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PubMed1996

Investigation of the interaction of m-calpain with phospholipids: calpain-phospholipid interactions.

Phosphatidyl inositol, phosphatidyl choline, phosphatidyl glycerol, phosphatidyl serine, phosphatidyl ethanolamine, phosphatidic acid and sphingomyelin were all found to be effective at reducing the Ca2+ requirement for m-calpain autolysis. In the absence of phospholipid, pig kidney m-calpain required 1.4 mM Ca2+ for 50% autolysis under the assay conditions used. Phospholipids caused a reduction in this Ca2+ requirement to a value between 0.45 mM Ca2+ for phosphatidyl glycerol and 1.1 mM Ca2+ for phosphatidyl ethanolamine. Previous studies (Crawford, C., Brown, N.R. and Willis, A.C. (1990) Biochem. J. 265, 575-579) have shown that the most probable site for phospholipid interaction in calpain is the N-terminal region between residues 39 to 62 of the small subunit of calpain (G17TAMRILGG). In this study we examine the possible role of this G17TAMRILGG region. Three synthetic peptides corresponding to parts of this sequence were used to examine the phospholipid binding sequence. Analysis of the phospholipid vesicle binding properties of these peptides suggested that both the TAMRIL and polyglycine sequences were required for binding to phosphatidyl inositol vesicles.

Amino Acid Sequence↗

PubMed1996

Structure of chymotrypsin variant B from Atlantic cod, Gadus morhua.

The amino-acid sequence of chymotrypsin variant B isolated from the pyloric caeca of Atlantic cod has been elucidated. The characterization of the primary structure is based on N-terminal Edman degradation and mass spectrometry of the native protein and enzymatically derived peptides. Chymotrypsin variant B showed 72% sequence identity with the A-variant and 64% and 62%, respectively, with the bovine counterparts A and B, all consisting of 245 amino acids. This new sequence contains a higher proportion of charged residues compared with bovine chymotrypsin but fewer polar hydrogen-bond forming residues which might contribute to its lower thermostability. It also shares the emerging characteristics of other fish serine proteinases which have relatively higher methionine content, including a conserved Met-134 in a loop leading into a domain-connecting strand. The inherent mobility in methionine side-chains may contribute to the maintenance of flexibility at low temperatures. Several amino-acid sequence differences adjacent to the catalytic site are observed in the two cod chymotrypsin variants which also differ in kinetic properties. Unlike the mammalian chymotrypsins, which contain several autolysis sites, cod variant B only contains a single autolysis site. The three-dimensional structures of the A- and B-variants of cod has been modelled on the known crystal structure of bovine alpha-chymotrypsin showing almost superimposable structures.

Amino Acid Sequence↗

PubMed1991

The amino terminus of the putative Drosophila choline acetyltransferase precursor is cleaved to yield the 67 kDa enzyme.

A putative precursor of the 67 kDa choline acetyltransferase (Acetyl-CoA: choline-O-acetyltransferase; EC 2.3.1.6) polypeptide from Drosophila was examined using polyclonal antibodies. The central purpose of the study was to probe the suspected precursor with anti-peptide antibodies that could identify a cleavable amino terminal domain, since such a structure could be responsible for targeting the enzyme to the presynaptic terminal. Antisera were produced to both a plasmid-expressed fusion-free enzyme protein and a 26-amino acid-long peptide reproducing sequence from the enzyme. Both antisera were capable of precipitating enzyme activity from crude supernatants. Western blotting with the antibody to the plasmid-expressed enzyme visualized a major polypeptide at 75 kDa and minor polypeptides at 67 and 54 kDa. Affinity-purified IgG to the synthetic peptide only recognized the 75 kDa component and was unable to recognize purified 67 kDa enzyme protein. Timed autolysis of the enzyme in crude homogenates demonstrated both a 67 kDa polypeptide that was present prior to homogenization and a species that appeared as a product of the autolysis. The evidence from this study is consistent with the expectation that the 75 kDa band, visualized on Western blots with antisera to the enzyme, is an authentic enzyme protein. These data further suggested that the 75 kDa protein is an amino-terminally extended precursor of the 67 kDa enzyme that can be cleaved to generate the 67 kDa species.

