Changes In Protease Activity Research Articles

Strain-dependent differences in the regulatory roles of sarA and agr in Staphylococcus aureus.

The accessory gene regulator (agr) and the staphylococcal accessory regulator (sar) are central regulatory elements that control the production of Staphylococcus aureus virulence factors. To date, the functions of these loci have been defined almost exclusively using RN6390, which is representative of the laboratory strain 8325-4. However, RN6390 was recently shown to have a mutation in rsbU that results in a phenotype resembling that of a sigB mutant (I. Kullik et al., J. Bacteriol. 180:4814-4820, 1998). For that reason, it remains unclear whether the regulatory events defined in RN6390 are representative of the events that take place in clinical isolates of S. aureus. To address this issue, we generated mutations in the sarA and agr loci of three laboratory strains (RN6390, Newman, and S6C) and four clinical isolates (UAMS-1, UAMS-601, DB, and SC-1). Mutation of sarA in the cna-positive strains UAMS-1 and UAMS-601 resulted in an increased capacity to bind collagen, while mutation of agr had little impact. Northern blot analysis confirmed that the increase in collagen binding was due to increased cna transcription. Without exception, mutation of sarA resulted in increased production of proteases and a decreased capacity to bind fibronectin. Mutation of agr had the opposite effect. Although mutation of sarA resulted in a slight reduction in fnbA transcription, changes in the ability to bind fibronectin appeared to be more directly correlated with changes in protease activity. Lipase production was reduced in both sarA and agr mutants. While mutation of sarA in RN6390 resulted in reduced hemolytic activity, it had the opposite effect in all other strains. There appeared to be reduced levels of the sarC transcript in RN6390, but there was no difference in the overall pattern of sar transcription or the production of SarA. Although mutation of sarA resulted in decreased RNAIII transcription, this effect was not evident under all growth conditions. Taken together, these results suggest that studies defining the regulatory roles of sarA and agr by using RN6390 are not always representative of the events that occur in clinical isolates of S. aureus.

Enhancement of preservation characteristics of Meju, an intermediate material for Korean legume-based fermented soy sauce, Kanjang, by irradiation

Meju, an intermediate product for making Korean traditional soy sauce, Kanjang, was prepared and irradiated at 0, 5, 10 and 20 kGy and then stored at 25°C for 12 months (mo). Mould and Lactobacillus spp. were nearly eliminated by gamma irradiation with a dose of 5–10 kGy, and the Bacillus spp. was decreased by 4 decimal reduction with a dose of 10 kGy. Changes of protease activity, NH 3-nitrogen, NH 2-nitrogen, pH and color in the gamma-irradiated Meju were stable compared to non-irradiated control. Sensory evaluation after 12-mo storage showed that soy sauce made from the Meju irradiated with 10 and 20 kGy was acceptable and scored as same as the freshly made one. Therefore, it was considered that the Meju can be stored up to 12 mo by treating with gamma irradiation without any adverse quality change to make soy sauce.

Changes in protease activity and Cry3Aa toxin binding in the Colorado potato beetle: implications for insect resistance to Bacillus thuringiensis toxins

Widespread commercial use of Bacillus thuringiensis Cry toxins to control pest insects has increased the likelihood for development of insect resistance to this entomopathogen. In this study, we investigated protease activity profiles and toxin-binding capacities in the midgut of a strain of Colorado potato beetle (CPB) that has developed resistance to the Cry3Aa toxin of B. thuringiensis subsp. tenebrionis. Histological examination revealed that the structural integrity of the midgut tissue in the toxin-resistant (R) insect was retained whereas the same tissue was devastated by toxin action in the susceptible (S) strain. Function-based activity profiling using zymographic gels showed specific proteolytic bands present in midgut extracts and brush border membrane vesicles (BBMV) of the R strain not apparent in the S strain. Aminopeptidase activity associated with insect midgut was higher in the R strain than in the S strain. Enzymatic processing of toxin did not differ in either strain and, apparently, is not a factor in resistance. BBMV from the R strain bound ∼60% less toxin than BBMV from the S strain, whereas the kinetics of toxin saturation of BBMV was 30 times less in the R strain than in the S strain. However, homologous competition inhibition binding of 125I-Cry3Aa to BBMV did not reveal any differences in binding affinity ( K d∼0.1 μM) between the S and R strains. The results indicate that resistance by the CPB to the Cry3Aa toxin correlates with specific alterations in protease activity in the midgut as well as with decreased toxin binding. We believe that these features reflect adaptive responses that render the insect refractory to toxin action, making this insect an ideal model to study host innate responses and adaptive changes brought on by bacterial toxin interaction.

