calpain; E-64 inhibits irreversibly while leupeptin inhibits reversibly the autolysis; and irreversibly inactivated by E-64 calpain is fully digested by native calpain. Autolysis of calpain in the presence of alkali denatured casein increases the Ca2+ sensitivity of the protease for its half maximal and maximal caseinolytic activity. Limited autolysis of calpain is also induced in the presence of the endogenous substrate G-actin, and the rate of autolysis is slower than that obtained in the absence of substrates.

Animals↗
PubMed1978

Influence of autolysis on rat gastric endocrine cells. Histochemical and electron microscopical study.

In the present study histochemical parameters of the rat gastric endocrine cells were followed up in the course of 24-h autolysis, and their ultrastructure was studied during autolysis lasting for 60 min. The autolysis occurred at 37 degrees C. In the light microscope, with the histochemical methods applied, only EC, ECL and G cells could be identified during the one-hour autolysis. With the autolysis proceeding for 6 and 12 h, only argyrophil method according to Grimelius (1968) enabled visualization of gastric argyrophilic cells. After 24 h of autolysis, none of the methods applied (not even the Grimelius method) proved to be adequate for successful demonstration of the gastric endocrine cells. In the course of 60-min autolysis, electron microscopic examination provided identification of the EC, ECL, AL, D1, and G cells with the characteristical ultrastructural appearance of granules. The granules of the endocrine cells (G cells included) were found to be considerably resistant to autolysis. The effect of 60-min autolysis did not induce granule "emiocytosis" or dissolution of granule content. Autolysis exceeding five minutes resulted in damage of the mitochondria of different degrees and in dilatation of the profiles of endoplasmic reticulum (particularly in G and AL cells). The results obtained in the present study demonstrate the feasibility of in vitro experimental stimulation since the endocrine granules have proved to be resistant to the effects of simultaneously developing autolysis.

Animals↗
PubMed1989

The role of autolysis in activity of the Ca2+-dependent proteinases (mu-calpain and m-calpain).

A recent hypothesis suggests that proteolytic activity of the micromolar and millimolar Ca2+-requiring forms of the Ca2+-dependent proteinases (mu- and m-calpain, respectively) is regulated in vivo by their association with a phosphatidylinositol-containing site on the plasma membrane followed by autolysis of the proteinases. Phosphatidylinositol association lowers the Ca2+ concentration needed for autolysis, and autolysis, in turn, lowers the Ca2+ concentration needed for proteolytic activity. To test this hypothesis, we have compared the Ca2+ concentrations needed for autolysis and for proteolytic activity of the calpains both in the presence and the absence of phosphatidylinositol. Bovine skeletal muscle mu-calpain required 40-50 microM Ca2+ for half-maximal rate of proteolysis of a casein substrate, 140-150 microM Ca2+ for half-maximal autolysis in the presence of 80 microM phosphatidylinositol, and 190-210 microM Ca2+ for half-maximal autolysis in the absence of phosphatidylinositol. Consequently, mu-calpain is an active proteinase and does not require autolysis for activation. Bovine skeletal muscle m-calpain required 700-740 microM Ca2+ for half-maximal rate of proteolysis of a casein substrate, 370-400 microM Ca2+ for half-maximal autolysis in the presence of 80 microM phosphatidylinositol, and 740-780 microM Ca2+ for half-maximal autolysis in the absence of phosphatidylinositol. These results are consistent with the idea that m-calpain functions in its autolyzed form, but the results do not demonstrate that unautolyzed m-calpain is inactive. 80 microM phosphatidylinositol had no effect on the Ca2+ requirement of the autolyzed forms of either mu- or m-calpain but lowered the specific activity of mu-calpain to 20% of its activity in the absence of phosphatidylinositol. Of the four forms of the calpains, unautolyzed m-calpain, autolyzed m-calpain, and unautolyzed mu-calpain would not be proteolytically active at the free Ca2+ concentrations of 300-1200 nM present inside normal cells, and neither mu- nor m-calpain would undergo autolysis at these Ca2+ concentrations, even in the presence of phosphatidylinositol. Cells must contain a mechanism other than or in addition to membrane association and autolysis to activate the calpains.

Animals↗
PubMed1997

Autolysis, Ca2+ requirement, and heterodimer stability in m-calpain.