Amino Acid Sequence↗

PubMed1989

Evaluation of eosin-fluorescence in the diagnosis of sudden cardiac death.

The diagnostic significance of the eosin-fluorescence method was evaluated and compared with the enzymehistochemical beta-hydroxybutyrate-dehydrogenase method (beta-HBDH) and the degree of hyperchromasia in 568 samples from 24 bets-HBDH-negative and beta-HBDH-positive sudden cardiac deaths (SCD) and 23 non-cardiac deaths as controls. The effect of autolysis was investigated separately on isolated human hearts at +4 degrees C and +22 degrees C. All samples were examined without any knowledge of clinical or autopsy data. Normal olive-green fluorescence was observed in only five individuals of the control group and two in the beta-HBDH-negative one. The frequency of the yellow-fluorescence and hyperchromasia increased from the control subjects towards the beta-HBDH-positive-ones. The difference in frequency distribution of fluorescence between the groups was statistically highly significant (P less than 0.001). The change in fluorescence did not correlate with the loss of beta-HBDH-reaction or the distribution pattern of hyperchromasia. Postmortem autolysis did not change the fluorescence significantly but the 78% wrong positive subjects in the control group render this method too sensitive and unreliable for medicolegal purposes.

Adult↗

PubMed2004

Activation of caspase-like activity and poly (ADP-ribose) polymerase degradation during sporulation in Aspergillus nidulans.

Mycelium vacuolization, protein degradation, and as the final stage autolysis, often accompanies developmental changes in fungi and similarities between autolysis and apoptosis have previously been suggested. Caspases are the key executors of apoptosis and in this study caspase-like activities were detected in protein extracts from Aspergillus nidulans during sporulation. This was shown by hydrolysis of the fluorescent DEVD- and IETD-AFC peptide substrates specific for caspase 3- and 8-like activities, respectively. These activities were repressed by the caspase 3 and 8 specific irreversible peptide inhibitors DEVD-fmk and IETD-fmk, but were not affected by the unspecific inhibitor E-64. Isoelectric focusing of protein extracts followed by activity staining revealed the presence of two bands with caspase-like activity. One of the proteins degraded both caspase 3 and caspase 8 specific substrates whereas the other only degraded the caspase 8 substrate. Searches in an A. nidulans genome database revealed two genes encoding metacaspase proteins with predicted sizes of 45 kDa that could be responsible for the measured caspase-like activities. The searches also found a single gene encoding a poly (ADP-ribose) polymerase (PARP) protein with a predicted size of 81 kDa. PARP is one of the known target proteins inactivated by caspase degradation in animal cells. Western blotting of fungal extracts using a bovine PARP antibody confirmed the presence of a fungal PARP-like protein of about 81 kDa. By Western blotting it was shown that this PARP-like protein band was present only at early time points until the start of conidia formation and the accompanying increase in caspase-like activity. Thereafter, a degradation product of about 60 kDa appeared indicating that the degradation of the fungal PARP-like protein was specific. The PARP antibody also recognized an 85 kDa protein band that was not degraded, and which conceivably represents a modified form of the 81 kDa PARP. Fungal extracts high in caspase-like activity could degrade both the fungal 81 kDa PARP and bovine PARP. In the presence of the caspase 3 inhibitor DEVD-fmk this degradation was delayed. Thus, as in animal apoptotic cells, caspase activities are involved in fungal mycelium self-activated proteolysis.

Animals↗

PubMed2004

Release of macromolecules by Saccharomyces cerevisiae during ageing of French flor sherry wine "Vin jaune".

The French flor sherry wine "Vin jaune" spends 6 years and 3 months in the same barrel under a yeast velum. Because of temperature variations in the cellars, this velum sinks partially into the wine and a deposit of dead yeasts cells accumulates in the bottom of the barrels, favouring the formation of new velum. Growth and autolysis occur simultaneously. This study investigated the evolution of macromolecules released by yeasts during the ageing of "Vin jaune" in a model system closely simulating winemaking. It was observed that the release of macromolecules during the formation of the velums by living yeasts was low but greatly increased when the velums fell and yeast viability decreased. The release of macromolecules was then due to the autolysis of dead cells. Analysis of macromolecules during ageing revealed that they contained 73.3-78.5% neutral sugars and 6-7% proteins according to the ageing stage. Their amino acid composition did not change during ageing. A high content of serine and threonine commonly involved in O-glycosidic linkages present in yeast mannoproteins was observed. Throughout ageing, the mannose and glucose contents of macromolecules increased but the ratio of polymeric mannose to glucose decreased. Size exclusion chromatography showed that mannoproteins released in wine were partially hydrolysed by yeast beta-1,3-glucanases freed in wine.

