Autolytic Reactions Research Articles

Changes of Enzymes Activity and Protein Profiles Caused by High-Pressure Processing in Sea Bass (Dicentrarchus labrax) Fillets

High-pressure processing (HPP) is a technology of growing interest for food preservation, due to its ability to control the activity of degradative enzymes. The effect of three variables (pressure levels of 100, 250, and 400 MPa; pressure holding times of 0, 5, 15, and 30 min; and pressurization rates of 8 and 14 MPa/s) on the activity of the enzymes acid phosphatase, cathepsins (B and D), lipase, and calpains was studied using sea bass fillets as a case study model. Additionally, the effect of HPP on sarcoplasmic proteins was studied by SDS-PAGE and isoelectric focusing electrophoreses. The increase in pressure level and holding time decreased the protein concentration in sarcoplasmic extracts, and also the activity of calpains. As compared to nontreated samples, acid phosphatase activity was lower at 400 MPa, and for cathepsin D lower activities were observed at 100 and 400 MPa. The increase in pressurization rate increased the activity of cathepsin D, lipase, and calpains, although it was not always significant. In contrast, cathepsin B and lipase activities were less affected by HPP treatments. Electrophoresis separation of sarcoplasmic proteins showed that the intensity of many protein bands changed mainly due to pressure level and holding time. The results of this study suggest that HPP causes lysosomes disruption and also denaturation, aggregation, and fragmentation of sarcoplasmic proteins, and this evidence might be related to the decrease in enzymes activity especially at 400 MPa. In conclusion, HPP can be a potential tool to control the activity of degradative enzymes, which might prevent the softening of sea bass muscle due to autolytic reactions.

Antemortem and postmortem biochemistry, drip loss and lipid oxidation of European sea bass muscle tissue

Slaughtering methods of farmed fish can influence swimming activity and stress levels with consequences for the flesh quality properties of fish fillets. For example, muscle metabolic activity prior to death can affect the concentration of oxygen, ATP and the pH of muscle. Handling stress of fish prior to harvesting is characterized by increased swimming activity which leads to anaerobic metabolism while energy stores are depleted and white muscle pH drops leading to acceleration of autolytic reactions after death with consequences for the organoleptic parameters of fish fillets and the filleting yield of farmed fish. In addition to the food quality aspects of the slaughtering method, the potential suffering of fish being handled during common aquaculture procedures or during slaughtering prompted scientific research in the welfare of farmed fish during harvesting. The purpose of this work was to investigate the effect of handing stress on post- mortem muscle pH and rigor mortis. European sea bass were harvested using two different levels of handling stress. Muscle pH was monitored shortly after death and after ice storage. Drip loss, and thiobarbituric acid reactive substances (TBARS) were measured in ice stored fillets. Antemortem stress resulted in rapid drop of muscle pH and onset of rigor mortis. Drip loss, and thiobarbituric acid reactive substances (TBARS) increased in the stressed fish. The results indicate that reducing handling stress during harvesting can delay rigor mortis and improve the quality of European sea bass fillets.

Spoilage potential of ice‐stored whole musky octopus (Eledone moschata)

SummaryThe spoilage potential of fresh musky octopus (Eledone moschata) was determined by evaluating the changes in biochemical and physical properties, microbial growth and sensory quality of the mantle and tentacles, over a period of 18 days. Whole musky octopus, stored in melting ice from the time of harvest, had a storage life of 10 days. Early deterioration in the sensory quality resulted primarily from autolytic reactions. Pseudomonas spp. dominated the spoilage flora, reaching an average count of 108 CFU g−1 at the end of the trial. The bacterial metabolic end products (trimethylamine, total volatile bases) were not useful as objective indicators of freshness, and measurements of pH would only provide an indicator of decomposition. Changes in the dielectric properties of the mantle were reflective of quality deterioration, but could not yield precise information on the storage history of the cephalopod. The common sulphide producer, Shewanella putrefaciens, was not a major spoiler of ice‐stored musky octopus.

Functional Screening of Serine Protease Inhibitors in the Medical Leech Hirudo medicinalis Monitored by Intensity Fading MALDI-TOF MS

The blood-feeding invertebrates are a rich biological source of drugs and lead compounds to treat cardiovascular diseases because they have evolved highly efficient mechanisms to feed on their hosts by blocking blood coagulation. In this work, we focused our attention on the leech Hirudo medicinalis. We performed, by "intensity fading" MALDI-TOF mass spectrometry, a comprehensive detection and functional analysis of pre-existent peptides and small proteins with the capability of binding to trypsin-like proteases related to blood coagulation. Combining "intensity fading MS" and off-line LC prefractionation allowed us to detect more than 75 molecules present in the leech extract that interact specifically with a trypsin-like protease over a sample profile of nearly 2,000 different peptides/proteins in the 2-20-kDa range. Moreover we resolved 232 individual components from the complex mixture, 13 of which have high sequence homology with previously described serine protease inhibitors. Our findings indicate that such extracts are much more complex than expected. Additionally, intensity fading MS, when complemented with LC separation strategies, seems to be a useful tool to investigate complex biological samples, establishing a new bridge between profiling, functional peptidomics, and subsequent drug discovery.

