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PubMed2005

mu-Calpain and calpain-3 are not autolyzed with exhaustive exercise in humans.

mu-Calpain and calpain-3 are Ca2+-dependent proteases found in skeletal muscle. Autolysis of calpains is observed using Western blot analysis as the cleaving of the full-length proteins to shorter products. Biochemical assays suggest that mu-calpain becomes proteolytically active in the presence of 2-200 microM Ca2+. Although calpain-3 is poorly understood, autolysis is thought to result in its activation, which is widely thought to occur at lower intracellular Ca2+ concentration levels ([Ca2+]i; approximately 1 microM) than the levels at which mu-calpain activation occurs. We have demonstrated the Ca2+-dependent autolysis of the calpains in human muscle samples and rat extensor digitorum longus (EDL) muscles homogenized in solutions mimicking the intracellular environment at various [Ca2+] levels (0, 2.5, 10, and 25 microM). Autolysis of calpain-3 was found to occur across a [Ca2+] range similar to that for mu-calpain, and both calpains displayed a seemingly higher Ca2+ sensitivity in human than in rat muscle homogenates, with approximately 15% autolysis observed after 1-min exposure to 2.5 microM Ca2+ in human muscle and almost none after 1- to 2-min exposure to the same [Ca2+]i level in rat muscle. During muscle activity, [Ca2+]i may transiently peak in the range found to autolyze mu-calpain and calpain-3, so we examined the effect of two types of exhaustive cycling exercise (30-s "all-out" cycling, n = 8; and 70% VO2 peak until fatigue, n = 3) on the amount of autolyzed mu-calpain or calpain-3 in human muscle. No significant autolysis of mu-calpain or calpain-3 occurred as a result of the exercise. These findings have shown that the time- and concentration-dependent changes in [Ca2+]i that occurred during concentric exercise fall near but below the level necessary to cause autolysis of calpains in vivo.

Animals↗
PubMed1998

[Phenomenon of karyolysis in cytophotomorphometric examinations].

Studies were performed on hepatocytes of ten rabbits' livers. The one-year-old animals were sacrificed by a blow in the suboccipital region. Under aseptic conditions each liver was divided into 9 parts. One of them was examined at once (zero time) the remaining parts were placed in sterile vessels lined with gauze moistened in Ringer's solution, which created an environment of 100% humidity. Autolysis was being conducted for 8 hours at temperature 37 degrees C, carrying out the investigations with hourly intervals. The cells for studying were obtained by preparing contact smears that stemmed from a fresh cross-section of the liver segment. The smears were fixed in formalin vapour 40% proceeding for 2 hours at temperature 21 degrees C, and subsequently in 96% ethanol for 0.5 hour. Feulgen's reaction was run in accordance with the routine principles by using the basic fuschin NI 42510 of Fischer Firm (USA). The morpho-photometric measurements of nuclei were taken by means of computed device "Morphoquant" of Carl Zeiss Firm, Jena, 1982. The following measurements of the cell nuclei were accomplished, namely: A. morphometric--surface, circumferential length, bulged surface, ratio of diameters, unstained surface; B. photometric--medium extinction, circumferential extinction, total extinction. The results of studies were documented by 95 diagrams. On the basis of measurements taken, it has been established that, during the 7 hour-long period of autolysis, nucleus of hepatocyte is subjected to dynamic transformations, whereas the plotted function does not follow a linear course. During the observation the circumference and the surface resemble a polynomial with three extremes (Fig. 12, 21, 39); similarly the mean extinction and circumference extinction (attention is attracted by its severe course as compared to surface and circumference) (Fig. 57, 66). At the time of experiment the differentiation of nuclei population undergoes changes in the direction of its uniformity which concerns, to greater or lesser degree, all the investigated features (compare figures of histograms). The isolated group of tetraploidal nuclei is characterised by occupying another place. It is defined by 3 variables in spatial arrangement (Fig. 76, 80). The analysis of correlation allowed for determination of the dynamic connections, between the studied variables, which seem to be specific for a given experimental time (Tab. 1, 2). On the ground of the performed studies 7 final conclusions have been formulated: 1. Autolysis of the nuclei of hepatocytes is non-linear phenomenon; (its course resembles multinominal graph 6 degrees with three extremes in 1, 4 and 6th hour. 2. Photomorphometric parameters allow for determining the status of the nucleus of hepatocyte in the course of autolysis. 3. The fourth hour of autolysis is a characteristic point after which no response of nucleus to deteriorating oxygen condition was observed. 4. In postmortem autolysis of the nuclei of hepatocytes pyknosis is found to appear, there is no phenomenon of karyorrhexis. 5. After a 7-hour-long experiment, Feulgen's diffusional reaction starts in cytoplasm. 6. A group of tetraploid nuclei is being isolated in the course of the autolysis of hepatocytes. 7. In the course of experiment there appeared a small quantitative loss of nuclear DNA.