Bacteriolysis↗

PubMed2006

Where do the immunostimulatory effects of oral proteolytic enzymes ('systemic enzyme therapy') come from? Microbial proteolysis as a possible starting point.

Enteric-coated proteolytic enzyme preparations like Wobenzym and Phlogenzym are widely used for the so-called 'systemic enzyme therapy' both in humans and animals. Numerous publications reveal that oral proteolytic enzymes are able to stimulate directly the activity of immune competent cells as well as to increase efficiency of some of their products. But origins of the immunostimulatory effects of oral proteolytic enzymes are still unclear. The hypothesis described here suggests that it may be proteolysis of intestinal microorganisms that makes the immune competent cells to work in the immunostimulatory manner. The hypothesis was largely formed by several scientific observations: First, microbial lysis products (lipopolysaccharides, muropeptides and other peptidoglycan fragments, beta-glucans, etc.) are well known for their immunostimulatory action. Second, a normal human being hosts a mass of intestinal microorganisms equivalent to about 1 kg. The biomass (mainly due to naturally occurring autolysis) continuously supplies the host's organism with immunostimulatory microbial cell components. Third, the immunostimulatory effects resulting from the oral application of exogenously acting antimicrobial (lytic) enzyme preparations, such as lysozyme and lysosubtilin, are likely to be a result of the action of microbial lysis products. Fourth, cell walls of most microorganisms contain a considerable amount of proteins/peptides, a possible target for exogenous proteolytic enzymes. In fact, several authors have already shown that a number of proteases possess an ability to lyse the microbial cells in vitro. Fifth, the pretreatment of microbial cells (at least of some species) in vitro with proteolytic enzymes makes them more sensitive to the lytic action of lysozyme and, otherwise, pretreatment with lysozyme makes them more susceptible to proteolytic degradation. Sixth, exogenous proteases, when in the intestines, may participate in final steps of food-protein digestion. The resulting food-borne peptides have recently been shown to be potential activators of microbial autolysis. The main question that needs to be answered in order to verify the hypothesis is whether oral proteases are able (and to what extent) to lyse/mediate lysis of intestinal microorganisms in situ. Methods based on up-to-date molecular biology techniques to allow investigation of the influence of exogenous proteases on microbial lysis processes in vivo (in the intestines) need to be developed. Research testing of this hypothesis may have an important impact in development of novel preparations for the systemic enzyme therapy.

Adjuvants, Immunologic↗

PubMed2005

Primary structure of brevilysin L4, an enzymatically active fragment of a disintegrin precursor from Gloydius halys brevicaudus venom.

Brevilysin L4 (L4) is a non-hemorrhagic P-I class metalloprotease (MP) isolated from Gloydius halys brevicaudus venom. Its complete amino acid sequence has been determined. L4 is a single-chain polypeptide and highly homologous to those of other snake venom MPs. A zinc-binding motif, HExxHxxGxxH, is located at residues 142-152. A characteristic feature of L4 is the presence of a spacer sequence (LRTDTVS) at the C-terminal that links metalloprotease and disintegrin domains and is usually removed by post-translational proteolysis, suggesting that L4 is expressed together with a spacer region and a disintegrin domain at the C-terminal. The nucleotide sequence of a cDNA clone encoding L4 has revealed that L4 is a disintegrin precursor and produced as a P-II class MP. The disintegrin coded after L4 sequence was brevicaudin 1, a disintegrin previously isolated from the same venom. P-II class MPs have been suspected to undergo autoproteolysis to release disintegrins. Although being P-I class MP, L4 itself autocatalytically degrades with a half-life of 30min at pH 8.5 and 37 degrees C in the absence of Ca(2+). Sequence analysis of several fragment peptides produced during the autolysis of L4 indicated that more than 40 peptide bonds were split, and the cleavages of Ser(60)-Asn(61), Thr(99)-Ala(100), and Phe(103)-Asp(104) bonds may trigger the autoproteolysis. Addition of Ca(2+) completely suppressed the cleavage of these particular bonds, resulting in a marked prevention of autoproteolysis. Thus, L4 provides a good model for the investigation of autolysis of some MPs.