Comparison of selected methods of assessing freshness quality and remaining storage life of iced gilthead sea bream ( Sparus aurata)

Quality changes of whole, iced gilthead sea bream were monitored by sensory evaluation, k 1 value, GR Torrymeter, and bacterial counts. The methods were tested for their suitability to determine freshness quality and remaining storage life in ice. Depending on the measured parameter, post-mortem age of the iced fish could be predicted with an accuracy of ±1.5–3.6 days. Although assessment of cooked fish flavour is the underlying basis for establishing the state of the fish, the quality index method can be more effective for routine freshness evaluations. The k 1 value provides a useful means of monitoring early storage change, resulting primarily from autolytic reactions. Counts of sulphide-producing bacteria can be used to determine the time to rejection, while total counts at 20 °C are only poor measures of freshness quality. The GR Torrymeter offers a unique practical tool for assessing freshness quality and remaining storage life of iced gilthead sea bream.

Acrylonitrile induces autolysis Bacillus subtilis.

Acrylonitrile (AN) is an industrial chemical used in the manufacture of plastics and other polymers. AN has been reported to be an acute toxin and is a known carcinogen in rodents. When AN was mixed with suspensions of Bacillus subtilis, the bacteria began autolysis. It was determined that AN is partially converted to cyanide, a strong protonophore in B. subtilis. Autolytic enzymes in B. subtilis become active when the protonmotive force is dissipated. The amount of cyanide produced from AN, however, was not enough to promote autolysis in exponential B. subtilis. This is the first report showing that AN may induce autolytic reactions in bacteria. It is suggested the autolysis of B. subtilis may be useful in the environmental monitoring of AN. In addition, the metabolism of AN by bacilli may be useful in bioremediation.

Acrylonitrile induces autolysis Bacillus subtilis

Acrylonitrile (AN) is an industrial chemical used in the manufacture of plastics and other polymers. AN has been reported to be an acute toxin and is a known carcinogen in rodents. When AN was mixed with suspensions of Bacillus subtilis, the bacteria began autolysis. It was determined that AN is partially converted to cyanide, a strong protonophore in B. subtilis. Autolytic enzymes in B. subtilis become active when the protonmotive force is dissipated. The amount of cyanide produced from AN, however, was not enough to promote autolysis in exponential B. subtilis. This is the first report showing that AN may induce autolytic reactions in bacteria. It is suggested the autolysis of B. subtilis may be useful in the environmental monitoring of AN. In addition, the metabolism of AN by bacilli may be useful in bioremediation.

Pectin Solubilization and Enzymic Activity of Tomato Fruit Locule Tissue Cell Wall

Hydrolytic activities in liquefying locule tissue of mature-green tomato (Lycopersicon esculentum Mill. `Solar Set') fruit were studied in pursuing the understanding of mechanisms involved in the rheological changes occurring in this tissue. Ethanol-insoluble solids (EIS) were prepared with and without enzyme-inactivating treatment. The release of uronic acids from enzymically active EIS incubated under autolysic conditions was 5-fold higher than recoveries from inactive EIS. Uronic acid release was partially inhibited by 1 mm Hg2+. Cell-free proteins extracts from active EIS exhibited hydrolytic activity against inactive EIS. Pectins released from active EIS showed no evidence of main-chain hydrolysis. Neutral sugars recovered as 80% ethanol-soluble products of autolytic reactions included glc, gal, rha, ara, xyl, and man. Gal was recovered at significant higher levels in autolysates of active EIS. Glycosidases present at high activities in locule tissue included α- and β-galactosidases, β-mannosidase, β-arabinosidase, and β-glucosidase. The results confirm our earlier findings that the metabolism of water, chelator, and alkali-soluble pectins in tomato locule tissue involves deglycosylation with no apparent depolymeriation. These changes alone appear to be inadequate in explaining the unique rheological characteristics in locule gel tissue.

