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PubMed1996

Relationship between phase variation in colony morphology, intrastrain variation in cell wall physiology, and nasopharyngeal colonization by Streptococcus pneumoniae.

Streptococcus pneumoniae undergoes phase variation in colony morphology, which has been implicated as a factor in the pathogenesis of pneumococcal disease. Phenotypic differences between opaque and transparent colony forms correlate with differences in rates of autolysis. This study examined whether differences in autolysis are caused by differences in expression of the major amidase, LytA, or the structure of its peptidoglycan substrate. No significant difference was detected by high-pressure liquid chromatography analysis of stem peptides released after treatment of purified peptidoglycan with amidase. Differences in the rate of digestion of purified cell walls, furthermore, did not correlate with susceptibility to autolysis. Lower levels of autolysis in opaque variants, however, was associated with decreased levels of immunodetectable LytA on colony immunoblots and Western blots (immunoblots). Diminished cell-surface-associated LytA in opaque variants was also demonstrated by whole-cell inhibition enzyme-linked immunosorbent assay. Since transparent variants have been shown both to colonize the nasopharynx more efficiently in an animal model and to express more surface-exposed LytA, it was determined whether LytA contributes to colonization in a neonatal rat model of pneumococcal carriage. Defined mutants in the lytA gene were used to show that there was no significant contribution by LytA to nasopharyngeal colonization in this model. Although the expression of LytA was shown to undergo phase variation in association with colony morphology, lytA mutants are still capable of phenotypic variation in colony morphology, which suggests that other factors are responsible for intrastrain differences which affect colonization.

Animals↗
PubMed1983

Cell wall-DNA association in Bacillus subtilis.

Autolysis of cell walls of Bacillus subtilis 168 resulted in solubilization of wall-associated DNA. Most of the DNA was solubilized only in the later stages of autolysis. Solubilization of up to 70% of the wall by autolysins resulted in only 25 to 30% solubilization of wall-associated DNA. When the wall fragments remaining after 70% autolysis were analyzed by electron microscopy, it was observed that the preparations were highly enriched for completed septa, or poles. Partial autolysis at pH 5.2 or pH 8.6, both of which reflect hydrogen ion levels that permit either N-acetylglucosaminidase or N-acetylmuramyl-L-alanine amidase, but not both, to act, gave rise to enrichment of cell poles. When walls were incubated with subtilisin, DNase, or RNase, release of DNA (or DNA fragments) was accelerated. Density gradient centrifugation patterns of lysates of cells pulse-labeled with N-[3H]acetylglucosamine and then chased revealed that a small, but significant, proportion of the radioactivity sedimented to a density position equivalent to that of DNA-membrane complexes. Because the pulse-chase sequence enriched for radioactivity in cell poles, the results suggest that at least some molecules from polar cell walls have an affinity for DNA-membrane complexes. We suggest that DNA binds strongly, possibly via a DNA-membrane complex, to cell poles of B. subtilis. The results provide support for a view offered previously (Koch et al., FEMS Microbiol. Lett. 12:201-208, 1981) that some special structure in or very near the poles of gram-positive bacilli is involved in the segregation of DNA during cell division.

Bacillus subtilis↗
PubMed1994

Mutations which alter the kinetics of calcium transport alter the regulation of competence in Streptococcus pneumoniae.

In Streptococcus pneumoniae, Ca2+ induces a stress response which is regulated by a proteic activator known as competence factor (CF). This stress response is expressed as the induction of competence for DNA uptake and genetic transformation in exponentially growing cultures and by autolysis in late exponential phase. DNA transport during competence can be described as a homeostatic response that prevents autolysis of the cultures. Electrogenic and cooperative calcium transport with a Hill number (nH) of 2 appears to mediate this Ca2+ response. Mutant strains altered in their kinetics for Ca2+ transport, with nHs of 1 and 4, were isolated and characterized in order to address the role of the kinetics of Ca2+ transport in the Ca2+ response. The reduced cooperativity of Ca2+ uptake in mutant strain Cp2200 was associated with an absolute requirement for added CF to develop competence and with resistance to autolysis. The enhanced cooperativity of Ca2+ uptake in mutant strain Cp3300 was associated with facilitated competence and hypersensitivity to autolysis. Moreover, the mutation carried by strain Cp3300 increases the CF response of previously described competence-defective mutants. The pleiotropic mutants Cp2200 and Cp3300 allowed us to demonstrate that cooperativity of transport determines the Ca2+ response in S. pneumoniae.