Amino Acid Sequence↗

PubMed1989

Autodegradation of rat liver proteasomes (large multicatalytic proteinase complexes).

Purified proteasomes (large multicatalytic proteinase complexes) were found to be very stable, showing no change in activities or structures during prolonged incubation in medium of pH 7.5 at 37 degrees C. However, on addition of urea they were degraded autocatalytically in a time- and dose-dependent manner, suggesting that destruction of the proteasomal complexes acts as a signal for their autolysis. ATP at a physiological concentration greatly stimulated the urea-dependent breakdown of proteasomes. The autolysis induced by urea was almost completely inhibited by hemin, but not by other protease inhibitors tested, such as leupeptin, chymostation and Ep-475. Thus, autolytic degradation of proteasomes appears to be important for the regulation of enzyme levels in eukaryotic cells.

Adenosine Triphosphate↗

PubMed1996

Alpha-1-proteinase inhibitor prolongs small intestinal graft preservation and survival.

Organ preservation solutions currently used for cold storage of human donor organs are less effective in preserving small intestinal grafts than other organ grafts. The maximal safe period of cold preservation for human small intestinal graft is only about 6 hours. The pathology of preserved and reperfused small intestinal grafts is characterized by mucosal autolysis and sloughing. The authors speculated that the preservation/reperfusion injury results from a proteinase/proteinase-inhibitor imbalance in the graft that favors tissue degradation. This study evaluates whether the addition of an alpha-1-proteinase inhibitor (alpha-1-PI) to the preservation solution can improve graft survival after small bowel transplantation. Forty small intestinal grafts (20 cm long) were harvested from Lewis rats. The grafts were divided randomly into three groups and were preserved in one of the following solutions: normal saline (NS) (n = 10), alpha-1-PI (25 mg/mL; n = 20), or proteinase-free bovine serum albumin (BSA) (25 mg/mL; n = 10). After 12-hour cold storage in the respective solutions, the grafts were transplanted orthotopically into syngeneic recipients. Full-thickness graft biopsies were performed before and 1 hour after revascularization. The effectiveness of preservation was judged by graft histopathology and recipient survival. Histological studies showed that there was less mucosal autolysis and sloughing of the grafts in the alpha-1-Pl group than in the other two groups. All recipients in the NS and BSA groups died of graft failure within 7 days (NS: median, 4 days; range, 2 to 5 days; BSA: median, 6 days; range, 4 to 7). However, 60% (12 of 20) of the recipients in the alpha-1-PI group survived more than 90 days (median, > 90 days; range, 4 to > 90 days; P < .005 v NS or BSA groups, log-rank method). These data suggest that the inclusion of alpha-1-PI in the preservation solution may enhance graft integrity and improve the surgical outcome after small bowel transplantation.

Animals↗

PubMed2003

Molecular cloning and expression of structural domains of bothropasin, a P-III metalloproteinase from the venom of Bothrops jararaca.

Mature P-III snake metalloproteinases are soluble venom components which belong to the Reprolysin sub family and are structurally related to the mammalian membrane-bound A Disintegrin And Metalloproteinase (ADAMs). Here we present the molecular cloning of bothropasin, a metalloproteinase with hemorrhagic and myonecrotic activities isolated from the venom of Bothrops jararaca. The full-length cDNA encoding the bothropasin precursor was cloned by immunoscreening and its authenticity was confirmed by the amino acid sequence of internal fragments obtained from an autolyzed sample of native bothropasin. The predicted bothropasin precursor is comprised of the elements of a P-III venom metalloproteinase: signal sequence, pro-, metalloproteinase, disintegrin-like and cysteine-rich domains. In the autolysis process of native bothropasin, the disintegrin-like and cysteine-rich domains remained intact while the metalloproteinase domain was cleaved at different sites. The attempts made to obtain the recombinant precursor form of bothropasin using bacterial, yeast and mammalian cell expression systems failed to produce it in an amount sufficient to analyze the activation of the zymogen. Nevertheless, the study of the expression of the individual domains of bothropasin using a bacterial system resulted in the production of recombinant pro-and disintegrin-like+cysteine-rich domains but not the metalloproteinase domain. These results along with the autolysis pattern of the native protein suggest a role for the metalloproteinase domain in the structural stability of bothropasin.