Inhibition of cell wall autolysis and auxin‐induced elongation of Cicer arietinum epicotyls by β‐galactosidase antibodies

Polyclonal antibodies were raised in response to βIII‐galactosidase purified from cell wall of Cicer arietinum epicotyls. The antibody preparation generated, bound to βIII protein giving a major protein band in the zone corresponding to Mr 45 000, the molecular mass previously estimated for βIII‐galactosidase. These antibodies clearly suppress autolytic reactions in isolated walls of Cicer arietinum epicotyl segments, while the preimmune serum had no effect on autolytic reaction. The results strongly support the idea that the autolytic degradation of the cell wall is carried out by the βIII‐galactosidase.The antibodies against β‐galactosidase were also able to inhibit cell wall hydrolysis mediated by both total cell wall protein extracted by LiCl and cell wall hydrolysis mediated by βIII‐galactosidase.Since autolysis is thought to be related to the process of cell wall loosening, the effects of the antibodies against the autolytic enzyme was also tested on epicotyl growth. β‐galactosidase antibodies consistently inhibited IAA‐induced elongation.

Inhibition of Auxin-Induced Cell Elongation of Maize Coleoptiles by Antibodies Specific for Cell Wall Glucanases

Polyclonal antibodies were raised in rabbits in response to the administration of purified exo- and endoglucanases extracted from cell walls of maize (Zea mays L. B37 x Mo17) coleoptiles. Since the antibodies formed specific conjugates when challenged with the glucanase antigens in immunoblot assays they were employed to evaluate the participation of glucanases in tissue growth. Indole-3-acetic acid induced cell elongation of abraded coleoptile segments was inhibited when the antibodies were supplied as a short term pretreatment (25-200 microgram/milliliter of serum protein). The extent of inhibition of IAA induced cell elongation was additive when endo- and exoglucanase antibodies were applied together. The results suggest that both enzymes have a role in mediating IAA-induced cell elongation. Pretreatment with exo- and endoglucanases antibodies also inhibited IAA induced degradation of noncellulosic beta-d-glucans and the increased level of cellulosic polymers in maize coleoptiles. Antibodies also inhibited the expression of the autohydrolytic degradation of glucans in isolated cell walls. The extent of inhibition was dependent on the antibody concentration applied. The results support the contention that enzymatic processes mediated by exo- and endoglucanases are responsible for cell wall autolytic reactions and that these reactions are linked to the mechanism for expressing auxin induced cell elongation in maize coleoptiles.

Selected cell wall proteins from Zea mays: Assessment of their role in wall hydrolysis

Coleoptile cell wall proteins from Zea mays L. hybrid B 37 × Mo 17 were extracted and fractionated. Three enzymes identified in that extract were examined to determine their role in cell wall hydrolysis with a goal of evaluating the extent to which they participated in autohydrolytic reactions. Two separate proteins were identified as endo‐ and exo‐glucanases. Incubation of these enzymes with heat inactivated cell walls, liberates products derived from the constitutive (1→3), (1→4)‐β‐d‐glucan. The release of sugars from walls resembles that of cell wall autolysis. A third cell wall protein degraded polysaccharides in a more general manner, releasing carbohydrates containing xylose, arabinose, galactose and glucose. Polyclonal antibodies raised against the exoglucanase protein suppressed autolytic reactions of isolated cell wall.

Effect of anti‐wall protein antibodies on auxin‐induced elongation, cell wall loosening, and β‐D‐glucan degradation in maize coleoptile segments

Antiserum raised against the LiCl extract of maize shoot cell walls suppresses auxin‐induced elongation of maize coleoptile segments. A series of polyclonal antibodies were raised against protein fractions separated from the LiCl extract of maize (Zea mays L. cv. B73 x Mo17) coleoptiles by SP‐Sephadex and Bio‐Gel P‐150 chromatography. To understand the role of cell wall proteins in growth regulation, the effect of these antibodies on auxin‐induced elongation and changes in the cell walls of maize coleoptiles was examined. Four of the fractions prepared reacted with the antiserum raised against the total LiCl extract and effectively suppressed its growth‐inhibiting activity. Only these fractions contained the proteins responsible for eliciting growthinhibiting antibodies. The antibodies capable of growth inhibition of auxin‐induced elongation of segments also inhibited auxin‐induced cell wall loosening (decrease in the minimum stress‐relaxation time of the cell walls) of segments. The antibodies raised against one of the protein fractions separated by SP‐Sephadex inhibited the autolytic reactions of isolated cell walls and the auxin‐induced decrease in (1→3), (1→4)‐β‐D‐glucans in the cell walls. Thus, the degradation of β‐D‐glucans by cell wall enzymes may be associated with the cell wall loosening that is responsible for cell elongation. Because the other antibodies did not influence the auxin‐induced degradation of (1→3), (1→4)‐β‐D‐glucanses, β‐D‐glucanases and other cell wall enzymes may cooperate in regulation of cell elongation in maize coleoptiles.

