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At least 811 records · Page 45Linked to original sources
PubMed2006

Biomass recycling from a riboflavin cultivation with B. subtilis: lysis, extract production and testing as substrate in riboflavin cultivation.

Autolysis of riboflavin-producing B. subtilis can be induced by pH, lack of carbon source, and the buffer system. Stress factors like temperature shift or oxygen dearth enhance the autolysis process. After cultivation of a riboflavin-producing strain, the pH of the whole culture broth was adjusted to 6.5-7.5. At a temperature of 40 degrees C, autolysis started after 1 h. Adding a defined amount of commercially available endo- and exo-proteases enhanced both auto- and proteo-lysis. Optimization of endo- and exo-protease concentrations and of the time increased the degree of proteolysis. Additionally, the amount of DNA and Protein trapped in the riboflavin crystals could be significantly reduced by autolysis. After autolysis, the cultivation broth was centrifuged and the supernatant was cross-flow filtrated with a cut off of 10 kDa. Using this autolysate instead of yeast extract as a medium component for riboflavin production with B. subtilis, a riboflavin yield of 77% was obtained in comparison with the standard cultivation on yeast extract.

Bacillus subtilis↗
PubMed2002

Activity of cell-wall degradation associated with differentiation of isolated mesophyll cells of Zinnia elegans into tracheary elements.

Cell walls were prepared from cultured mesophyll cells of Zinnia elegans L. that were transdifferentiating into tracheary elements and incubated in a buffer to undergo autolysis. The rate of autolysis of cell walls was determined by measuring the amount of carbohydrate released from the cell walls into the buffer during incubation. During the course of culture of mesophyll cells, the autolysis rate increased markedly at the time when thickenings of secondary cell walls characteristic of tracheary elements became visible (after 48-72 h of culture), and thereafter the rate remained at a high level. Comparative studies on the autolysis rate of cell walls using various control cultures, in which tracheary element differentiation did not take place, revealed a close relationship between the autolysis rate around the 60th hour of culture and differentiation. Sugar analysis by colorimetric assays and gas chromatography of carbohydrates released from the cell walls detected uronic acid, arabinose, galactose, glucose, xylose, rhamnose, fucose, and mannose. Among these sugars, uronic acid was the most abundant, and accounted for approximately half of the total released sugars. The decrease of acidic polysaccharides in the primary cell walls during tracheary element differentiation was visualized by staining cultured cells with alcian blue at pH 2.5. These results suggest that active degradation of components of primary cell walls, including pectin, is integrated into the program of tracheary element differentiation.

Alcian Blue↗
PubMed1985

Immunological analysis of rat pancreatic prokallikrein activation.

The present study shows that tissue kallikrein is present in rat pancreas as a proenzyme that can be converted by autolysis to a 38 000 Da active enzyme. The activation of pancreatic prokallikrein was examined by direct radioimmunoassay, enzymatic assays, active-site labeling with immunoprecipitation, and Western blot analyses. A monoclonal antibody (V1C3), which binds only active kallikrein, was used in a direct radioimmunoassay to monitor the appearance of the active enzyme. During a 22-h autolysis of pancreatic extract, a time-dependent increase in active kallikrein concentration paralleled the increase of kallikrein activities measured by both TosArgOMe esterase and kininogenase assays. The activation process was further analyzed by labeling the pancreatic extract with [14C]diisopropylphosphorofluoridate [( 14C]DFP) followed by immunoprecipitation with sheep anti-kallikrein antiserum. Pancreatic prokallikrein was not labeled by [14C]DFP; however, upon autolysis, a 38 000 Da active kallikrein can be labeled with [14C]DFP and increase in quantity with time. Western blot analysis, using a monoclonal antibody (V4D11) which recognizes both latent and active tissue kallikreins, identified a 39 000 Da pancreatic prokallikrein prior to autolysis and a 38 000 Da active kallikrein after 7 h of autolysis. The results indicate that the pancreatic prokallikrein exists as a 39 000 Da protein which may be converted to a 38 000 Da active kallikrein, indistinguishable from purified urinary, brain, spleen or submandibular gland kallikrein.