Animals↗
PubMed2004

Insertion sequence 1 of muscle-specific calpain, p94, acts as an internal propeptide.

The physiological role of the skeletal muscle-specific calpain 3, p94, is presently unknown, but defects in its gene cause limb girdle muscular dystrophy type 2A. This calcium-dependent cysteine protease resembles the large subunit of m-calpain but with three unique additional sequences: an N-terminal region (NS), and two insertions (IS1 and IS2). The latter two insertions have been linked to the chronic instability of the whole enzyme both in vivo and in vitro. We have shown previously that the core of p94 comprising NS, domains I and II, and IS1 is stable as a recombinant protein in the absence of Ca(2+) and undergoes autolysis in its presence. Here we show that p94I-II cannot hydrolyze an exogenous substrate before autolysis but is increasingly able to do so when autolysis proceeds for several hours. This gain in activity is caused by cleavage of IS1 during autolysis because a deletion mutant lacking the NS region (p94I-II DeltaNS) shows the same activation profile. Similarly, the calpain inhibitors E-64 and leupeptin have almost no inhibitory effect on substrate hydrolysis by p94I-II soon after calcium addition but cause complete inhibition when autolysis progresses for several hours. As autolysis proceeds, there is release of the internal IS1 peptide, but the two portions of the core remain tightly associated. Modeling of p94I-II suggests that IS1 contains an amphipathic alpha-helix flanked by extended loops. The latter are the targets of autolysis and limited digestion by exogenous proteases. The presence and location of the alpha-helix in recombinant IS1 were confirmed by circular dichroism and by the introduction of a L286P helix-disrupting mutation. Within p94I-II, L286P caused premature autoproteolysis of the enzyme. IS1 is an elaboration of a loop in domain II near the active site, and it acts as an internal autoinhibitory propeptide, blocking the active site of p94 from substrates and inhibitors.

Amino Acid Sequence↗
PubMed1994

Cell-Wall Autohydrolysis in Isolated Endosperms of Lettuce (Lactuca sativa L.).

Cell walls prepared from the endosperm tissue of hydrated lettuce (Lactuca sativa L.) seeds undergo autohydrolysis. Release of carbohydrates is most rapid (0.4-0.6 [mu]g per endosperm) within the 1st h of incubation in buffer, but substantial autolysis is sustained for at least 10 h. Autolysis is temperature sensitive, and the optimum rate occurs at pH 5. The rate of autolysis increases markedly in the period just prior to radicle emergence. The cell-wall polysaccharide composition in micropylar and lateral endosperm regions differs significantly; the micropylar walls are rich in arabinose and glucose with substantially lower amounts of mannose. Although walls prepared from both micropylar and lateral regions undergo autolysis, micropylar walls release carbohydrates at a higher rate than lateral walls. Autolysis products elute as large polymers when subjected to size-exclusion chromatography, suggesting that endo-enzyme activity is responsible for release of fragments containing arabinose, galactose, mannose, and uronic acids. Arabinose, galactose, mannose, and glucose are also released as monomers. As a function of time, the ratio of polymers to monomers decreases, indicating that exo-enzyme activity is also present. Thermoinhibition or treatment with abscisic acid suppresses germination and reduces the rates of autolysis of walls isolated from the endosperm by about 25%. Treatments that alleviate thermoinhibition (kinetin and gibberellic acid) increase the rates of autolysis by 20 to 30% when compared to thermoinhibited controls.

Journal Article↗
PubMed1982

Induction and control of the autolytic system of Escherichia coli.