Differential autolysis of human plasmin at various pH levels

Denaturation of human plasmin in solutions of various pH levels was studied. The denaturation and loss of catalytic activity of plasmin in solutions of between pH 3.5 and 10.5 is a second-order kinetics. In alkaline solutions of pH levels >11.5, plasmin undergoes a first-order denaturation. The second-order denaturation of plasmin is mainly due to the autolytic reactions between plasmin molecules. Two autolytic processes of human plasmin in aqueous solution were observed. In a slightly acidic solution (pH 6.5) the light (B) chain was found to be cleaved faster than the heavy (A) chain of plasmin. On the other hand, the heavy (A) chain was cleaved in an alkaline solution of pH near 11.0. A cleaved heavy (A) chain of molecular weight 58,000 was observed. Both the heavy (A) chain and the light (B) chain were found to be cleaved at pH levels between 6.5 and 11.0. The loss of the esterase activity of plasmin samples in the autolytic process is in parallel with the decline of intact light (B) chain. The autolytic cleavage of the heavy (A) chain led to the formation of a new type of catalytically active plasmin.

Uronic Acid Products Release from Enzymically Active Cell Wall from Tomato Fruit and Its Dependency on Enzyme Quantity and Distribution

Isolated cell wall from tomato (Lycopersicon esculentum Mill. cv Rutgers) fruit released polymeric (degree of polymerization [DP] > 8), oligomeric, and monomeric uronic acids in a reaction mediated by bound polygalacturonase (PG) (EC 3.2.1.15). Wall autolytic capacity increased with ripening, reflecting increased levels of bound PG; however, characteristic oligomeric and monomeric products were recovered from all wall isolates exhibiting net pectin release. The capacity of wall from fruit at early ripening (breaker, turning) to generate oligomeric and monomeric uronic acids was attributed to the nonuniform ripening pattern of the tomato fruit and, consequently, a locally dense distribution of enzyme in wall originating from those fruit portions at more temporally advanced stages of ripening. Artificial autolytically active wall, prepared by permitting solubilized PG to bind to enzymically inactive wall from maturegreen fruit, released products which were similar in size characteristics to those recovered from active wall isolates. Extraction of wall-bound PG using high concentrations of NaCl (1.2 molar) did not attenuate subsequent autolytic activity but greatly suppressed the production of oligomeric and monomeric products. An examination of water-soluble uronic acids recovered from ripe pericarp tissue disclosed the presence of polymeric and monomeric uronic acids but only trace quantities of oligomers. The significance in autolytic reactions of enzyme quantity and distribution and their possible relevance to in vivo pectin degradation will be discussed.

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)

Wall‐protein antibodies as inhibitors of growth and of autolytic reactions of isolated cell wall

Antiserum raised against protein prepared from corn (Zea mays L. hybrid B73 X Mo17) seedling cell wall exhibits antigrowth activity and inhibits autolytic reactions of isolated cell wall. The growth of corn coleoptile segments pretreated in wall‐protein antiserum was inhibited 35% compared to tissue which had been exposed to normal serum. Since the cuticle constituted a barrier to the entry of serum protein the coleoptile surface was abraded prior to exposure to the serum. Growth inhibition observed in nonabraded sections (8 to 10%) was apparently the result of the entry of serum protein via the cut ends of the tissue. Subsections recut from the central region of antiserum‐treated, nonabraded coleoptiles responded normally in growth experiments. Antiserum brought in contact with isolated cell wall prior to its use with intact coleoptiles was less effective as a growth inhibitor, which suggested that the antibodies which impede growth responses bind to cell wall.Wall‐protein antiserum also strongly inhibited autolytic reactions of isolated cell wall. Wall treated in normal serum or buffer liberated sugars at a rate of 8.6 μg‐ (mg wall dry‐weight)−1·h−1 while antiserum‐treated wall autolyzed at a rate of 2.6 μg·mg−1·h−1 representing a 70% reduction in autolytic potential. After 24 h yields from antiserum‐treated wall were 50% lower than yields obtained from control wall. The antiserum had no effect on the molecular‐weight distribution of the autolytically‐liberated products, and direct assays of wall‐bound and solubilized endo‐ and exo‐β‐d‐glucanases involved in autolytic reactions demonstrated no significant impairment of their catalytic activity. However, the addition to antiserum‐treated wall of a wall‐protein preparation containing autolytic enzymes resulted in a significant enhancement (132%) in autolytic potential. The evidence suggests that enzyme mobility may be critical in the autolytic hydrolytsis of plant cell wall.