Animals↗
PubMed1981

The effects of surface and macromolecular interactions on the kinetics of inactivation of trypsin and alpha-chymotrypsin.

The autolysis of trypsin and alpha-chymotrypsin is accelerated in the presence of colloidal silica and glass surfaces. It is proposed that adsorption of the enzymes (favoured by electrostatic factors) results in a conformational change that renders the adsorbed enzyme more susceptible to proteolytic attack. Although the adsorbed enzymes are more susceptible to proteolysis, their activity towards low-molecular-weight substrates is not affected, indicating a relatively minor conformational change on adsorption. The rates of autolysis in solution (i.e. in ;inert' vessels) are second-order for both trypsin and alpha -chymotrypsin, with rate constants of 13.0mol(-1).dm(3).s(-1) for trypsin (in 50mm-NaCl at pH8.0 at 25 degrees C) and 10.2mol(-1).dm(3).s(-1) for alpha-chymotrypsin (in 0.1m-glycine at pH9.2 at 30 degrees C). In glass vessels or in the presence of small areas of silica surface (as colloidal silica particles), the autolysis of both trypsin and alpha-chymotrypsin can show first-order kinetics. Under these conditions, saturation of the surface occurs and the fast surface proteolytic reaction controls the overall kinetic order. However, when greater areas of silica surface are present, saturation of the surface does not occur, and, since for a considerable portion of the adsorption isotherm the amount adsorbed is approximately proportional to the concentration in solution, second-order kinetics are again observed. A number of negatively charged macromolecules have been shown similarly to increase the rate of autolysis of trypsin: thus this effect, observed initially with glass and silica surfaces, is of more general occurrence when these enzymes adsorb on or interact with negatively charged surfaces and macromolecules. These observations explain the confusion in the literature with regard to the kinetics of autolysis of alpha-chymotrypsin, where first-order, second-order and intermediate kinetics have been reported. A further effect of glass surfaces and negatively charged macromolecules is to shift the pH-activity curve of trypsin to higher pH values, as a consequence of the effective decrease in pH in the ;microenvironment' of the enzyme associated with the negatively charged surface or macromolecule.

Adsorption↗
PubMed1994

Calpain (Ca(2+)-dependent thiol protease) in erythrocytes of young and old individuals.

Limited proteolysis by calpain (Ca(2+)-activated protease; EC 3.4.22.17) is believed to regulate the function of membrane enzymes and modify the behavior of membrane structural proteins. Calpain is activated by autolysis. The degradation of band 3 protein by mu-calpain is known to be enhanced in erythrocyte membranes from human individuals > 70 years old (old) as compared with that from individuals 20-30 years old (young). In the present study, monoclonal antibody to mu-calpain was used to study the behavior of calpain in erythrocytes of young and old individuals. Less calpain was found in erythrocyte cytosol and membranes from old than in those from young. Increasing the erythrocyte Ca2+ induced translocation of calpain to the cell membrane and autolysis of the enzyme. Alkylation of erythrocyte thiols also promoted translocation of calpain to the membrane, especially in the presence of Ca2+. When calpain was added to erythrocyte membranes, initial binding was greater and subsequent autolysis faster in old than in young individuals, possibly arising from alterations in cell membranes of old individuals. The enhanced calpain autolysis was accompanied by enhanced degradation of band 3 protein in the old. The results suggest that calpain in old individuals is translocated to the cell membrane and is activated by autolysis, resulting in degradation of certain membrane proteins and loss of calpain. Enhanced calpain-induced membrane proteolysis may play a role in abnormal cell destruction (e.g., shortening the life span of erythrocytes in the aged, neuronal degeneration, etc). The erythrocyte membrane provides a convenient model for the study of age-associated alterations in cell membranes and in calpain behavior.

Adult↗
PubMed2004

Early postmortem biochemical factors influence tenderness and water-holding capacity of three porcine muscles.