Allosteric Regulation↗
PubMed1967

Autolytic enzyme system of Streptococcus faecalis. 3. Localization of the autolysin at the sites of cell wall synthesis.

Cell walls from exponential-phase cultures of Streptococcus faecalis ATCC 9790 autolyzed in dilute buffers. Walls were isolated from cultures grown in the presence of (14)C-lysine for about 10 generations and then on (12)C-lysine for 0.1 to 0.8 of a generation (prelabeled). These walls released (14)C to the soluble fraction more slowly than they lost turbidity during the initial stages of autolysis. Walls isolated from cultures grown in the presence of (14)C-lysine for only the last 0.1 to 0.4 of a generation (postlabeled) released (14)C to the supernatant fluid more rapidly than they lost turbidity. Autolysin in both pre- and postlabeled walls was inactivated, and such walls were then incubated in the presence of unlabeled walls containing active autolysin. The inactivated walls lost their (14)C label only very slowly until autolysis of the unlabeled walls was virtually complete and release of soluble autolysin was expected. When this experiment was done in the presence of trypsin, a fourfold increase in the autolysis rate resulted, but the same pattern of (14)C release was observed. A parallel release of (14)C and loss of turbidity from pre- or postlabeled walls was observed upon trypsin "activation" and by addition of isolated soluble autolysin to inactivated walls. We conclude that the wall-bound autolysin acts first on the more recently synthesized portion of the wall. Trypsin appears to speed wall autolysis by activating additional latent autolysin in situ at sites in the older portion of the wall.

Antibodies↗
PubMed1988

Activation mechanism of calcium-activated neutral protease. Evidence for the existence of intramolecular and intermolecular autolyses.

The activation mechanism through limited autolysis of a calcium-activated neutral protease (CANP) with a high sensitivity to calcium ions (microCANP) was analyzed. The rate of autolysis was dependent on microCANP concentration. The reaction was inhibited by high concentrations of digestible substrates but not by a nondigestible substrate. Incubation of microCANP inactivated by N-ethylmaleimide with a small amount of activated microCANP caused the degradation of the former in a manner similar to the autolysis of native microCANP. Immobilized microCANP bound to an anti-microCANP immunoglobulin G column autolyzed on addition of calcium ions. These results show that activation of microCANP through limited autolysis involves both intramolecular and intermolecular reactions.

Animals↗
PubMedDate not supplied

[Effect of starvation on the intensity of proteolysis and amidation of proteins in tissues of rats of different ages].

Rates of autolysis and degree of amidation of tissue proteins were studied in rats under conditions of ageing and after starvation during 7 days. Proteins with decreased content of amide groups accumulated in brain, liver tissues and skeletal muscles. This phenomenon occurred apparently due to decrease in the rate of protein autolysis observed in ageing. In testes, where the rate of protein autolysis was similar both in young and old animals, the protein amidation was not altered on the course of ageing. Under conditions of severe starvation activation of proteolysis was observed in cells. At the same time, the amide content was restored in liver and muscle proteins of old rats up to the level, estimated in tissues of young animals. These data suggest stimulation of selective degradation of the deamidated proteins. The activation of autolysis and normalization of the protein amide groups content were not observed in brain of old animals under conditions of starvation.

Aging↗
PubMed1983

The effects of fibrinogen and its cleavage products on the kinetics of plasminogen activation by urokinase and subsequent plasmin activity.

