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PubMed1998

Autolysis of Lactococcus lactis is influenced by proteolysis.

The autolysin AcmA of Lactococcus lactis was shown to be degraded by the extracellular lactococcal proteinase PrtP. Autolysis, as evidenced by reduction in optical density of a stationary-phase culture and concomitant release of intracellular proteins, was greatly reduced when L. lactis MG1363 cells expressed the cell wall-anchored lactococcal proteinase PrtP of the PI-type caseinolytic specificity (PI). On the other hand, lactococcal strains that did not produce the proteinase showed a high level of autolysis, which was also observed when the cells produced the secreted form of PI or a cell wall-anchored proteinase with PIII-type specificity. Autolysis was also increased when MG1363 expressed the cell wall-anchored hybrid PI/PIII-type proteinase PIac. Zymographic analysis of AcmA activity during stationary phase showed that AcmA was quickly degraded by PI and much more slowly by PrtP proteinases with PIII-type and intermediate specificities. Autolysis of L. lactis by AcmA was influenced by the specificity, amount, and location of the lactococcal proteinase. No autolysis was observed when the various proteinases were expressed in an L. lactis acmA deletion mutant, indicating that PrtP itself did not cause lysis of cells. The chain length of a strain was significantly shortened when the strain expressed a cell wall-anchored active proteinase.

Bacteriolysis↗
PubMed1983

Mitochondrial complexes I, II, III, IV, and V in myocardial ischemia and autolysis.

Ischemic myocardium was produced by occluding the left circumflex coronary artery in anesthetized dogs. Autolyzed myocardium was produced by incubating transmural samples of canine left ventricle at 37 degrees C. Tissue pH was recorded continuously in each model using a microcombination pH electrode impaled into the midmyocardium. The activities of the five mitochondrial inner membrane enzyme complexes of electron transport and coupled oxidative phosphorylation were assayed as a function of time of ischemia or autolysis. While the activities of complex II (succinate-CoQ reductase) and IV (cytochrome c oxidase) were completely stable, that of complex I (NADH-CoQ reductase) decreased markedly, but largely only after 20 min of ischemia or autolysis. At 20 min and beyond, the decrease in the activity of complex I paralleled closely the decrease in whole mitochondrial oxygen uptake with NAD-linked substrates in both models. The activity of complex III (CoQH2-c reductase) decreased at a more gradual rate during ischemia or autolysis, and its rate of decrease paralleled that of succinate-supported oxygen uptake. The activity of complex V (oligomycin-sensitive ATPase) decreased most rapidly (by 40% in only 5 min of autolysis) but nearly leveled off beyond 20 min in the two models. A strikingly similar pattern of differential enzyme lability was observed in isolated control mitochondria incubated at lowered pH values. The results demonstrate 1) differential enzyme lability within the mitochondrial inner membrane, 2) a connection between severity of acidosis and the degree of enzyme activity loss, and 3) the usefulness of simple tissue autolysis as an analogue of in situ myocardial ischemia.

Adenosine Triphosphatases↗
PubMedDate not supplied

Effect of vitamin E on autolysis and sporulation of Aspergillus nidulans.

The morphologic and physiologic effects of vitamin E, a powerful antioxidant, on the autolysis and sporulation of Aspergillus nidulans FGSC26 were studied. In carbon-depleted submerged cultures, reactive oxygen species (ROS) accumulated in the cells and, concomitantly, progressing autolysis was observed, which was characterized by decreasing dry cell masses and pellet diameters as well as by increasing extracellular chitinase activities. Vitamin E supplemented at a concentration of 1 g/L hindered effectively the intracellular accumulation of ROS, the autolytic loss of biomass, the disintegration of pellets, and the release of chitinase activities. In surface cultures, vitamin E inhibited autolysis of both A. nidulans FGSC26 and a loss-of-function FlbA autolytic phenotype mutant. In addition, supplementation of the culture medium with this antioxidant also had a negative effect on the sporulation of strain FGSC26 and the FadAG203R hypersporulating phenotype mutant. These results suggest that accumulation of ROS was involved in the initiation of both sporulation and autolysis in this filamentous fungus, but that FadA/FlbA signaling was not involved in this vitamin E-dependent regulation. Vitamin E can be recommended as a supplement in fermentations in which the disintegration of pellets and gross autolysis should be avoided.

Antioxidants↗
PubMed1982

Limited autolysis reduces the Ca2+ requirement of a smooth muscle Ca2+-activated protease.