Various methods of inducing autolysis of Escherichia coli cells were investigated, some being described here for the first time. For the autolysis of growing cells only induction methods interfering with the biosynthesis of peptidoglycan were taken into consideration, whereas with harvested cells autolysis was induced by rapid osmotic or EDTA shock treatments. The highest rates of autolysis were observed after induction by moenomycin, EDTA, or cephaloridine. The different autolyses examined shared certain common properties. In particular, regardless of the induction method used, more or less extensive peptidoglycan degradation was observed, and 10(-2) M Mg2+ efficiently inhibited the autolytic process. However, for other properties a distinction was made between methods used for growing cells and those used for harvested cells. Autolysis of growing cells required RNA, protein, and fatty acid synthesis. No such requirements were observed with shock-induced autolysis performed with harvested cells. Thus, the effects of Mg2+, rifampicin, chloramphenicol, and cerulenin clearly suggest that distinct factors are involved in the control of the autolytic system of E. Coli. Uncoupling agents such as sodium azide, 2,4-dinitrophenol, and carbonyl-cyanide-m-chlorophenyl hydrazone used at their usual inhibiting concentration had no effect on the cephaloridine or shock-induced autolysis.

Anti-Bacterial Agents↗
PubMed1997

Molecular analysis of the feline immunodeficiency virus protease: generation of a novel form of the protease by autoproteolysis and construction of cleavage-resistant proteases.

The feline immunodeficiency virus (FIV) protease is essential for virion maturation and subsequent viral replication in that it cleaves the Gag and Gag/Pol polyproteins at eight sites to release the respective structural proteins and enzymes. During purification of a recombinant FIV protease (PR), we noted that it underwent autoproteolysis (autolysis) to give discrete cleavage products. These additional PR cleavage sites were defined using N-terminal amino acid sequence analysis and mass spectrometry. Protease breakdown products were also found in FIV virions and were of the same apparent molecular weights as the in vitro autolysis products. Four primary PR autolysis sites were blocked via substitution of either the P1 amino acid with a beta-branched amino acid or the P1' amino acid with lysine. Cleavage-resistant PRs which had Km and k(cat) values similar to those of FIV PR were constructed. An autolysis time course determined that blocking all four primary autolysis sites yielded a cleavage-resistant PR which was enzymatically stable. Concomitant with autolysis is the generation of an N-terminally truncated form of the PR (Thr6/PR) which has enhanced stability with respect to that of FIV PR. A structural basis for the Thr6/PR activity is presented, as are the possible roles of autolysis in the viral replication cycle.

Animals↗
PubMed1993

Effect of monoclonal antibodies specific for the 28-kDa subunit on catalytic properties of the calpains.

Nine monoclonal antibodies (mAbs) specific for the 28-kDa subunit common to mu- and m-calpains have been assayed for their effects on mu- and m-calpains. All nine react with the COOH-terminal part (domain VI) of the 28-kDa subunit, and all nine affect the Ca2+ concentration required for autolysis of m-calpain, but have little effect on the Ca2+ concentration required for autolysis of mu-calpain. None of the nine affect the specific proteolytic activity of mu- or m-calpain. Two of the mAbs, 5B9 and 5B3, were selected for further study. mAb 5B9 decreased the Ca2+ concentration required for autolysis to one-fifth of that required in its absence; sequencing of chymotryptic fragments showed that the epitope for mAb 5B9 is between amino acid residues 92 and 104 of the 28-kDa subunit. mAb 5B3 increased the Ca2+ concentration required for autolysis; the epitope for mAb 5B3 is located between amino acid residues 148 and 178 of the 28-kDa subunit, which is the region that contains the first EF-hand Ca(2+)-binding sequence in this subunit. Although it increases the Ca2+ concentration required for autolysis, mAb 5B3 has no effect on the Ca2+ concentration required for proteolytic activity of m-calpain, and unautolyzed m-calpain is not a proenzyme. That all nine mAbs react with domain VI and not with the NH2-terminal domain V of the 28-kDa subunit suggests that domain VI (and not domain V) is involved in autolysis, contrary to the view that phosphatidylinositol lowers the Ca2+ concentration required for autolysis by binding to domain V.

Amino Acid Sequence↗
PubMedDate not supplied

[Changes in the proteolytic activity of tissues during aging].