The effects of fibrinogen and its plasmic cleavage fragments on the activation of Glu-, Lys-, and Val442- plasminogen by urokinase were investigated. A possible explanation for the large variations in the published steady state parameters for Glu-plasminogen activation is the undetected formation of Lys-plasminogen and its subsequent more rapid activation to plasmin. When Lys-plasminogen formation was avoided, the Km for Glu-plasminogen activation by urokinase was 2.5 microM with or without lysine present and the catalytic rate constant (kcat) was 3.4 min-1 in the absence of lysine, but increased to 49.0 min-1 in its presence. For Lys-plasminogen activation, both the Km of 2.7 microM and the kcat of 57.8 min-1 were only slightly increased by lysine. With Val442-plasminogen, the absence of the first 4 kringle structures of Lys-plasminogen resulted in a 6-fold higher Km and a 3-fold higher kcat, both of which were relatively unchanged by lysine. The specificity of urokinase for Val442-plasminogen, as measured by the quotient kcat/Km was thus half that for Lys-plasminogen. Fibrinogen, Fragment D, and Fragment E enhanced the rate of activation of Glu-plasminogen to Glu-plasmin as measured by the irreversible binding of plasmin to fluorescently labeled bovine pancreatic trypsin inhibitor. Both fibrinogen and Fragment D increased the value of kcat/Km about 4-fold whereas Fragment E caused a 2-fold enhancement. In contrast to Glu-plasminogen activation, the urokinase activation of Lys-plasminogen was not affected by fibrinogen or its fragments, yet a marked inhibition of Lys-plasmin autolysis occurred in their presence, with the half-life of plasmin being increased 13-fold by fibrinogen, 5-fold by Fragment D, and 3-fold by Fragment E. The K4 kringle region may be particularly involved in the plasmin-plasmin interaction that results in autolysis, since it significantly reduced degradation when incubated with Lys-plasmin. Val442-plasmin displayed essentially no autolysis, which further implicates the first 4 kringles in the autolytic reactions. In addition to these effects, the rate of Glu-plasminogen conversion to Lys-plasminogen by plasmin was increased 4-fold by fibrinogen or Fragment E, but only 2-fold by Fragment D. This augmentation was not merely due to inhibition of Lys-plasmin autolysis since Fragment D has a greater effect in that regard. The sum of these interactions indicates that Glu-plasminogen binds to the Fragment D region of fibrinogen/fibrin through its low affinity binding site(s) and, as when lysine binds at these sites, the activation to Glu-plasmin is then accelerated.(ABSTRACT TRUNCATED AT 400 WORDS)

Enzyme Activation↗
PubMed1993

A clinico-neuropathological study on brain death.

A clinico-neuropathological study was conducted on 60 cases of brain death, 36 males and 24 females, ranging in age from 11 to 81 years, the average being 49.4 years. Of these, 29 patients died of cerebrovascular disease. The average duration of brain death was 99 hours. The mean weight of the brain was 1466 g. Neuropathological findings were brain edema, congestion, herniation and various subarachnoid hemorrhages. Histologically, the cytoplasm of neurons was pale and ghost-like. In the white matter, myelin staining was pale, and nuclei of the glial cells were shrunken and piknotic. Autolysis of the cerebellar granular layer and the pituitary gland was evident in all cases. No reactive astrocytosis or infiltration of the cells in or around necrotic tissue could be seen. In eight cases, there was laminar infiltration of neutrophils in the superficial area of the cerebrum and brain stem, possibly due to temporary or partial recirculation. Correlation between the degree of autolysis and duration of brain death was observed, but no relationship between the degree of autolysis and the difference of underlying disease could be found. Autolysis in the cerebral cortex, thalamus, tegmentum of the brain stem, cerebellar granular layer and pituitary gland was most prominent. However, neuropathological diagnosis of underlying diseases could be made even in brain death. Histologically, the cases of brain death differed from those of cardiac arrest-induced encephalopathy and from those of long postmortem autopsy.