Chicken gizzard smooth muscle contains large amounts of Ca2+-activated protease activity. Approximately 15 mg of purified enzyme can be obtained from 1 kg of fresh muscle. The enzyme consists of two subunits (Mr = 80,000 and 30,000) present in a 1:1 molar ratio. In the presence of CaCl2, the 80,000/30,000-dalton heterodimer (form I) is rapidly converted by limited autolysis to a 76,000/18,000-dalton species (form II). Both the 80,000- and 30,000-dalton subunits are degraded simultaneously. Moreover, the Ca2+ dependence for autolysis (K0.5 = 300 microM) is identical for both subunits. Neither the time course nor the Ca2+ dependence of the autolytic conversion reaction is altered by 10- and 20-fold molar excesses of substrate. Limited autolysis markedly reduces the Ca2+ requirement for substrate degradation. Using N-[ethyl-2-3H]maleimide-labeled 27,000-dalton cardiac myosin light chains as substrate, the Ca2+ requirement of form I was found to be quite high (K0.5 = 150 microM). Under similar conditions, the Ca2+ requirement of form II was 30-fold lower (K0.5 = 5 microM). Limited autolysis did not alter the specific activity of the enzyme. Our results demonstrate that smooth muscle contains an abundant amount of Ca2+-activated protease. Moreover, autolysis of this enzyme may play an important regulatory role by converting the native form to a species that is fully active at physiological levels of intracellular calcium ion.

Animals↗
PubMedDate not supplied

[Alterations in acid and alkaline phosphatase activity in some steroid-producing organs of rats in autolysis].

Alterations in activity of acid and alkaline phosphatases as well as in concentration of inorganic phosphate were studied in tissue and homogenates of adrenal glands and testicles of rats within 48 hrs of the postmortal autolysis. During the autolysis acid phosphatase of homogenate and tissue of adrenal glands as well as alkaline phosphatase from the gland were more stable than acid and alkaline phosphatases from testicles. Activities of acid and alkaline phosphatases decreased gradually in homogenate and tissue of testicles during autolysis. Within the first day of autolysis distinct inhibition of alkaline phosphatase was also observed in the homogenate of testicles and adrenal glands. An increase in amount of inorganic phosphate in tissue and homogenate of adrenal gland and in tissue of testicles did not depend on alterations in activity of the phosphatases on autolysis. Prehomogenization of these tissues altered markedly the enzymatic activity both in adrenal glands and in testicles.

Acid Phosphatase↗
PubMed1998

Selective coupling of mu-calpain activation with the NMDA receptor is independent of translocation and autolysis in primary cortical neurons.

Excessive mu-calpain activation has been linked to several cellular pathologies including excitotoxicity and ischemia. In erythrocytes and other non-central nervous system (CNS) cells, calpain activation is thought to occur following a Ca2+-induced translocation of inactive cytosolic enzyme to membranes and subsequent autolysis. In the present report, we show that transiently exposing primary rat cortical neurons to lethal (50 microM) N-methyl-D-aspartic acid (NMDA) caused protracted calpain activation, measured as increased spectrin hydrolysis, but this was independent of translocation or autolysis of the protease. An anti-mu-calpain antibody showed that calpain was largely membrane associated in cortical neurons, and, consequently, neither translocation nor autolysis of the protease was observed following ionomycin or lethal NMDA treatment. By contrast, in rat erythrocytes, calpain was largely cytosolic and underwent rapid translocation and autolysis in response to ionomycin. Calpain-mediated spectrin hydrolysis was specifically coupled to Ca2+ entry through the NMDA receptor because nonspecific Ca2+ influx via ionomycin or KCl-mediated depolarization failed to activate the enzyme. Thus, calpain appears selectively linked to glutamate receptors in cortical neurons and regulated by mechanisms distinct from that occurring in many non-CNS cells. The data suggest that intracellular signals coupled to the NMDA receptor are responsible for activating calpain already associated with cellular membranes in cortical cells.

Animals↗
PubMed1993

Biochemical, morphological and cytochemical studies of enhanced autolysis of Saccharomyces cerevisiae. 2. Morphological and cytochemical studies.

Morphological and cytochemical observation of Saccharomyces cerevisiae undergoing of induced autolysis were done in response to various chemical inducers of autolysis (NaCl, ethanol, fresh autolyzate). Changes in the inner structure of yeast cells were monitored by transmission electron microscopy and the surface of the cell wall was observed by scanning electron microscopy during autolysis. Cytochemical characterization of autolyzed cells was performed using four synthetic substrates for determination of proteinase activities but only carboxypeptidase Y could be detected in the vacuolar membranes. The morphological studies supported the data obtained from biochemical studies and confirmed that optimized conditions of autolysis have a significant effect on the structural changes of autolyzed yeast.