The intensity of proteolytic processes and qualitative composition of autolysis products of the brain, liver and testicle tissues of young and old rats were studied. The gel-chromatographic analysis (Sephadex G-15 and G-50) revealed no considerable amount of high-molecular peptides (1500 Da and over) before and after autolysis. The measurement of the quantity of free amino groups in the gel-chromatographic fraction after the complete acid hydrolysis has confirmed that result. The low-molecular peptides and free amino acids, are the main products of the tissue autolysis. The intensity of proteolytic processes, determined by an increase in the amount of amino acids depends on the autolysis duration and age of animals. The total increment of amino acids in the brain and liver tissues of old animals for the first hour of autolysis has been higher by 102 and 219% as compared to young ones. The autolysis of testicles of the young and old animals after the first hour of incubation is characterized by the same intensivity. Such a regularity is not revealed when analyzing the same processes by the Lowry method.

Aging↗
PubMedDate not supplied

[Autolytic changes in serum lipids of normal rats and following acute oxygen deficiency].

Alterations in content of main classes of lipids and phospholipids were studied in blood serum during autolysis in normal rats and in acute oxygen deficiency. In the control animals the most distinct alterations were found in content of phospholipids, which was decreased down to 1/2 of the initial level within 24 hrs of autolysis with simultaneous increase in concentration of fatty acids within 10 hrs. A decrease in content of free cholesterol together with an increase in concentration of its esters were observed in hypoxic animals within 24 hrs of autolysis. During autolysis concentration of phosphatidylcholines was decreased both in control and hypoxic animals. At the same time, an increase in lysophosphatides within 4 hr of autolysis and a decrease in concentration of phosphatidic acids within 24 hrs were found in the control group. In animals with hypoxia phosphatidic acids were only slightly increased and polyglycerophosphatides were found during subsequent periods of autolysis.

Acute Disease↗
PubMed2003

Evidence for heterogeneous forms of the snake venom metalloproteinase jararhagin: a factor contributing to snake venom variability.

The reprolysin subfamily of metalloproteinases includes snake venom metalloproteinases (SVMP) and mammalian disintegrin/metalloproteinase. These proteins are synthesized as zymogens and undergo proteolytic processing resulting in a variety of multifunctional proteins. Jararhagin is a P-III SVMP isolated from the venom of Bothrops jararaca. In crude venom, two forms of jararhagin are typically found, full-length jararhagin and jararhagin-C, a proteolytically processed form of jararhagin that is composed of the disintegrin-like and cysteine-rich domains of jararhagin. To better understand the structural and mechanistic bases for these forms of jararhagin in the venom of B. jararaca and the source of venom complexity in general, we have examined the jararhagin forms isolated from venom and the autolysis of isolated jararhagin under the conditions of varying pH, calcium ion concentration, and reducing agents. From our results, jararhagin isolated from venom appears as two forms: a predominant form that is stable to in vitro autolysis and a minor form that is susceptible to autolysis under a variety of conditions including alkaline pH, low calcium ion concentrations, or reducing agent. The autolysis site for production of jararhagin-C from isolated jararhagin was different from that observed for jararhagin-C as isolated from crude venom. Taken together, these data lead us to the conclusion that during the biosynthesis of jararhagin in the venom gland at least three forms are present: one form which is rapidly processed to give rise to jararhagin-C, one form which is resistant to processing in the venom and autolysis in vitro, and one minor form which is susceptible to autolysis under conditions that promote destabilization of its structure. The presence of these different forms of jararhagin contributes to greater structural and functional complexity of the venom and may be a common feature among all snake venoms. The biological and biochemical features in the venom gland responsible for these jararhagin isoforms are currently under investigation.

Amino Acid Sequence↗
PubMed1967

Nutrition and lysosomal activity. The influence of vitamin E deficiency and its duration on the stability of lysosomes in the kidneys of rats.