The objective of this study was to determine whether differences in pork tenderness and water-holding capacity could be explained by factors influencing calpain activity and proteolysis. Halothane-negative (HAL-1843 normal) Duroc pigs (n = 16) were slaughtered, and temperature and pH of the longissimus dorsi (LD), semimembranosus (SM), and psoas major (PM) were measured at 30 and 45 min and 1, 6, 12, and 24 h postmortem. Calpastatin activity; mu-calpain activity; and autolysis and proteolysis of titin, nebulin, desmin, and troponin-T were determined on muscle samples from the LD, SM, and PM at early times postmortem. Myofibrils from each muscle were purified to assess myofibril-bound (mu-calpain. Percentage drip loss was determined, and Warner-Bratzler shear (WBS) force was analyzed. Myosin heavy-chain (MHC) isoforms were examined using SDS-PAGE. The pH of PM was lower (P < 0.01) than the pH of LD and SM at 30 and 45 min and 1 h postmortem. The PM had a higher (P < 0.01) percentage of the MHC type IIa/IIx isoforms than the LD. The-LD had the greatest proportion of (P < 0.01) MHC IIb isoforms of any of the muscles. The PM had the lowest (P < 0.01) percentage of MHC IIb isoforms and a greater (P < 0.05) percentage of type I MHC isoforms than the LD and SM. The PM had less (P < 0.01) drip loss after 96 h of storage than the SM and LD. The PM had more desmin degradation (P < 0.01) than the LD and SM at 45 min and 6 h postmortem. Degradation of titin occurred earlier in the PM than the LD and SM. At 45 min postmortem, the PM consistently had some autolysis of mu-calpain, whereas the LD and SM did not. At 6 h postmortem, some autolysis of mu-calpain (80-kDa subunit) was observed in all three muscles. The rapid pH decline and increased rate of autolysis in the PM paralleled an earlier appearance of myofibril-bound mu-calpain. The SM had higher calpastatin activity (P < 0.05) at 45 min, 6 h, and 24 h and had higher WBS values at 48 h (P < 0.01) and 120 h (P < 0.05) postmortem than the LD. At 48 and 120 h postmortem, more degradation of desmin, titin, and nebulin were observed in the LD than in the SM. These results show that mu-calpain activity, mu-calpain autolysis, and protein degradation are associated with differences in pork tenderness and water-holding capacity observed in different muscles.

Animals↗
PubMedDate not supplied

[Effect of arginine on the activity of proteolytic enzymes in the rat brain and liver].

The influence of arginine on autolysis and proteolysis was studied. Arginine at the concentration of 0.5 and 1.0 microM/ml was added to the incubation mixture. Proteolytic processes were studied in the acid, neutral and alkaline media (pH 4.5; 7.4; 8.5). Autolysis was determined by incubation of the brain and liver homogenates and proteolysis by the use of bovine serum albumin as a substrate. Autolytic and proteolytic activities were calculated as an increase of Folin positive compounds or amino nitrogen in the samples. It was established that the influence in vitro of arginine on the proteolytic processes depended on pH, type of the peptide-hydrolases, to a lesser extent, on the arginine concentration and did not depend on the tissue type. Arginine displayed its regulative action in the brain and liver by the same way. The addition of arginine had an effect on autolysis and proteolysis in the neutral and alkaline media. Determination of autolytic and proteolytic activities by Folin positive compounds has shown that arginine addition into the samples decreased autolysis and proteolysis. At the same time determination of autolysis and proteolysis by amino nitrogen in the presence of arginine has shown that autolytic and proteolytic activities increased.

Animals↗
PubMed1998

Hydroxyapatite induces autolytic degradation and inactivation of matrix metalloproteinase-1 and -3.