Adolescent↗
PubMed2001

Type I collagen stabilization of matrix metalloproteinase-2.

The activity of matrix metalloproteinase-2 (MMP-2) is regulated stringently on the posttranslational level. MMP-2 efficiently undergoes autolysis into inactive polypeptides in vitro, prompting the hypothesis that MMP-2 autolysis may function as an alternative mechanism for posttranslational control of MMP-2 in vivo. Moreover, MMP-2 binds to intact type I collagen fibrils; however, the functional consequences of this interaction have not been fully elucidated. To test the hypothesis that MMP-2 binding to type I collagen functions as a positive regulator of MMP-2 proteolytic potential, the effect of type I collagen on MMP-2 activity, inhibition by tissue inhibitor of metalloproteinase-2 (TIMP-2), and enzyme stability was examined. Here, we report that purified MMP-2 binds but does not cleave intact type I collagen. The presence of type I collagen affects neither enzymatic activity against a quenched fluorescent peptide substrate nor the kinetics of inhibition by TIMP-2. However, MMP-2 is stabilized from autolysis in the presence of type I collagen, but not by elastin, fibrinogen, or laminin. These data provide biochemical evidence that MMP-2 exosite interactions with type I collagen may function in the posttranslational control of MMP-2 activity by reducing the rate of autolytic inactivation.

Autolysis↗
PubMed2000

Effect of mu-calpain on m-calpain.

The free Ca(2+) concentrations required for half-maximal proteolytic activity of m-calpain are in the range of 400-800 microM and are much higher than the 50-500 nM free Ca(2+) concentrations that exist in living cells. Consequently, a number of studies have attempted to find mechanisms that would lower the Ca(2+) concentration required for proteolytic activity of m-calpain. Although autolysis lowers the Ca(2+) concentration required for proteolytic activity of m-calpain, 90-400 microM Ca(2+) is required for a half-maximal rate of autolysis of m-calpain, even in the presence of phospholipid. It has been suggested that mu-calpain, which has a lower Ca(2+) requirement than m-calpain, might proteolyze m-calpain and reduce its Ca(2+) requirement to a level that would allow it to be active at physiological Ca(2+) concentrations. We have incubated m-calpain with mu-calpain for 60 min at a ratio of 1:50 mu-calpain:m-calpain, in the presence of 50 microM free Ca(2+); this Ca(2+) concentration is high enough for more than half-maximal activity of mu-calpain, but does not activate m-calpain. Under these conditions, mu-calpain caused no detectable proteolytic degradation of the m-calpain polypeptide and did not change the Ca(2+) concentration required for proteolytic activity of m-calpain. mu-Calpain also did not degrade the m-calpain polypeptide at 1000 microM Ca(2+), which is a Ca(2+) concentration high enough to completely activate m-calpain. It seems unlikely that mu-calpain could act as an "activator" of m-calpain in living cells. Because m-calpain rapidly degrades itself (autolyzes) at 1000 microM Ca(2+) and because the subsite specificities of mu- and m-calpain are very similar if not identical, failure of mu-calpain to rapidly degrade m-calpain at 1000 microM Ca(2+) suggests a unique role of autolysis in calpain function.

Animals↗
PubMed1979

Crystalloid inclusions reminiscent of Hirano bodies in autolyzed peripheral nerve of normal wistar rats.

In a study of experimental autolysis of peripheral nerves of normal Wistar rats, crystalloid inclusions similar to Hirano bodies were observed. They occurred in the nuclei and cytoplasms of Schwann cells and in myelinated axons. They first developed after a 12-h autolysis and increased in number and size with time. Their fine structure, localization, and morphogenesis are discussed in relation to the autolysis of the cellular organelles.

Animals↗
PubMed1987

The potential use of implanted radiolabelled bovine nasal cartilage in dialysis tubing to evaluate agents affecting cartilage degradation in vivo.