1. Experiments on rats were made to find whether the increased liability of the kidney-cortex tubules to autolysis post mortem, which is a well-established abnormality in vitamin E deficiency, can be correlated with changes in lysosomal activity. Parallel observations were made on the development of certain other abnormalities characteristic of avitaminosis E. 2. In rats killed after long periods (8-10 months) of subsistence on a standard vitamin E-deficient diet, containing lard, both the rate of kidney autolysis post mortem and the enzyme activity of lysosome preparations from the fresh tissues were much greater than in controls. A greater percentage difference was usually found in the ;free' enzyme fraction than in ;bound' or ;total' activity. 3. In rats killed after graded periods (3-8 months) of deficiency, two abnormalities (decreased resistance of the erythrocytes to haemolysis, and brown discoloration of the uterus) were already evident at a stage (3-4 months) when the liability to rapid kidney autolysis had not begun. At this point the enzymic activity of kidney extracts differed little between deficient animals and controls given alpha-tocopherol. As the duration of deficiency advanced, parallel increases occurred in the rate of kidney autolysis and in lysosomal instability. 4. When cod-liver oil, rich in polyunsaturated fatty acids but freed from vitamin A, was substituted for lard in the diet, the time (1(1/2) months) required for the inducement of both rapid kidney autolysis and decreased lysosomal stability was greatly shortened. The time for the inducement of brown discoloration of the uterus was not shortened and the kidney abnormalities appeared while the uterus was still normal. 5. Confirmation was thus obtained for the view that the various tissues of the rat respond differently to the relationship between the adequacy of the vitamin E status and the intake of polyunsaturated fatty acids. The kidney-cortex tubules, as evidenced by autolysis post mortem and the corresponding decrease in lysosomal stability, may be classed among those tissues that are most sensitive to the effect of high intakes of polyunsaturated acids.

Animals↗
PubMed2006

Suppressed disassembly of autolyzing p94/CAPN3 by N2A connectin/titin in a genetic reporter system.

p94/calpain 3 is a skeletal muscle-specific member of the Ca(2+)-regulated cytosolic cysteine protease family, the calpains. Defective p94 protease activity originating from gene mutations causes a muscular dystrophy called calpainopathy, indicating the indispensability of p94 for muscle survival. Because of the existence of the p94-specific regions IS1 and IS2, p94 undergoes very rapid and exhaustive autolysis. To elucidate the physiological relevance of this unique activity, the autolytic profiles of p94 and the effect of the p94 binding protein, connectin/titin, on this process were investigated. In vitro analysis of p94 autolysis showed that autolysis in IS1 proceeds without immediate disassembly into fragments and that the newly identified cryptic autolytic site in IS2 is critical for disassembling autolyzed fragments. As a genetic system to assay p94 autolysis semiquantitatively, p94 was expressed in yeast as a hybrid protein between the DNA binding and activation domains of the yeast transcriptional activator Gal4. Transcriptional activation by the Gal4-p94:WT hybrid protein is precluded by p94 autolysis. Complete or partial loss of autolytic activity by C129S active site mutation, limb girdle muscular dystrophy type 2A pathogenic missense mutations, or PCR-based random mutagenesis could be detected by semiquantitative restoration of Gal4-dependent beta-galactosidase gene expression. Using this system, the N2A connectin fragment that binds to p94 was shown to suppress p94 autolytic disassembly. The proximity of the IS2 autolytic and connectin-binding sites in p94 suggested that N2A connectin suppresses IS2 autolysis. These data indicate the importance of p94-connectin interaction in the control of p94 functions by regulating autolytic decay of p94.

Animals↗
PubMed1978

Effects of temperature on the autolytic enzyme system of Streptococcus faecalis.

The cellular autolytic reaction system in Streptococcus faecalis ATCC 9790 was analyzed for relative increases in reaction rates with increasing temperature by determination of Arrhenius activation energies (E). The systems examined were: (i) an isolated wall-enzyme complex in 0.01 M sodium phosphate, pH 6.9; (ii) exponential-phase cells suspended in 0.01 or o.3 M sodium phosphate pH 6.8, or in 0.04 M ammonium acetate, pH 6.8, (iii) growing cultures deprived of glucose or lysine; and (iv) cultures treated in growth media with the nonionic detergent, Triton X-100. For detergent-treated cells, E values were between 23.9 and 27.4 kcal/mol (ca. 100.1 to 174.7 kJ/mol) at concentrations of Triton X-100 between about 0.03 and 0.072 mg/ml. E values dropped sharply to 11.5 to 13.0 kcal/m-l (ca. 48.2 to 54.4 kJ/mol) at Triton X-100 concentrations of 0.12 mg/ml or higher. For the remaining systems, E values ranged from 16 to 20 kcal/mol (ca. 67.0 to 83.7 kJ/mol) (wall lysis, cellular autolysis in 0.01 M sodium phosphate or in 0.04 M ammonium acetate, and autolysis of glucose-starved cells) to 31 to 38 kcal/mol (ca 129.8 to 159.1 kJ/mol) (cellular autolysis in 0.3 M sodium phosphate or autolysis of lysine-starved cells). High concentrations of Triton X-100 appear to lower the E values below the 16 to 20 kcal/mol observed for the autolysis of isolated walls. This effect may be related to disruption by the detergent of a hydrophobic complex regulating cellular autolysis in vivo.