In the course of studies to identify a protease capable of producing a long-lived 50 kDa fragment of bone acidic glycoprotein-75 (BAG-75), it was observed that incubation of matrix metalloproteinase (MMP)-3 (stromelysin 1) with preparations of BAG-75 led to inactivation of proteolytic function, e.g., an inability to fragment 125I-labeled BAG-75 added subsequently. MMP-1 (interstitial collagenase) was also inactivated by exposure to BAG-75 preparations. Investigation of the mechanism revealed that BAG-75 preparations contained millimolar levels of inorganic phosphate which formed hydroxyapatite crystals under digestion conditions. Hydroxyapatite crystals alone and in BAG-75-hydroxyapatite complexes induced the autolytic degradation of both active and precursor forms of MMP-1 and MMP-3. Autolytic degradation in the presence of hydroxyapatite was demonstrated by a loss in catalytic function assayed with peptide and/or protein substrates, and, by fragmentation into polypeptides of <10 kDa. The fate of MMP-3 incubated with hydroxyapatite depends upon the time of incubation, the free calcium concentration, and the concentration of crystals. Specifically, hydroxyapatite-induced autolysis requires a near physiological free calcium concentration of 0.5-1.0 mM. Autolysis was maximal in the presence of 150 microg/ml hydroxyapatite where MMP-3 was only partially bound to crystals. However, autolysis also occurred at higher crystal concentrations where all input MMP-3 was bound (>1000 microg/ml), suggesting that autolysis may be mediated by bound enzyme. The effect of hydroxyapatite appears to be specific for MMP-1 and MMP-3 since the catalytic activity of chymotrypsin, trypsin, papain, and thermolysin remained unchanged after exposure to hydroxyapatite. These results document for the first time a novel catalytic role for hydroxyapatite crystals in vitro and provide an initial biochemical characterization of the intermolecular, autolytic, calcium ion-dependent, matrix metalloproteinase-specific degradative mechanism.

Animals↗
PubMed1999

Auto-inactivation by cleavage within the dimer interface of Kaposi's sarcoma-associated herpesvirus protease.

An autolysis site of functional and structural significance has been mapped within the dimer interface of Kaposi's sarcoma-associated herpesvirus protease. Cleavage 27 residues from the C terminus of the 230 amino acid residue, 25 kDa protein was observed to cause a loss of dimerization and proteolytic activity, even though no active site moieties were lost. Gel-filtration chromatography and analytical ultracentrifugation were used to analyze the changes in oligomerization upon autolysis. The selective auto-disruption of this essential protein-protein interface by proteolytic cleavage resulted in a 60 % loss in mean residue ellipticity by circular dichroism as well as a 20 % weaker, 10 nm red-shifted intrinsic protein fluorescence emission spectrum. These apparent conformational changes induced a strict inhibition of enzymatic activity. An engineered substitution at the P1' position of this cleavage site attenuated autolysis by the enzyme and restored wild-type dimerization. In addition to retaining full proteolytic activity in a continuous fluorescence-based enzyme assay, this protease variant allowed the determination of the enzyme's dimerization dissociation constant of 1.7 (+/-0.9) microM. The structural perturbations observed in this enzyme may play a role in viral maturation, and offer general insight into the allosteric relationship between the dimer interface and active site of herpesviral proteases. The functional coupling between oligomerization and activity presented here may allow for a better understanding of such phenomena, and the design of an enzyme variant stabilized to autolysis should further the structural and mechanistic characterization of this viral protease.

Amino Acid Sequence↗
PubMed1982

Effect of leukocyte hydrolases on bacteria XVI. Activation by leukocyte factors and cationic substances of autolytic enzymes in Staphylococcus aureus: modulation by anionic polyelectrolytes in relation to survival of bacteria in inflammatory exudates.

The mechanisms involved in the activation of autolytic enzymes in Staphylococcus aureus, by leukocyte extracts, cationic proteins, phospholipase A2, amines, and membrane-damaging agents was studied in a resting cell system as well as by growing staphylococci. The bacteria were labeled with [14C]N-acetylglucosamine and were subjected to a variety of agents either in 0.1 M acetate buffer, pH 5.0, or in phosphate buffer, pH 7.4. While intact log-phase cultures were found to undergo partial autolysis at pH 5.0 and almost complete lysis at pH 7.4, both heat-killed bacteria and bacterial cell walls were completely resistant to autolysis in buffers. Autolysis at pH 5.0 can be further activated by leukocyte extracts, nuclear histone, crystalline ribonuclease, egg-white and human lysozyme, phospholipase A2, as well as by spermine, spermidine, and polymyxins B and E. The addition of viable log-phase bacteria to radiolabeled heat-killed staphylococci or to radiolabeled cell walls which had been cleaned off autolytic enzymes resulted in degradation of the radiolabeled targets. The data suggest that the various inducers of autolysin activation caused leakage of autolytic enzymes from the intact bacteria which attacked the depolymerized the bacterial cell walls. Anionic polyelectrolytes like heparin, dextran sulfate, suramine, polyglutamic acid, and liquid (polyanethole sulfonic acid) markedly inhibited both spontaneous and induced lysis. Staphylococci which had grown in the presence of anionic polyelectrolytes became highly resistant to lysis triggered by any of the inducers of autolysis. Since inflammatory exudates are known to be rich in anionic polyelectrolytes, it is suggested that the prolonged survival of intact bacterial cells in such a milieu may be due to the inactivation of autolytic enzymes. It is also postulated that the degradation of certain bacterial species following phagocytosis or extracellular degradation may not be the result of the action of hydrolytic enzymes but rather the result of activation by leukocyte factors of autolytic enzymes which lead to bacteriolysis.