Autolysis↗
PubMed1975

Ultrastructural nuclear changes of extranucleolar ribonucleoprotein structures during autolysis of normal liver cells.

The ultrastructural changes of extranucleolar nuclear ribonucleoprotein structures during autolysis of normal liver cells were studied by means of usual and cytochemical procedures. The results revealed that the earliest changes were characterized by the fragmentation of the perichromatin fibrils and disappearance of the perichromatin granules. This was followed by the formation of numerous dense granules among the altered perichromatin fibrils and by the disappearance of dense fibrillar structures from the remaining interchromatin areas. In the later stages of autolysis the altered perichromatin fibrils loosed their density and the dense granules located among them disappeared. In addition, fine filamentous and cross-striated bodies appeared in the nucleoplasm as well as in the cytoplasm of some hepatocytes. The results obtained by the EDTA treatment and digestion of the sections with Pronase and RNA-ase indicated that the altered perichromatin fibrils contained ribonucleoprotein which together with that of the perichromatin granules represent nuclear components most sensitive to the autolysis. The chemical nature of the dense granules located among the altered perichromatin fibrils was different from that of the latter as well as from that of the nuclear microspherules [6]. The dense fine filamentous and cross-striated nucleoplasmic and cytoplasmic bodies are considered to represent a nonspecific structural phenomenon accompanying regressive cellular alterations since these bodies were present only in the later stages of autolysis.

Animals↗
PubMed1986

Coomassie brilliant blue G-250 dye-binding technique for determination of autolytic protein breakdown in Euglena gracilis and comparison to other methods of autolysis measurement.

The Coomassie brilliant blue G-250 dye-binding technique of M. M. Bradford (1976, Anal. Biochem. 72, 248-254) for protein quantification has been used to measure autolytic protein breakdown in cell-free extracts of Euglena gracilis. Specific autolysis rates were calculated from the difference between initial and actual absorbances at different incubation times of Coomassie brilliant blue-stained protein. They were found to depend linearly on time and initial protein concentration. Calibration against another method of protein determination is necessary due to different color yields with various protein mixtures. The high sensitivity and reproducibility of this method permit determination of specific autolysis rates below 0.1% mg-1 h-1 over a pH range between 3 and 8, without protein precipitation or pH adjustment, and in the presence of high amounts of amino acids and/or small peptides. Results obtained by this method are comparable to those of other autolysis measurements and to proteolytic activity determination by azocaseinolysis. Proteolytic autolysis has been observed in both the soluble and the particulate fractions of E. gracilis cell-free extracts, but displays different pH optima and specific activities in these fractions, as is also the case for azocaseinolysis. The method described is easy to perform, inexpensive, time saving, and should be applicable to other biological systems as well.

Animals↗
PubMed2002

The protease core of the muscle-specific calpain, p94, undergoes Ca2+-dependent intramolecular autolysis.

Limb girdle muscular dystrophy type 2A is linked to a skeletal muscle-specific calpain isoform known as p94. Isolation of the intact 94-kDa enzyme has been difficult to achieve due to its rapid autolysis, and uncertainty has arisen over its Ca2+-dependence for activity. We have expressed a C-terminally truncated form of the enzyme that comprises the protease core (domains I and II) along with its insertion sequence, IS1, and N-terminal leader sequence, NS. This 47-kDa p94I-II mini-calpain was stable during purification. In the presence of Ca2+, p94I-II cleaved itself within the NS and IS1 sequences. Mapping of the autolysis sites showed that NS and IS1 have the potential to be removed without damage to the protease core. Ca2+-dependent autolysis must be an intramolecular event because the inactive p94I-II C129S mutant was not cleaved by incubation with wild-type p94I-II. In addition, the rate of autolysis of p94I-II was independent of the concentration of the enzyme.

Autolysis↗
PubMed1994

Autolysis and extension of isolated walls from growing cucumber hypocotyls.