Partial purification of endo- and exo-?-D-glucanase enzymes from Zea mays L. seedlings and their involvement in cell-wall autohydrolysis

The proteins dissociated from isolated Zea seedling cell wall using high-ionic-strength salt solutions have been found to include a number of enzymes which appear to participate in autolytic reactions of the cell wall. These enzymes caused extensive degradation of enzymatically inactive cell wall, liberating as much as 100 μg/mg dry weight over a 48-h period. Lithium chloride (3M) was shown to be most effective in yielding protein and wall-degrading activities.Molecular-sieve chromatography of the cell-wall protein resolved endo-β-D-glucanase and exo-β-1,3-glucanase (EC 3.2.1.58) activities when Avena glucan and laminarin, respectively, were employed as substrates. The exoenzyme (molecular weight around 60,000) was strongly inhibited by inorganic mercury at a concentration which suppressed the release of monosaccharide from autolytically active cell wall. The endo-β-D-glucanase (MW around 26,000), which showed a marked preference for substrates of mixed-linkage, exhibited features indicating that it initiates the autolytic solubilization of wall glucan.Cell-wall β-D-glucan, recovered as a component of an alkali-soluble cell-wall fraction, served as a substrate for the purified glucanases. Their hydrolysis pattern, assessed using gel exclusion chromatography and product analysis, confirmed that they hydrolyze β-D-glucan. The products generated by the endoglucanase were similar in molecular-size distribution to those liberated from autolytically active-wall. Exoglucanase activity was required for extensive hydrolysis of β-D-glucan in vitro.During coleoptile development the autolytic activity of the cell wall increased dramatically. This increased activity, however, did not parallel the growth potential of the tissue, but more closely reflected an increase in cell-wall β-D-glucan, the primary substrate for autolytic reactions.

Experimentelle Induktion von „rotund bodies”︁ bei Thermus aquaticus

With several isolates of T. aquaticus rotund bodies (rbs) have been artificially induced. The most active agents were lysozyme, pronase (not trypsin), cellulase (not alpha-amylase or beta-glucosidase), penicillin and some mineral salts. Two modifications of rbs were observed: the vesticular type arising from single cells or filaments and the aggregate-rbs resulting from secondary associations of several cells or filaments. In both cases the primary event is a partial detachment of the outer layer of cell envelopes followed by its swelling to form a hyaline bleb. Within these osmotically stable blebs cells and/or filaments involved in the ribs production become irreversibly trapped. Appearance of filaments and rbs in stationary phase cultures are discussed as a consequence of unbalanced growth culminating in abnormal autolytic reactions.

Studies on soil fumigation—III: Effects on enzyme activities, bacterial numbers and extractable ninhydrin reactive compounds

Fumigation of a field soil with chloropicrin and methyl bromide, either singly or in combination, differentially decreased soil enzyme activities and viable bacterial numbers and increased the amounts of ninhydrin reactive compounds extractable with acidified Tris buffer. Chloropicrin treatment was more effective than methyl bromide. The rates of hydrolysis of both an amide and a peptide derivative were decreased by chloropicrin treatment and remained relatively low despite changes in activities over 325 days. By contrast, caseinase activity initially was decreased by both chloropicrin and methyl bromide fumigation, but activities of the fumigated soils recovered to exceed those of untreated soils. Thereafter, caseinase activities of fumigated and untreated soils exhibited relatively large fluctuations, which were partly associated with seasonal drying of the soils in the field. Chloropicrin but not methyl bromide fumigation markedly depressed the viable bacterial populations, which subsequently increased to be above those of the untreated soils. There was no consistent relationship between the release of ninhydrin reactive compounds following fumigation and changes in bacterial numbers or changes in enzyme activity. Autolytic reactions are probably important in the early stages of amino-nitrogen release in fumigated soils. Net gains in caseinase activity may be partly due to the production de novo of extracellular proteases by microorganisms or to the release of intracellular proteases from killed cells.

Cooperative transitions between active α- and β-trypsin conformations

The temperature dependence of hydrolytic and autolytic reactions catalyzed by free and immobilized α- and β-trypsin has been studied. A reversible cooperative transition was found to occur in the temperature interval 40–45°. The transition is probably an unfolding — refolding process involving only part of the molecule, as the enzyme function is retained. This indicates that the complete unfolding of these enzymes consists of several independent unfolding processes — multistate unfolding. Some implications of multistate unfolding in proteins are discussed.