Amidohydrolases↗
PubMed2000

Neuronal nuclear DNA fragmentation in the aged canine brain: apoptosis or nuclear DNA fragility?

Neuronal DNA fragmentation, as revealed with the method of in situ end-labeling of nuclear DNA fragmentation (TUNEL), has been reported in both the canine and human brains in normal ageing, and in some human age-related neurodegenerative diseases. These results have suggested that apoptosis plays an important role in age-related neuronal loss. It is not clear, however, whether the TUNEL method is highly specific for apoptosis, as DNA fragmentation also occurs in the late stages o necrosis. In this study we have examined 27 dogs aged from 8 to 18 years, to investigate the occurrence of nuclear DNA fragmentation. An autolysis index based on current histological criteria was assigned to each animal to evaluate the effects of autolysis on nuclear DNA integrity. Our results have shown that neuronal nuclear DNA fragmentation is frequent in aged dogs, although it is not accompanied by apoptotic morphology. Yet, a positive relation between TUNEL labelling and the degree of tissue autolysis was observed. In contrast, no TUNEL labelling was detected in young control dogs despite autolysis indices being similar to those in aged dogs. Taken together, these results suggest that neuronal nuclear DNA fragmentation is an age-related phenomenon, not due to apoptosis, whenever other factors render neuronal DNA more susceptible to autolytic fragmentation. We confirm the effect of autolysis in a subpopulation of neurons in the aged canine brain, inducing nuclear DNA fragmentation.

Aging↗
PubMed1988

Phospholipid composition and amphiphile content of isolated sarcolemma from normal and autolytic rat myocardium.

Sarcolemmal vesicles were purified to a similar extent, 50- to 60-fold on a protein basis, from normal rat hearts and hearts subjected to 30 or 60 min of autolysis at 37 degrees C (total ischemia in vitro). Electron microscopic examination of the autolytic hearts revealed sarcolemmal discontinuities and other morphological characteristics typical of irreversible cell injury. Total contents and percentage composition of phospholipid classes did not differ between normal and autolytic hearts or between sarcolemmal preparations from these hearts. There was no increase in lysophospholipid contents of whole hearts or of purified sarcolemma after autolysis. Long chain acyl-CoAs or acylcarnitines did not accumulate in autolytic hearts under our experimental conditions. The molar long chain acyl-CoA: phospholipid ratio in isolated sarcolemma was extremely low (1:100,000). It increased 3-fold after autolysis but the increase was most probably the result of an increase in mitochondrial contamination of the sarcolemmal preparations from autolytic hearts. The molar long chain acylcarnitine: phospholipid ratio of isolated sarcolemma was much larger (1:100), but it did not change after autolysis. Experiments, in which radioactive amphiphiles were incorporated in isolated sarcolemma that was subsequently repeatedly washed, indicated that the lysophospholipid and acylcarnitine contents of isolated sarcolemma reflect the contents of sarcolemma in situ, but that sarcolemmal acyl-CoA is used for re-acylation reactions during purification, explaining the low acyl-CoA content of isolated sarcolemma. Na/K-ATPase and Na/Ca-exchange activities were markedly depressed in isolated sarcolemma from autolytic hearts. Our results suggest that sarcolemmal phospholipid breakdown and sarcolemmal amphiphile accumulation are not responsible for the structural and functional defects of the sarcolemma after autolysis.

Acetylcarnitine↗
PubMed2001

Structural determinants of the half-life and cleavage site preference in the autolytic inactivation of chymotrypsin.