Walls isolated from cucumber hypocotyls retain autolytic activities and the ability to extend when placed under the appropriate conditions. To test whether autolysis and extension are related, we treated the walls in various ways to enhance or inhibit long-term wall extension ('creep') and measured autolysis as release of various saccharides from the wall. Except for some non-specific inhibitors of enzymatic activity, we found no correlation between wall extension and wall autolysis. Most notably, autolysis and extension differed strongly in their pH dependence. We also found that exogenous cellulases and pectinases enhanced extension in native walls, but when applied to walls previously inactivated with heat or protease these enzymes caused breakage without sustained extension. In contrast, pretreatment of walls with pectinase or cellulase, followed by boiling in methanol to inactivate the enzymes, resulted in walls with much stronger expansin-mediated extension responses. Crude protein preparations from the digestive tracts of snails enhanced extension of both native and inactivated walls, and these preparations contained expansin-like proteins (assessed by Western blotting). Our results indicate that the extension of isolated cucumber walls does not depend directly on the activity of endogenous wall-bound autolytic enzymes. The results with exogenous enzymes suggest that the hydrolysis of matrix polysaccharides may not induce wall creep by itself, but may act synergistically with expansins to enhance wall extension.

Autolysis↗
PubMed1990

Postmortem autolysis in the pancreas: multivariate statistical study. The influence of clinicopathological conditions.

The pancreases of 92 autopsied patients were evaluated to determine the influence of clinicopathological conditions upon the extent of postmortem autolysis. The postmortem interval was the most important factor influencing postmortem autolysis. In order to more precisely evaluate the influence of associated factors upon the extent of postmortem autolysis in the pancreas, quantification I of multivariate statistical analysis was used. Twelve factors such as postmortem interval, mode of death, malignant neoplasia as an underlying cause of death, and history of abdominal surgery were chosen for analysis. The multiple correlation coefficient (R2) was 0.71. In the case of unexpectedly severe or unexpectedly mild postmortem autolysis, we suggest that these 12 factors should be taken into close consideration whether or not the postmortem interval is long or short.

Acute Disease↗
PubMed1992

Autolysis of methicillin-resistant and -susceptible Staphylococcus aureus.

The autolytic activities, including unstimulated, Triton X-100-stimulated, and daptomycin-induced, of various sets of methicillin-resistant and related methicillin-susceptible strains were compared. Faster rates of autolysis were noted in two heterogeneous methicillin-resistant transductants than in their methicillin-susceptible parental recipients, in a heterogeneous resistant strain than in a susceptible derivative created by chemical mutagenesis, and in a homogeneous resistant strain than in a derivative that had decreased methicillin resistance and was created by transposon Tn551 mutagenesis. These results suggest that the presence of the methicillin resistance region, mec, either directly or indirectly through an interaction with other host genes, confers a faster rate of autolysis on strains. Various auxilliary genes are known to affect methicillin resistance expression, and one of these genes, femA, was necessary for the expression of this faster rate of autolysis. These differences in autolytic activities were not observed in isolated crude cell walls retaining autolytic activities, suggesting different modes of regulation of autolysins in intact cells and isolated walls. In contrast, one homogeneous, highly resistant strain, DU4916, had a lower autolytic activity than did derived heterogeneous resistant and susceptible strains created by chemical mutagenesis and a strain that had decreased resistance and was created by transposon mutagenesis. Our observations suggest that methicillin resistance expression is associated with an enhanced rate of autolysis, in heterogeneous resistant strains at least.

Autolysis↗
PubMed1977

Role of teichuronic acid in Bacillus licheniformis: defective autolysis due to deficiency of teichuronic acid in a novobiocin-resistant mutant.

nov-12, a novobiocin-resistant mutant of Bacillus licheniformis ATCC 9945, grows as long chains of cells, a characteristic of autolytic-deficient (Lyt-) mutants. Isolated walls from nov-12 autolyzed at a rate equal to 5% of that displayed by wild-type walls, thus confirming the Lyt- phenotype. Protein-free nov-12 walls displayed marked resistance to, and also failure to bind, added autolysin solubilized from wild-type walls. Comparison of isolated cell walls revealed a deficiency in teichuronic acid in the mutant. Lesser differences were observed in walls of this strain, including a reduction in galactose, an increase in the proportion of peptidoglycan, and small quantitative differences in peptidoglycan composition though the proportions of protein and teichoic acid were similar in walls of both strains. Autolytic sensitivity was studied in walls in which protein, teichoic acid, and teichuronic acid were removed successively by selective extraction procedures. Autolysis of wild-type walls was unaffected by removal or protein or teichoic acid, but teichuronic acid removal rendered wild-type walls as insensitive to autolysis as mutant walls had been throughout. Therefore, in this mutant, deficiency in teichuronic acid alone leads to the Lyt- phenotype and, hence, activity and binding of autolysin(s) are dependent upon teichuronic acid but not teichoic acid. Also, the potential rate of autolysis of cell walls in this organism was correlated with the proportion of teichuronic acid in the wall. The possible significance of these findings with respect to control of autolysis and cell separation is discussed.