Defining a Link between Gap Junction Communication, Proteolysis, and Cataract Formation

Disruption of the connexin alpha 3 (Cx46) gene (alpha 3 (-/-)) in mice results in severe cataracts within the nuclear portion of the lens. These cataracts are associated with proteolytic processing of the abundant lens protein gamma-crystallin, leading to its aggregation and subsequent opacification of the lens. The general cysteine protease inhibitor, E-64, blocked cataract formation and gamma-crystallin cleavage in alpha 3 (-/-) lenses. Using a new class of activity-based cysteine protease affinity probes, we identified the calcium-dependent proteases, m-calpain and Lp82, as the primary targets of E-64 in the lens. Profiling changes in protease activities throughout cataractogenesis indicated that Lp82 activity was dramatically increased in alpha 3 (-/-) lenses and correlated both spatially and temporally with cataract formation. Increased Lp82 activity was due to calcium accumulation as a result of increased influx and decreased outflux of calcium ions in alpha 3 (-/-) lenses. These data establish a role for alpha 3 gap junctions in maintaining calcium homeostasis that in turn is required to control activity of the calcium-dependent cysteine protease Lp82, shown here to be a key initiator of the process of cataractogenesis.

Contribution of a metal-peroxide adduct to neurodegeneration is due to its oxidative protease activity.

Many hypotheses have been developed to explain aging and age-related neurodegenerative disorders; one of the most compelling is the role of oxidative stress to induce changes in protease activity in brains of patients of Alzheimer's disease and prion disease. At the moment however, there is no clear answer how protein degradation may be achieved in the brain. We have observed that several metal compounds can degrade proteins in the presence of hydrogen peroxide, and elucidated the reaction scheme based on the new theoretical point for the reactivity of a metal-peroxide adduct with eta 1-coordination mode. In this article we would like to point out the importance of a copper(II)-peroxide adduct to promote neurodegenerative diseases such as prion disease and amyotrophic lateral sclerosis through its oxidative protease function.

ACTIVITY OF CATHEPSIN G, ELASTASE, AND THEIR INHIBITORS IN PLASMA DURING METHANOL INTOXICATION

Methanol oxidation in the liver is accompanied by formation of formaldehyde and free radicals. These compounds can react with biologically active proteins, including proteolytic enzymes and their inhibitors. The activity of cathepsin G and elastase and their inhibitors such as alpha-1-antitrypsin and alpha-2-macroglobulin in plasma of rats given methanol orally in doses of 1.5, 3, and 6 g/kg was investigated for 7 days. The activity of cathepsin G and elastase was increased from 12 h to 5 d, proportionally to methanol dose. At the same time, activity of their inhibitors was reduced. Methanol ingestion in humans caused changes in activities of proteases and their inhibitors with similar direction as in rats. These changes in activity of proteases and their inhibitors produce significant disturbances in proteolytic-antiproteolytic balance after methanol administration.

Effect of gamma irradiation on proteases activities in Musca domestica.