The roles of N-terminal autolysis of the large (80 kDa) and small (28 kDa) subunits in activation of rat m-calpain, in lowering its Ca2+ requirement, and in reducing its stability have been investigated with heterodimeric recombinant calpains containing modified subunits. Both autolysis and [Ca2+]0.5 were influenced by the ionic strength of the buffers, which accounts for the wide variations in previous reports. Autolysis of the small subunit (from 28 to 20 kDa) was complete within 1 min but did not alter either the Ca2+ requirement ([Ca2+]0.5) or the stability of the enzyme. Autolysis of the NHis10-80k large subunit at Ala9-Lys10 is visible on gels, was complete within 1 min, and caused a drop in [Ca2+]0.5 from 364 to 187 microM. The lower value of [Ca2+]0.5 is therefore a property of the Delta9-80k large subunit. Autolysis at Ala9-Lys10 of the unmodified 80-kDa large subunit is not detectable on gels but was assayed by means of the fall in [Ca2+]0.5. This autolysis was complete in 3.5 min and was inhibited by high [NaCl]. The autolysis product of these calpains, which is essentially identical to that of natural m-calpain, was unstable in buffers of high ionic strength. Calpain in which the large subunit autolysis site had been mutated was fully active but did not undergo a drop in [Ca2+]0.5, showing that m-calpain is active prior to autolysis. The main physiological importance of autolysis of calpain is probably to generate an active but unstable enzyme, thus limiting the in vivo duration of calpain activity.

Animals↗
PubMedDate not supplied

[Effect of regulators on bacterial autolysis].

The aim of this work was to study the effect of autolysis regulators (the fraction of microbial teichoic acids) on the rate of autolysis and the activity of bacterial extracellular lytic enzymes. The regulators of autolysis isolated from 23 cultures belonging to 10 microbial species regulated the rate of autolysis in Bacillus, E. coli and Streptococcus lactis. The regulators either activated or inhibited autolysis depending on the substrate (of a bacterium to be subjected to autolysis). The quantitative dependence of the autolysis rate on the regulator concentration was specific for each pair 'regulator--substrate'. The regulatory properties of the fraction of teichoic acids varied depending on the age of a culture from which they had been isolated. The regulators of autolysis, with an exception of the preparation from E. coli, inhibited the activity of B. subtilis extracellular lytic enzymes in the course of their action on E. coli cells. The possibility for using the regulators of autolysis in microbiological processes is discussed.

Bacillus↗
PubMed1994

Effect of physiological conditions on the autolysis of Staphylococcus aureus strains.

The effect of physiological conditions on autolysis and autolytic activity in various strains of Staphylococcus aureus was determined. The rate of whole cell autolysis of S. aureus was growth phase dependent and a maximum rate was observed in early stationary phase cultures. However, the autolysins extracted by the freeze-thaw method (cell-wall bound autolytic activity) did not show any significant increase in activity. The addition of NaCl to the growth medium enhanced the rate of autolysis with the highest rate being displayed by cultures grown in 1.5 M NaCl. However, lower autolytic activity was found in the freeze-thaw extracts of cultures grown at higher concentrations of NaCl. The rate of autolysis of cultures grown at 30 degrees C was higher than cultures grown at 37 or 43 degrees C. Thus, the rate of autolysis seems to be independent of the bacterial growth rate. Cultures grown in slightly acidic conditions showed a faster rate of autolysis compared to cultures grown under alkaline conditions. SDS-polyacrylamide gel containing 0.2% crude cell-wall of S. aureus did not show any obvious correlation with the appearance of any particular lytic band in the zymogram to autolytic activity or rate of autolysis of cultures grown under various environmental conditions. A nonhemolytic phenotype, mutations in the accessory gene regulator, and lysogeny (phages phi 11, phi 12, phi 13) had no obvious effect either on the rate of autolysis or on the pattern of lytic bands in the zymograms.

Autolysis↗
PubMed1976

Electron microscopical and enzyme histochemical changes in the rat myocardium during prolonged autolysis.

The effect on rat myocardium of autolysis at 19 degrees C, for up to 20 days, was studied by electron microscopy and enzyme histochemistry. The enzymes studied included monoamine oxidase (MAO), sytochrome oxidase (CytO), non-specific esterase ((Ns.E.), phosphorylase (P-ase), succinate dehydrogenase (SDH) and various NAD- and NADP-linked dehydrogenases. The myocardium lost its histochemical P-ase activity within a few hours of autolysis, whereas the activity of all other enzymes remained quite normal for at least about 4 days, except that of MAO and SDH, which were normal for about 8 and 12 days, respectively. The myocardial cells lost activity of various enzymes in a patchy manner during prolonged autolysis and practically all histochemical enzyme activity disappeared within 20 days. The early period of autolysis was accompanied by rapid ultrastructural changes of myocardial cells. During prolonged autolysis the gross architecture of the myocardium was lost gradually by the 12th to 20th days. Mitochondria were the organelles most resistant to the effects of autolysis, and numerous mitochondria with morphologically solid inner and outer membranes were seen among the totally disintegrated myocardium 20 days after death. The loss of P-ase activity coincided with the loss of glycogen. The loss of MAO, SDH and CytO activities was not closely related to the morphological preservation of mitochondria, but, in accordance with other enzymes, was more closely related to the disintegration of the over-all myocardial structure. The present results showed that the architecture of the myocardium, and especially that of the mitochondria, was surprisingly resistant to the effects of autolysis at room temperature. Also several enzymes of the myocardium other than those examined so far maintained quite stable histochemically demonstrable activity during prolonged autolysis. These observations give support to the possibility of making the diagnosis of myocardial infarction at postmortem more accurate than with the present morphological and histochemical routine methods.