Cartilage which undergoes extensive autolysis in vitro (spontaneous or stimulated) is characterized by proteoglycan loss. Experimental conditions and inhibitor profils studies suggest neutral metalloproteinases induce the autolysis. In these preliminary studies we compared the degradation of Na2 35SO4 labeled bovine nasal cartilage (BNC) plugs placed in dialysis tubing in vitro and in vivo. The dialysis tubing was used to exclude large molecules (molecular weights greater than 2000) like proteinases, and proteinase inhibitors (e.g. alpha 2-macroglobulin) but not potential test agents from the implanted cartilage. Cartilage autolysis occurred with live tissue but not with heat-killed tissue in both the in vitro and in vivo systems. In addition retinoic acid and phenanthroline were effective when placed inside or outside the dialysis tubing. A potentially useful procedure to evaluate agents which affect cartilage degradation is described.

Animals↗
PubMed1977

Early post-mortem change of the crista ampullaris. A light and electron microscopic study of the Guinea-pig.

Post-mortem change (autolysis) of the sensory and the secretory epithelia was studied by means of the light and the electron microscope. The first ultrastructurally recognizable sign of autolysis occurred 10-15 min after death in the mitochondria of the hair cells and their adjacent nerve endings. The morphology of hair cells was occasionally well preserved 30 min post-mortem, but after 90 min or more autolytic changes were invariably severe. The secretory epithelium, however, was considerably less affected by autolysis than the hair cells during the first 5 h after death.

Animals↗
PubMed2001

Molecular pathology and evolutionary and physiological implications of pancreatitis-associated cationic trypsinogen mutations.

Since the identification in 1996 of a "gain of function" missense mutation, R122H, in the cationic trypsinogen gene (PRSS1) as a cause of hereditary pancreatitis, continued screening of this gene in both hereditary and sporadic pancreatitis has found more disease-associated missense mutations than expected. In addition, functional analysis has yielded interesting findings regarding their underlying mechanisms resulting in a gain of trypsin. A critical review of these data, in the context of the complicated biogenesis and complex autoactivation and autolysis of trypsin(ogen), highlights that PRSS1 mutations cause the disease by various mechanisms depending on which biochemical process they affect. The discovery of these mutations also modifies the classical perception of the disease and, more importantly, reveals fascinating new aspects of the molecular evolution and normal physiology of trypsinogen. First, activation peptide of trypsinogen is under strong selection pressure to minimize autoactivation in higher vertebrates. Second, the R122 primary autolysis site has further evolved in mammalian trypsinogens. Third, evolutionary divergence from threonine to asparagine at residue 29 in human cationic trypsinogen provides additional advantage. Accordingly, we tentatively assign, in human cationic trypsinogen, the strongly selected activation peptide as the first-line and the R122 autolysis site as the second-line of the built-in defensive mechanisms against premature trypsin activation within the pancreas, respectively, and the positively selected asparagine at residue 29 as an "amplifier" to the R122 "fail-safe" mechanism.

Amino Acid Sequence↗
PubMed1996

Hepatocyte vacuolation and autolytic changes in the liver of pilot whales, Globicephala melas, stranded on Cape Cod, MA, USA.

Most cetacea available for internal sampling in recent times have died through mass or single stranding events. It is important to know how the time elapsed between death and sampling affect quality of tissues. This study evaluated histological quality in the liver of long-finned pilot whales that either died or were euthanased after mass stranding events. Histological detection of significant autolysis was found in animals when 2 or more hours elapsed between death and sampling. In addition, hepatocytes often had marked idiopathic cytoplasmic vacuolation that did not stain with hematoxylin and eosin. The extent of this vacuolation did not show any correlation with time between death and sampling, but did appear more often in animals of greater total length. These observations suggest that when animals die or are euthanased at a single or mass stranding, every effort should be made to obtain samples as soon as possible, although meaningful histological observations can still be made in the presence of significant autolysis. These data also suggest that a multi-disciplinary study should be conducted to determine whether increasing autolysis is associated with changes in the organic chemical residues, molecular biology, histopathology and microbiology of those tissues.