The treatment of foodstuffs with relatively low doses of gamma(y)-irradiation to reduce infestation by stored product insect pests has become increasingly widespread in many countries (1). In attempting to determine optimum yirradiation dosages to effect the above where insecticideresistant and susceptible individuals may be present in a population, the question arises as to how susceptibility to yirradiation between insects of different species andor insecticide-resistant and susceptible strains may be biochemically determined; in particular whether this susceptibility may be mediated by proteolytic enzymes, which are responsible for intracellular protein catabolism in all cells. The rational for this hypothesis is as follows (a) previous work has shown a link between y-irradiation and insecticide susceptibility in the flour beetle (Tribolium castaneum), although biochemical mechanism was not elucidated (2); (b) Substantially increased activity levels for a range of protease types in a pesticide resistant strain ofM. domestica, compared to susceptible (3); (c) y-irradiation of mammalian tissue extracts in vitro resulted in reduced activities for a number of protease types (4). In order to hrther elucidate this hypothesis effect of the yirradiation on the biochemical changes in organisms, the objective of present work was therefore to compare changes in protease activities in insecticide resistant and susceptible strains of M. dornestica following y-irradiation. A DDT resistant (17bb) and a susceptible (Cooper) strains of M. domestica were selected for investigation. Insects (approx. 25) in nutrient impregnated containers were subjected to yirradiation for 3 hrs (equivalent to 300 k Radd3 k Gray). This irradiation dosage was chosen to fall within the range ( m a 10 k Gray) normally used for irradiation for insect disinfection Samples of resistant and susceptible insects were placed in identical containers for 3 hrs, but not irradiated. AAer irradiation, insects were immediately frozen and stored at 40°C. After thawing insects were homogenized and proteolytic enzymes assays were carried out as described previously (5). The activity levels of cytoplasmic and lysosomal proteases in insects of resistant and susceptible strains following exposure to y-irradiation is shown in Table 1 For both the DDT resistant and susceptible strains of M. domestrcu, it is apparent that there was no significant difference in activity levels for any of the cytoplasmic and lysosomal proteases between irradiated and non-irradiated insects (note the increased levels of protease activity in 17bb strain compared to Cooper, as reported previously (3). We conclude from the above data (a) there is no difference in susceptibility to inactivation by y-irradiation for any protease type in 17bb and Cooper strains of M. domesticu; (b) there is no evidence for a difference in susceptibility to irradiation between DDT resistant and susceptible strains. Table I Protease actlvltles in 17bb and Coooer Stram of M donit!st~ca L following r i d a t i o n

Amniotic membrane collagen content and type distribution in women with preterm premature rupture of the membranes in pregnancy.

Amniotic membrane samples were taken following preterm delivery from 15 women with preterm prelabour rupture of the membranes (PPROM) and 14 with unrelated reasons, and from 25 delivering at term. Fibrillar collagens were extracted and analysed by interrupted gel electrophoresis. Collagens were decreased in PPROM relative to controls, but showed similar sub-type distribution, with one exception. In this case an increased ratio of Type I to Type III collagen was observed, with normal collagen content. In conclusion, PPROM is accompanied by generalised reduction in amnion collagen content, involving all fibrillar sub-types monitored. This may reflect general changes in protease activity or in collagen expression in affected membranes.

Profiles of insulin-like growth factor binding proteins and the protease activity in the maternal circulation and its local regulation between placenta and decidua.