Bacteriolysis↗
PubMed1997

Influence of postmortem changes on immunohistochemical reactions in skin.

The influence of postmortem damage of tissues on the immunohistochemical diagnosis of wound age has not as yet been clarified. We utilized antibodies against the proteinase inhibitors alpha-1-antichymotrypsin and alpha-2-macroglobulin, fibronectin and lysozyme to study samples of skin which had been intact intravitally, but were damaged postmortem either by autolysis or compression with a surgical clamp at the time of dissection. Even in the absence of autolysis, antibodies against the proteinase inhibitors and fibronectin exhibited staining of tissue margins. Autolysis caused an increase in false positive results. In contrast, antibodies against lysozyme did not give false positive staining. There were no antigens sensitive to postmortem clamping and false positive results were not observed. Antibodies against proteinase inhibitors are not useful for the diagnosis of wound age because of a high number of false positive reactions in marginal areas. Fibronectin also showed false positive band-shaped staining patterns at the tissue margin. In addition, autolytic processes increase the number of false positives. The antibody against lysozyme is much less sensitive to autolysis and no false positive reactions were observed in our series of tests.

Fibronectins↗
PubMed2003

Members of the cystatin superfamily interact with MMP-9 and protect it from autolytic degradation without affecting its gelatinolytic activities.

Matrix metalloproteinases (MMPs), like other proteinases, can undergo autolytic degradation once activated in vivo. Whereas the activities of these enzymes are tightly regulated by tissue inhibitors of matrix metalloproteinases (TIMPs), it is not clear mechanistically how these enzymes are protected from autolysis in their active state. We previously reported that MMPs particularly MMP-9 and MMP-2 interact with the serum glycoprotein fetuin-A [Arch. Biochem. Biophys. (1995) 322, 250], a member of the cystatin superfamily. In the present analyses, we demonstrate that this interaction protects MMP-9 from autolytic degradation without interfering with its enzymatic activity, allowing it to efficiently digest gelatin. Our data demonstrate that MMP-9 binds to members of the cystatin family with K(diss) ranging from 25 to 58 nM for fetuin-A and 1.5-1.9 microM for cystatin C. The ability of fetuin-A to protect MMP-9 from autolysis requires a molar ratio of at least 8:1 (fetuin-A/MMP-9). More interestingly, our data show that the other members of the cystatin also have the ability to protect MMP-9 from autolysis, provided they are in molar excess relative to MMP-9. Taken together, our data suggest that cystatins, particularly fetuin-A, in any cellular compartment including the circulatory system, efficiently protect MMP-9 and possibly other MMPs from autolysis. This mechanism ensures the digestion of the preferred substrate for MMP-9 without sacrificing the enzyme in the process.

Animals↗
PubMed1997

Age- and radiation-dependent changes in carbonyl content, susceptibility to proteolysis, and antigenicity of soluble rat liver proteins.