Acetylgalactosamine↗
PubMed1986

Induction of autolysis in nongrowing Escherichia coli.

Unless relaxation of the stringent response is achieved, all nongrowing bacteria rapidly develop resistance to autolysis induced by a variety of agents, including all classes of cell wall synthesis inhibitors. We now describe inhibitors of cell wall synthesis which were unusual in that they could continue to effectively induce autolysis in relA+ Escherichia coli even after prolonged amino acid starvation. The process of cell wall degradation seems to be catalyzed by similar hydrolytic enzymes in nongrowing and growing cells, yet the activity of these new agents capable of inducing autolysis in the nongrowing relA+ cells did not involve relaxation of RNA or peptidoglycan synthesis. We propose that the suppression of autolysis characteristic of nongrowing cells can be bypassed by a novel mechanism of autolytic triggering which is independent of the relA locus.

Amino Acids↗
PubMed1988

Autolysis of the millimolar Ca2+-requiring form of the Ca2+-dependent proteinase from chicken skeletal muscle.

The millimolar Ca2+-requiring form of the Ca2+-dependent proteinase from chicken breast skeletal muscle contains two subunit polypeptides of 80 and 28 kDa, just as the analogous forms of this proteinase from other tissues do. Incubation with Ca2+ at pH 7.5 causes rapid autolysis of the 80-kDa polypeptide to 77 kDa and of the 28-kDa polypeptide to 18 kDa. Autolysis of the 28-kDa polypeptide is slightly faster than autolysis of the 80-kDa polypeptide and is 90-95% complete after 10 s at 0 degrees C. Autolysis for 15 s at 0 degrees C converts the proteinase from a form requiring 250-300 microM Ca2+ to one requiring 9-10 microM Ca2+ for half-maximal activity, without changing its specific activity. The autolyzed proteinase has a slightly lower pH optimum (7.7 vs. 8.1) than the unautolyzed proteinase. The autolyzed proteinase is not detected in tissue extracts made immediately after death; therefore, the millimolar Ca2+-requiring proteinase is largely, if not entirely, in the unautolyzed form in situ.

Amino Acids↗
PubMedDate not supplied

[Changes in phospholipids of the brain grey and white matter during in vitro autolysis in rats subjected to acute hypobaric hypoxic hypoxia].

The development of autolysis in grey brain matter of albino rats was accompanied by desintegration of aminophospholipids with parallel increase of glycerophosphates (GLP) and phosphatidic acids (PA) on early stages of incubation and lysophospholipids (LPL) on later stages. Acute hypobaric hypoxic hypoxia decreased the level of phosphatidylethanolamines (PE) with simultaneous accumulation of PA. Previous hypoxia altered the character of autolytic reorganizations of phospholipids. Oscillatory reciprocal reorganizations in the system PE > PS (phosphatidylserine) were observed at early stage (1 h) and at late stages of autolysis (24 h). At the same time increased transformation of phosphatidylcholines (PC) into sphingomyelins (SM) with simultaneous accumulation GLP was registered. During autolysis of brain white matter of control rats opposite oscillatory reorganizations of PE, PC, SM, PA with reduction of PE and simultaneous increase of LPL and PA level after 1 hour of incubation were observed. Reciprocal reactions of biotransformation in system PS > PE were revealed at 4th hour. Previous hypobaric hypoxic hypoxia reduced the level of total phospholipids as well as PS at simultaneous increase of LPL. Acute hypobaric hypoxic hypoxia increased autolytic transformations in system PC > SM and induced hydrolysis of PE, PC into LPL at late stages of autolysis.

Altitude↗
PubMed1986

[Electron microscopic cytochemical and morphometric study of the cerebral cortex synapses in postmortem autolysis].

Brain tissue staining with phosphotungstic acid was performed to assay neurofilament accumulations in synapses in the molecular layer of the rat cerebral cortex at different intervals after the animals' death. It was found that autolysis began in the dense projections of presynaptic grid. Within 30 min autolysis developed in mature and very young (immature) synapses. By the 90th min autolysis in asymmetric synapses was considerably enhanced. 6 hours later autolysis involved mature and indefinite synapses.

Animals↗