Animals↗
PubMed2005

Malonate-assisted purification of human caspases.

Caspases have in the past decade become some of the most intensely pursued targets for the design of small-molecule inhibitors. Two significant technological roadblocks to developing caspase-binding molecules are the poor solubility of a subset of the bacterially expressed proteins and the instability of the renatured proteins that results from rapid inactivating autolysis at high protein concentrations. In this report, we present a generalized method of renaturing human caspases and inhibiting the self-proteolytic activity of the enzymes without a need for covalent active-site inhibitors. Our method, which involves blocking the S1 region of the active site with malonate, enables one to inhibit fully the inactivating autolysis in human caspases and increases the yields of renatured active enzyme. It furthermore does not necessitate removal of malonate prior to setting up enzymatic assays since as high as 100-mM concentrations of malonate do not compete efficiently with caspase substrates or larger caspase inhibitors for binding to the active site. The method described in this report simplifies greatly caspase purification and makes it possible to stabilize the enzymes against autolysis without a need for costly, and frequently synthetically challenging, small-molecule inhibitors.

Autolysis↗
PubMed2003

Structure of human pro-chymase: a model for the activating transition of granule-associated proteases.

Human chymase is a protease involved in physiological processes ranging from inflammation to hypertension. As are all proteases of the trypsin fold, chymase is synthesized as an inactive "zymogen" with an N-terminal pro region that prevents the transition of the zymogen to an activated conformation. The 1.8 A structure of pro-chymase, reported here, is the first zymogen with a dipeptide pro region (glycine-glutamate) to be characterized at atomic resolution. Three segments of the pro-chymase structure differ from that of the activated enzyme: the N-terminus (Gly14-Gly19), the autolysis loop (Gly142-Thr154), and the 180s loop (Pro185A-Asp194). The four N-terminal residues (Gly14-Glu15-Ile16-Ile17) are disordered. The autolysis loop occupies a position up to 10 A closer to the active site than is seen in the activated enzyme, thereby forming a hydrogen bond with the catalytic residue Ser195 and occluding the S1' binding pocket. Nevertheless, the catalytic triad (Asp102-His57-Ser195) is arrayed in a geometry close to that seen in activated chymase (all atom rmsd of 0.52 A). The 180s loop of pro-chymase is, on average, 4 A removed from its conformation in the activated enzyme. This conformation disconnects the oxyanion hole (the amides of Gly193 and Ser195) from the active site and positions only approximately 35% of the S1-S3 binding pockets in the active conformation. The backbone of residue Asp194 is rotated 180 degrees when compared to its conformation in the activated enzyme, allowing a hydrogen bond between the main-chain amide of residue Trp141 and the carboxylate of Asp194. The side chains of residues Phe191 and Lys192 of pro-chymase fill the Ile16 binding pocket and the base of the S1 binding pocket, respectively. The zymogen positioning of both the 180s and autolysis loops are synergistic structural elements that appear to prevent premature proteolysis by chymase and, quite possibly, by other dipeptide zymogens.

Autolysis↗
PubMed1991

High-yield extraction and purification of glutathione reductase from baker's yeast.

Glutathione reductase was extracted from toluene-treated baker's yeast cells by a two-stage buffer autolysis method. The yeast cells were treated with toluene for 1 h at 40 degrees C. After removal of the toluene, the cells were then allowed to autolysis in buffer for 72 h at 4 degrees C. The cells were collected and resuspended in buffer. A second stage autolysis was carried out for another 96 h at 4 degrees C. The enzyme was purified to 786-fold from the second stage cell autolysate by using two steps of affinity chromatography with triazine dyes (Yellow H-E4G and Yellow H-E6G) coupled to Sepharose CL-4B. By using this simplified method, 1.44 mg (165 units/mg) of glutathione reductase was obtained from 65 g (wet weight) of yeast cells, equivalent to 80% enzyme recovery.

Autolysis↗