Soluble liver proteins (SLP) from old and gamma-irradiated young rats were studied with respect to their carbonyl content, the rates of autolysis and degradation by proteinase K, and their antigenicity for mice and compared with SLP from non-irradiated young animals. A significant increase in the carbonyl level was found in SLP from old and gamma-irradiated young rats as compared to SLP from intact young rats. The rates of SLP autolysis and proteolysis by proteinase K were increased in the same animal groups but did not correlate the carbonyl level. At the same time, whereas the antigenicity for mice of SLP from old rats was significantly higher than that of SLP from young rats, the antigenicity of SLP from gamma-irradiated rats did not differ from non-irradiated animals. Enrichment of the diet with antioxidant and vitamin supplements (AVS) during one month before the irradiation caused a decrease in the radiation-induced carbonyl level in rat SLP. However, this raised antioxidant level in animal diet did not influence the rates of SLP autolysis and degradation by proteinase K and also did not alter the antigenicity of these proteins. The data allow us to suggest that the increase in autolysis, degradation by the exogenous proteinase, and antigenicity of SLP from old rats are determined not only by carbonyl formation in these proteins due to action of oxygen radicals but also by other age-specific protein modifications.

Age Factors↗
PubMed2002

Stimulation of microbial autolytic system by tryptic casein hydrolysate.

Antimicrobial activity of a tryptic casein hydrolysate (TCH), its mode of antimicrobial action and its efficacy in treatment using a newborn calf colibacillosis model are described. The antimicrobial activity of TCH activated the autolytic system of the microbial cell and was expressed mathematically as the autolysis activation index. TCH stimulated the autolytic enzyme system of the 19 bacterial and five fungal strains tested. The autolysis activation index for naturally-autolyzing microorganisms was 1.45-22.0. Autolysis in three bacterial and one fungal strain that did not lyse in the absence of TCH was increased in its presence by 3.9-56.7% showing activation of latent autolysis. The in vivo trials on 800 newborn calves revealed 93.0% therapeutic and 93.5% prophylactic efficacy for TCH. These levels were similar to the 95.0 and 95.5% attained when Fermosorb, an authorized antimicrobial drug, was used.

Animals↗
PubMed1992

Incorporation of a stabilizing Ca(2+)-binding loop into subtilisin BPN'.

A rational approach was taken to improve the stability of subtilisin BPN' to autoproteolysis. Two sites of autoproteolysis were identified by isolation of early autolysis products and amino-terminal sequence analysis. These studies showed that subtilisin rapidly cleaves Ala48-Ser49 and Ser163-Thr164 peptide bonds at elevated temperatures. These two sites appear in regions of high mobility as estimated from crystallographic B-factors and are in extended surface loops. To improve the resistance to thermal-induced autolysis, we replaced sequences around these two sites with sequences derived from a thermophilic homologue of subtilisin, thermitase. Thermitase contains a Ca(2+)-binding site in the region surrounding Ser49. When the Ca(2+)-binding segment of thermitase corresponding to residues 45-63 of subtilisin BPN' was installed into subtilisin BPN', the chimeric protein gained the ability to bind another Ca2+ with moderate affinity (Kd approximately 100 microM). This enzyme had the same kcat as wild-type, had a KM value 8-fold larger than wild-type, and was slightly less stable to thermal inactivation in EDTA. However, in 10 mM CaCl2, the mutant subtilisin BPN' was 10-fold more stable to irreversible inactivation at 60 degrees C than wild-type subtilisin BPN' as measured by residual activity against the substrate sAAPF-pna. Next, mutations and deletions derived from thermitase were introduced near the second autolysis loop in subtilisin BPN' (residues 158-165). However, all of these mutants were less stable than wild-type subtilisin. Thus, some (but not all) mutations derived from a thermophilic homologue near sites of autolysis can be stabilizing to a mesophilic protease.

Amino Acid Sequence↗
PubMed1993

Studies of the active site of m-calpain and the interaction with calpastatin.