Insulin-like growth factors (IGF) and their specific binding proteins (IGFBPs) are believed to be important regulators of fetal growth. IGFBP protease which proteolyzes IGFBPs and changes the biochemical properties of IGFBPs is also presumed to be involved in fetal growth. The aim of this study is to elucidate the physiological significance of IGFBP protease in fetal growth and regulators of protease in placenta and decidua. The intact IGFBP-3 was proteolyzed into fragments when pregnant serum was incubated with 125I-IGFBP-3. IGFBP-3 protease activity showed a marked increase at 5 weeks of gestation and reached a plateau in maternal circulation at 15 weeks of gestation. These changes in protease activity correlated with the profiles of IGFBPs in the maternal circulation analyzed by Western ligand blot, where the IGFBP-1 is only the dominant IGFBP. The intact IGFBP-3 was proteolyzed when culture media of decidual cells were incubated with 125I-IGFBP-3, but was not proteolyzed when culture media of trophoblast cells were incubated with 125I-IGFBP-3. Decidual protease activity was slightly increased by IGF-I and completely inhibited by progesterone. The protease activity was more increased in the mothers with growth retarded infant than in those in the mothers with normal growth infants, suggesting that the protease activity is elevated in compensation for the impaired fetal growth. These results suggest that increased protease activity in maternal blood may be involved in the fetal growth indirectly by reducing the binding activity of IGFBP-3 to IGF-I, and that protease activity in maternal blood may be derived from decidua that is regulated by placental hormones.

Changes in brain protease activity in aging.

We measured changes in protease activity with aging, conducting assays of cathepsin D and calpain II activities and the rate of degradation of cytoskeletal proteins, preparing the enzymes and substrates from young and aged brains. Calpain preparations added to the young and to the aged substrates were standardized with casein as substrate so that age-related changes in calpain specificity and substrate susceptibility were measured. Several age-related differences were observed in substrate susceptibility and in enzyme activity. With respect to substrate, the neurofilament protein from young animals was somewhat more susceptible to calpain action than that from older animals. With respect to enzyme activity, calpain from aged brain cleaved neurofilament protein at a faster rate than did calpain from young. With neurofilaments, the most rapid breakdown usually occurred when enzyme from aged tissue was incubated with substrate from young. Kidney enzyme of aged rats incubated with neurofilament substrate of aged rats resulted in a more rapid breakdown than enzyme of young kidney incubated with substrate of young. The age dependence of tubulin breakdown was somewhat different from that of neurofilament breakdown. The most rapid breakdown usually occurred when using enzyme from young with tubulin from young. Incubation of neurofilament protein or tubulin with cathepsin D did not reveal any differences with aging. These studies suggest that an increase in enzyme activity observed previously during aging may also include changes in the properties of the enzyme (substrate specificity) and/or in the properties of their endogenous substrates (susceptibility to breakdown).

Metabolic adaptations supporting anoxia tolerance in reptiles: Recent advances

Animal survival during severe hypoxia and/or anoxia is enhanced by a variety of biochemical adaptations including adaptations of fermentative pathways of energy production and, most importantly, the ability to sharply reduce metabolic rate by 5–20 fold and enter a hypometabolic state. The biochemical regulation of metabolic arrest is proving to have common molecular principles that extend across phylogenetic lines and that are conserved in different types of arrested states (not only anaerobiosis but also estivation, hibernation, etc.). Our new studies with anoxia-tolerant vertebrates have identified a variety of regulatory mechanisms involved in both metabolic rate depression and in the aerobic recovery process using as models the freshwater turtle Trachemys scripta elegans and garter snakes Thamnophis sirtalis parietalis. Mechanisms include: 1) post-translational modification of cellular and functional proteins by reversible phosphorylation and changes in protein kinase (PKA, PKC) and/or phosphatase activities to regulate this, 2) reversible enzyme binding associations with subcellular structural elements, 3) differential gene expression and/or mRNA translation producing new mRNA variants and new protein products, 4) changes in protease activity, particularly the multicatalytic proteinase complex, and 5) both constitutive and anoxia-induced modifications to cellular antioxidant systems to deal with oxidative stress during the anoxic-aerobic transition of recovery.