The molecular mechanism of the autolysis of rat alpha-chymotrypsin B was investigated. In addition to the two already known autolytic sites, Tyr146 and Asn147, a new site formed by Phe114 was identified. The former two sites and the latter one are located in the autolysis and the interdomain loops, respectively. By eliminating these sites by site-directed mutagenesis, their involvement in the autolysis and autolytic inactivation processes was studied. Mutants Phe114-->Ile and Tyr146-->His/Asn147-->Ser, that had the same enzymatic activity and molecular stability as the wild-type enzyme, displayed altered routes of autolytic degradation. The Phe114-->Ile mutant also exhibited a significantly slower autolytic inactivation (its half-life was 27-fold longer in the absence and sixfold longer in the presence of Ca2+ ions) that obeyed a first order kinetics instead of the second order displayed by wild-type chymotrypsin inactivation. The comparison of autolysis and autolytic inactivation data showed that: (a) the preferential cleavage of sites followed the order of Tyr146-Asn147 --> Phe114 --> other sites; (b) the cleavage rates at sites Phe114 and Tyr146-Asn147 were independent from each other; and (c) the hydrolysis of the Phe114-Ser115 bond was the rate determining step in autolytic inactivation. Thus, it is the cleavage of the interdomain loop and not of the autolysis or other loops that determines the half-life of chymotrypsin activity.

Amino Acid Sequence↗
PubMed1971

Autolytic activity associated with competent group H streptococci.

Competent cells of group H streptococci strains Wicky and Challis autolyzed markedly when placed at 37 C in 0.05 m tris(hydroxymethyl)methyl-amino-propane sulfonic acid buffer (pH 9.0 to 9.1) containing 0.02 m 2-mercaptoethanol, whereas noncompetent cells autolyzed slightly. Autolysis of competent Wicky cells did not occur at 0 C or after the cells were heated at 100 C for 5 min. Culture fluids derived from strain Challis that contained competence factor (CF) activity did not contain lytic activity. Addition of native deoxyribonucleic acid (DNA) to competent Wicky cells caused a retardation in the rate of autolysis; ribonucleic acid and alkali-denatured DNA had less of an effect. Supernantant fluids derived from competent cell lysates lysed noncompetent Wicky cells but were inactive against cells of Hydrogenomonas eutropha, a group A Streptococcus, and against a commercial lysozyme substrate (Micrococcus lysodeikticus). This lytic activity was inactivated by heat (5 min at 100 C). Electron microscopic observations of autolyzed cells showed that autolysis occurs only at the site of cross-wall formation. A close relationship between the development of competence and autolysis is suggested by the fact that certain conditions that prevent the establishment of the competent state in Wicky populations (such as no CF, addition of CF simultaneously with chloramphenicol, and addition of trypsin-inactivated CF) also prevent autolysis. This observation emphasizes the indirect or inductive nature of CF on these processes.

Autolysis↗
PubMed1994

Characterisation of an oxidative response inhibitor produced by Streptococcus pneumoniae.

BACKGROUND: Pneumonia caused by infection with Streptococcus pneumoniae is still a major clinical problem. Reactive oxygen species contribute to the killing of these bacteria by polymorphonuclear leucocytes (PMNs). Defence mechanisms of Str pneumoniae which counter reactive oxygen species are characterised. METHODS: PMNs were stimulated with phorbol myristate acetate (PMA) in the presence and absence of Str pneumoniae and supernatants from them, and superoxide (O2-) production was measured by the reduction of ferricytochrome c. RESULTS: Streptococcus pneumoniae, but not Klebsiella pneumoniae or Staphylococcus aureus, inhibited PMA stimulated superoxide production by PMNs. Washed PMNs which had been preincubated with Str pneumoniae autolysis phase supernatants also exhibited depressed H2O2 production in response to PMA. The inhibitory activity was not attributable to non-specific cytotoxicity as assessed by release of the cytoplasmic enzyme lactate dehydrogenase, nor did the supernatants inhibit PMA stimulated degranulation of PMNs. Fractionation of the autolysis phase supernatants revealed inhibitory activity in both the fractions greater than and less than 10 kD. Like pneumolysin the inhibitory activity was heat sensitive. However, both a parent and pneumolysin negative mutant Str pneumoniae, and autolysis phase supernatants from them, inhibited PMN superoxide production. Antisera to pneumolysin failed to abrogate the inhibitory effect of intact Str pneumoniae or autolysis phase supernatants from types 1 or 14 Str pneumoniae. CONCLUSIONS: The inhibitory effect of Str pneumoniae on the respiratory burst of PMNs is not shared by two other common lung pathogens. The existence of a novel inhibitor of the PMN respiratory burst, distinct from pneumolysin, has been demonstrated. The inhibitor is specific for the respiratory burst and is active both in the logarithmic phase of growth and during autolysis.

Autolysis↗