Calpain autolyses in the presence of Ca2+. In the case of m-calpain (80 + 30 kDa) the first product is an 80 + 18 kDa species which has an intact large subunit and the C-terminal Ca(2+)-binding domain of the small subunit. It was possible to bind E64 into the active site of calpain in the presence of Ca2+ before cleavage of either calpain subunit. This suggests that the active site is functional before any autolysis has occurred and that calpain is not a proenzyme. Prolonged autolysis generates several fragments including a 42 kDa active-site domain fragment that showed no proteolytic activity and Ca(2+)-binding domain fragments. Some of the Ca(2+)-binding domain fragments were found to exist as heterodimers (23 + 18 kDa and 22 + 18 kDa), with the Ca(2+)-binding domain of the large subunit interacting with the Ca(2+)-binding domain of the small subunit. These species were true heterodimers, as they showed co-elution of the two Ca(2+)-binding domains on ion-exchange and gel-filtration chromatography, and immunoprecipitation of both polypeptides with an antiserum specific for the small-subunit Ca(2+)-binding domain. The generation of the dimer species after only 15 min autolysis suggests that the interaction between the Ca(2+)-binding domains is present in the native calpain structure. The interaction of calpain with calpastatin was investigated using an assay based on binding to calpastatin-Sepharose and a competitive binding assay. Calpain, active-site-blocked calpain and calpain fragments generated by autolysis were studied. Calpain bound to calpastatin in the presence of Ca2+; however, the isolated active-site-containing 80 kDa large subunit (proteolytically inactive), a 42 kDa active-site-containing fragment (proteolytically inactive) and Ca(2+)-binding domain fragments of calpain did not. Active-site-blocked calpain bound to calpastatin, but with an affinity reduced by approximately two orders of magnitude when compared with native calpain.

Amino Acid Sequence↗
PubMed1999

Calcium signaling in Streptococcus pneumoniae: implication of the kinetics of calcium transport.

The kinetics and pharmacological characterization of a Na+/Ca2+ exchange system, essential for the growth of the extracellular pathogen Streptococcus pneumoniae in high-calcium media, demonstrated that calcium transport, in addition to its role in calcium homeostasis, is involved in the induction of autolysis and of competence for genetic transformation. These responses are expressed respectively in cultures entering the stationary phase and growing with exponential rates. Experimental virulence also appears to be modulated by the kinetics of calcium transport. Calcium transport in S. pneumoniae is electrogenic and shows sigmoidicity, indicating a cooperative mechanism with an inflexion point at 1 mM Ca2+. Mutant strains with Hill number values of 4 and 1, compared to 2 in the wild-type strain, were isolated. These changes were associated with altered regulation of competence and autolysis, and also with reduced experimental virulence. By contrast, they could not be related to a specific calcium requirement for growth. This indicates that the cooperativity of Ca2+ transport is not involved in vegetative growth, but rather regulates competence and autolysis. Competence and autolysis represent two growth-phase-dependent responses to an oligopeptide-activator exported to the medium, the competence-stimulating peptide. Addition of this activator to noncompetent cells, triggers net and transient 45Ca2+ influx. One effect of the activator might be to activate a calcium transporter by enhancing its cooperativity. In addition to an increase in intracellular calcium, a transient membrane depolarization induced by electrogenic calcium influx may be part of the signaling mechanism. The competence activator is a quorum-sensing molecule whose synthesis is autoregulated. This regulation might involve calcium-mediated signaling. As an extracellular pathogen, S. pneumoniae probably develops in niches with variable calcium concentration. Interestingly, virulence depends strongly upon the kinetics of Ca2+ transport. Regulation of calcium influx may represent a common mechanism of sensing the environment, if the Na+/Ca2+ exchanger is the target for external mediators including the competence activator.

Calcium↗
PubMed1999

Development of bacterial contamination during production of yeast extracts.

Baker's yeast suspensions having bacterial populations of 10(6) and 10(8) CFU/ml were subjected to autolysis processes designed to obtain yeast extracts (YE). The bacterial contaminants added to the yeast cell suspensions were produced with spent broths obtained from a commercial yeast production plant and contained 59% cocci (Leuconostoc, Aerococcus, Lactococcus) as well as 41% bacilli (Bacillus). Autolyses were conducted at four different pH levels (4.0, 5.5, 7.0, and 8.5) and with two autolysis-promoting agents (ethyl acetate and chitosan). Processing parameters were more important than the initial bacterial population in the development of contaminating bacteria during manufacture of YE. Drops in the viable bacterial population after a 24-h autolysis were observed when pH was adjusted to 4.0 or when ethyl acetate was added. A significant interaction was found between the effects of pH and autolysis promoters on the bacterial population in YE, indicating that the activity of ethyl acetate, as opposed to that of chitosan, was not influenced by pH.

Acetates↗