Amino Acid Sequence↗

PubMed1997

Partially purified Echis carinatus venom cleaves active-site-mutated bovine prothrombin at two sites.

Partially purified snake venom of Echis carinatus (ECV) is a well-known enzyme used to generate meizothrombin from prothrombin. In this reaction, meizothrombin is slowly converted to two smaller fragments. It is not certain if this slow reaction is catalyzed by meizothrombin or by ECV. Using previously constructed active site mutant bovine prothrombin, we eliminated meizothrombin autolysis and examined ECV reactions. ECV did slowly generate two fragments during activation of the active site mutant prothrombin. This result indicates that ECV cleaved bovine prothrombin at two sites: at Arg323-Ile to expose the active site, at another bond at or near the known meizothrombin autolysis site. Because the former cleavage is faster than the latter one, ECV is still a reasonable tool to generate meizothrombin under proper conditions.

Animals↗

PubMed2000

Characterization of Streptococcus thermophilus strains that undergo lysis under unfavourable environmental conditions.

The autolysis of starter lactic acid bacteria appears as a promising way to enhance the flavour of fermented dairy products. The present work was aimed at investigating the autolysis phenomenon in Streptococcus thermophilus, a thermophilic lactic acid bacteria involved in the starters used for the production of yoghurts, Italian and Swiss-type cheeses. Out of 146 strains screened for their aptitude to spontaneously lyse at the end of growth in M17 medium containing lactose in limited concentration, six strains, among which is the type strain CNRZ 1358, were found to be highly autolytic. These autolytic strains are characterized by a typical bell-shaped growth curve. Lysis of the type strain, which was studied as the model, was triggered under unfavourable environmental conditions, such as lactose depletion and NaCl or organic solvents addition. The lysogenic character of this strain was evidenced. Taken together, our results indicate that the autolytic phenotype in S. thermophilus is linked to the lysogenic character but does not result from the massive prophage induction under stressing conditions.

Bacteriolysis↗

PubMed1986

Regional variations in the "adenine/oxypurine" pool of the heart in normoxia and oxygen deficiency.

The regional distribution of energy-related metabolites was determined for the normal dog heart by using high performance liquid chromatography to analyse the levels of ATP, ADP, AMP, IMP, adenosine, inosine, hypoxanthine/xanthine, uric acid and NAD in tissue samples from 7 defined areas of the myocardium and 2 of the conduction system, and calculating the overall concentration of these metabolites in each area. From 8 of the 20 hearts used, additional samples were taken and allowed to undergo autolysis in vitro at 37 degrees C for up to 2 h before being analysed in the same manner. The total concentration of the metabolites assayed ("adenine/oxypurine" pool) did differ from one area to another, most notably between ventricles (left = 6.08, right = 5.93, apex = 6.08 microM/g wet tissue weight) and atria (left wall = 4.44, left appendage = 4.12, right wall = 4.34, right appendage = 3.57 microM/g wet tissue weight), but for each area remained essentially constant during the period of autolysis studied.

Adenine Nucleotides↗

PubMed1986

Morphologic diagnosis of acute tubular necrosis (ATN) by autofluorescence.