Alcohol Oxidoreductases↗
PubMed1986

Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells. 3. Interplay between limited cell-wall autolysis, pectin methyl esterase activity and electrostatic effects in soybean cell walls.

Soybean cell walls display a process of autolysis which results in the release of reducing sugars from the walls. Loosening and autolysis of cell wall are involved in the cell-wall growth process, for autolysis is maximum during both cell extension and cell-wall synthesis. Autolysis goes to completion within about 50 h and is an enzymatic process that results from the activity of cell wall exo- and endo-glycosyltransferases. The optimum pH of autolysis is about 5. Increasing the ionic strength of the bulk phase where cell-wall fragments are suspended, results in a shift of the pH profile towards low pH. This is consistent with the view that at 'low' ionic strength, the local pH in the cell wall is lower than in the bulk phase. One of the main ideas of the model proposed in a preceding paper, is that pectin methyl esterase reaction, by building up a high fixed charge density, results in proton attraction in the wall. Low pH must then activate the wall loosening enzymes involved in autolysis and cell growth. This view may be directly confirmed experimentally. The pH of a cell-wall suspension, initially equal to 5, was brought to 8 for 20 min, then back to 5. Under these conditions, the rate of cell-wall autolysis was enhanced with respect to the rate of autolysis obtained with cell-wall fragments kept at pH 5. The pH response of the multienzyme plant cell-wall system basically relies on opposite pH sensitivities of the two types of enzymes involved in the growth process. Pectin methyl esterase, which generates the cell-wall Donnan potential, is inhibited by protons, whereas the wall-loosening enzymes involved in cell growth are activated by protons.

Autolysis↗
PubMed1986

The role of subunit autolysis in activation of smooth muscle Ca2+-dependent proteases.

Ca2+-dependent proteases isolated from chicken gizzard and bovine aortic smooth muscle were compared with respect to subunit autolysis and the role of autolysis in modulating enzyme activity. The protease isolated from chicken gizzard was a heterodimer consisting of 80,000- and 30,000-dalton subunits. The protease isolated under identical conditions from bovine aorta consisted of 75,000- and 30,000-dalton subunits. In the presence of Ca2+, both enzymes underwent autolysis of their 30,000-dalton subunits with conversion to an 18,000-dalton species. In addition, the 80,000-dalton subunit of the gizzard protease was degraded to a 76,000-dalton form. The Ca2+ concentrations required for autolysis of the 30,000-dalton subunits were different for the two enzymes (i.e. gizzard: K0.5 Ca2+ = 335 microM; aortic: K0.5 Ca2+ = 1,250 microM) although in both cases, stimulation of autolysis by Ca2+ exhibited positive cooperativity. When compared with respect to kinetics of substrate degradation, the native forms of the smooth muscle Ca2+-dependent proteases (gizzard, GIIa = 80,000/30,000-dalton heterodimer; bovine aortic, IIa = 75,000/30,000-dalton heterodimer) exhibited a lag phase in product appearance. On the other hand, the autolyzed forms (gizzard, GIIb = 76,000/18,000-dalton heterodimer; bovine aortic, IIb = 75,000/18,000-dalton heterodimer) exhibited linear rates of substrate degradation. These results were analyzed in terms of autolysis of the 30,000-dalton subunits as determined by the conversion of this subunit to its 18,000 dalton form. For both enzymes, the time course for the autolytic transition, 30,000----18,000 daltons, and Ca2+-dependence of the apparent rate constants for this transition were found to correlate well with the lag phase in enzymatic activity. No such correlation could be established for the 80,000----76,000 dalton autolytic transition of the high molecular mass subunit of the gizzard protease. Our results suggest that catalytic activity of the Ca2+-dependent proteases isolated from gizzard and bovine aortic smooth muscle requires autolysis of the 30,000-dalton subunit. The native or unautolyzed forms of these enzymes appear to be proenzymes that can be activated by autolysis.

Animals↗