Chilling temperature storage induces changes in protein patterns and protease activity in cherimoya fruit

Storage at 6 °C inhibited the ripening process and caused severe damage in cherimoya fruit (Annona cherimola Mill. cv. ‘Fino de Jete’). In the present study, we analyzed the modifications in protein pattern, free amino acid content and protease activity of cherimoyas during storage at this chilling temperature. SDS-PAGE analysis revealed non-accumulation of some polypeptides related to the ripening process due to storage at 6 °C, and two-dimensional electrophoresis confirmed the appearance of specific low-temperature polypeptides. While many polypeptides observed in freshly harvested fruit persisted during storage, several acid polypeptides were detected only during the first few days of storage at 6 °C. A substrate-dependent change in protease activity was also found in fruit under chilling temperature storage, as compared to ripening fruit. After a decrease to barely detectable levels during the early phase of cold storage, the proteolytic activity then increased, mainly hydrolizing endogenous proteins to free amino acid components.

Effect of phosphate on protease activity of senescing rice leaves

Effect of phosphate on proteolysis of senescing leaf segments of rice ( Oryza sativa L. cv Chilsung) seedlings was studied. Sodium phosphate buffer, pH 7, promoted proteolysis of the leaves and amino acid efflux into the medium. The buffer increased the activity of proteases against ribulose-1,5-bisphosphate carboxylase/oxygenase, whereas it inhibited the activity against hemoglobin. Incubation of leaf segments in phosphoric acid promoted proteolysis, suggesting that phosphate played a role in increasing proteolysis. However, the difference in the extent of proteolysis between sodium phosphate or sodium phosphate-citric acid buffer and phosphoric acid suggested that Na + also played a role toward the increased proteolysis of the buffer-incubated leaves. All three phosphate-containing compounds exhibited similar changes in protease activity against hemoglobin throughout the incubation.

Peptidases are affected differently in neo-cortical regions of brains from patients with Alzheimer's disease: D. Terwel, M. Markerink, J. Jolles, Department of Neuropsychology and Psychobiology; University of Limburg, P.O. Box 616, 6200 MD Maastricht, The Netherlands

Although it is recognized that changes in protease activities may be involved in the etiology of pathological changes in the human brain, there have been few studies on normal and pathological protein

Peptidases Are Affected Differently in Neocortical Regions of Brains from Patients with Alzheimer's Disease

Although it is recognized that changes in protease activities may be involved in the etiology of pathological changes in the human brain, there have been few studies on normal and pathological protein catabolism. In the present study the activities of major cortical aminopeptidase and prolylendopeptidase were determined in frontal and medial temporal cortical samples of brains from patients with Alzheimer''s disease (AD) and control patients matched for age. Prolylendopeptidase activity was reduced by 45–50% in the frontal and medial temporal cortex of AD patients as compared with controls, whereas major aminopeptidase activity was unaltered. The possibility that the changes in peptidase activities in AD are age-dependent was studied in postmortem neocortical samples from 18 AD patients who had died between 55 and 90 years of age. Prolylendopeptidase activity was lower in patients who died at a relatively early age than in patients who died at a relatively old age. This underscores the notion that, with respect to age, there may be heterogeneity in neurochemical changes in AD.

Extracellular protease production by Drosophila imaginal discs

We are investigating the role of extracellular proteases in imaginal disc eversion to understand the mechanism that controls cell rearrangements within epithelia. We have identified three cation-dependent neutral proteases released by Drosophila leg discs everting in culture. Serine protease inhibitors block disc eversion and inhibit activity of disc proteases. The pattern of extracellular proteases changes when eversion is blocked with added protease inhibitors. Changes in protease activity occur when released disc proteases are treated with trypsin. Trypsin treatment of intact imaginal discs releases protease and inhibitor activities to the medium, indicating their presence on the cell surface before release. Our results suggest that extracellular proteases are required for imaginal disc morphogenesis and are regulated by more than one mechanism.