The histologic diagnosis of acute tubular necrosis (ATN) frequently poses considerable difficulty, especially in its early phases. This is particularly true in postmortem material in which autolysis is commonly present. This study was designed to assess the value of enhanced autofluorescence in the diagnosis of ATN. The method has previously been shown to be useful in the identification of myocardial infarcts in both humans and experimental animals. We studied 12 patients with a histologic and clinical diagnosis of ATN. In all, severe hypotension of diverse etiology was detected from 15 hours to seven days prior to death. In all cases, a bright yellow autofluorescence was observed in the necrotic tubules in ordinary H & E sections. In addition, enhanced autofluorescence was observed in the necrotic tubular epithelium in the unstained, paraffin-embedded sections. Nine kidney sections from five medico-legal autopsy cases undergoing autolysis from 11 to 48 hours after death were used as controls. These kidneys invariably exhibited a faint dull olive green fluorescence, quite different from the fluorescence of the necrotic tubules. No effort was made to distinguish between ischemic (tubulorrhexis) and toxic (tubulonecrosis) changes by the fluorescent method. The mechanism of the enhanced autofluorescence is not clear. We conclude that ultraviolet light examination of H & E kidney sections allows accurate recognition of ATN.

Acute Kidney Injury↗

PubMed2003

How plants make tubes.

The plant body requires the transport of various materials over large distances. Two cell types that bear a striking resemblance morphologically are the cells specialized for water transport and those responsible for the transport of oxygen: xylem and lysigenous aerenchyma, respectively. Each of these cell types undergoes programmed cell death and cellular autolysis, resulting in the production of a functional space within the plant body. The major morphological difference observed is the presence of the lignified secondary wall in water-conducting tissues. The prevalence of tubular structures in other plant tissues suggests that the ability to form spaces through cellular autolysis is a fundamental paradigm in plant development and evolution.

Biological Evolution↗

PubMed1999

Stability of immobilized thermolysin in organic solvents.

Various factors affecting the stability of thermolysin immobilized by cross-linking with glutaraldehyde were elucidated, particularly in the water-immiscible organic solvents such as ethyl acetate and tert-amyl alcohol. The main reason for enzyme inactivation in water-immiscible organic solvents was found to be autolysis in the water phase, which may surround the enzyme immobilized inside the support. By contrast, in water-miscible organic solvents thermal denaturation was the predominant cause of enzyme inactivation. Courses of inactivation were expressed by second-order kinetics in the initial stage, after which inactivation proceeded at a slower rate. The extent of autolysis was found to strongly depend on the kind of organic solvent, the water content, and type of support and these dependencies were explained by the difference in the amount and state of water inside the support. Thermolysin was immobilized onto Amberlite XAD-7 as a compact aggregate inside the support which may increase the stability of the enzyme. Finally, it was shown that the stability of the immobilized enzyme could be correlated with the logP value for water-miscible organic solvents and with the solubility of water for water-immiscible organic solvents.

Journal Article↗

PubMed2003

Towards quantitative mRNA analysis in paraffin-embedded tissues using real-time reverse transcriptase-polymerase chain reaction: a methodological study on lymph nodes from melanoma patients.

Improved extraction techniques combined with sensitive real-time reverse transcriptase-polymerase chain reaction may allow detection of mRNA in formalin-fixed, paraffin-embedded (FFPE) materials, but the factors affecting mRNA quantification in clinical material using these methods have not been systematically analyzed. We designed analyses using real-time reverse transcriptase-polymerase chain reaction for quantification of MART-1, beta-actin, and beta(2)-microglobulin mRNAs. The analytical intra- and interassay imprecision (coefficient of variation) was in the range 10 to 20% for all three genes studied. Using these protocols, we studied the influence of tissue autolysis and length of formalin-fixation on mRNA detection in metastatic melanoma. Delay in freezing reduced detectable mRNA, although this was less than predicted and mostly occurred early in autolysis. MART-1, beta-actin, and beta(2)-microglobulin mRNAs were consistently detected in FFPE metastatic melanoma even after fixation for up to 3 weeks, although the total mRNA detected was markedly reduced in fixed compared with fresh tissues (up to 99%). Quantification of MART-1 was, however, possible if this was expressed relative to a housekeeping gene. The polymerase chain reaction product from FFPE tissues could be increased up to 100-fold amplifying short (<136 bp) compared with long amplicons. Variations in time before tissue processing and in fixation length seem to be less important sources of imprecision than previously assumed. Our findings suggest that quantitative analysis of mRNA in archive and routine diagnostic tissues may be possible.

Actins↗