Changes in Protease Activity in Leaves during Natural Development and Accelerated Ageing upon Detachment

Summary When detached oat leaves were aged in darkness, loss of protein and chlorophyll was similar whether leaf segments were placed horizontally in Petri dishes on wet filter paper, or held vertically in plastic holders with their bases in water. In horizontally placed leaves, the activities of the two proteases tended to increase but in leaves kept vertically protease activities decreased as in attached leaves. In light, both chlorophyll and protein loss occurred at about half the rate observed in darkness. However, in vertically held leaves the acidic protease started to increase by day 2, whereas the neutral protease first declined but increased later. These increases were not mimicked by incubation on sucrose in darkness and neither could they be attributed to a greater accumulation of free amino acids in the light. Kinetin and, to a lesser extent zeatin, maintained chlorophyll and protein levels and counteracted both the increases and decreases in protease activities observed during ageing in light or darkness, respectively. Since accelerated loss of protein in darkness was not accompanied by increases in protease activities, these observations are at variance with those of Martin and Thimann (Plant Physiol. 49: 64, 1972) and suggest that additional synthesis of the major proteases is not a prerequisite for protein breakdown during senescence.

Perinatal leupeptin retards subsequent acquisition of a visual discrimination task in chicks

Recent discoveries that calcium sensitive proteases (calpains) may play an important role in memory led to investigation of the effects of leupeptin, a calpain inhibitor, on learning. Intracerebral injections of either 0.9% saline, 80 micrograms aprotinin, 306 micrograms glutamate, or 8 or 80 micrograms leupeptin were administered to 95 two-day-old chicks. On Day 10 posthatch, animals were then tested on a task requiring discriminations between pebbles and food pellets. Chicks receiving 8 or 80 micrograms leupeptin or 306 micrograms glutamate were found to be deficient in acquiring the task. Aprotinin, a control serine protease inhibitor, did not retard discrimination learning. None of the drugs tested altered body weight or gross visual skills. These results suggest that retardation produced by leupeptin is not due to nonspecific changes in protease activity, visual competency, or growth. The reported results confirm earlier reports of leupeptin-induced memory impairments in rats.

Purification and characterization of a cytosol protease from dormant cysts of the brine shrimp Artemia.

A thiol protease has been isolated and purified from the postribosomal fraction of encysted embryos of the brine shrimp Artemia using a six-step procedure. The purified enzyme has a molecular weight of 55,000 +/- 4,200 and is composed of subunits of Mr 31,500 +/- 559 and 25,867 +/- 1,087. Isoelectric focusing revealed two discrete bands, one at pH 4.6 and the other at pH 5.1. The protease appears to be a member of the thiol group of proteases based on its inhibition by leupeptin, antipain, chymostatin, Ep-475, and several other thiol protease inhibitors. The enzyme was stimulated by heavy metal chelators and thiol reagents. At pH 3.5-4.0 the thiol protease hydrolyzed a wide range of proteins including bovine serum albumin, hemoglobin, Artemia embryo soluble proteins, Artemia lipovitelline, and protamine, whereas at pH 6.0-6.5 the enzyme showed a high degree of specificity for Artemia elongation factor 2 and lipovitelline alpha 1. The total amount of protease activity in crude homogenates of Artemia embryos decreased by about 50% during the first 24 h of development, while the amount of free, active enzyme decreased proportionally for 9 h of development then remained constant during the next 26-27 h of development. These changes in protease activity appear to reflect changing levels of an endogenous protease inhibitor during development.

Effect of pregnancy and diabetes on myofibrillar protease activity in maternal and fetal muscles.

The activity of protease was investigated by cleavage of 14C-labelled globin by the isolated myofibrillar fraction of maternal and fetal muscles of nonpregnant, pregnant, and pregnant diabetic rats, on days 15 and 20 of pregnancy. Diabetes was produced by i.p. streptozotocin injection on day 5 or 12 of pregnancy. There was no change in muscle protease activity on day 15 of pregnancy in nondiabetic rats, compared to nonpregnant control rats, whereas on day 20 protease activity rose in the gastrocnemius by 25%, in the diaphragm by 30%, and only slightly in the heart. Diabetes, imposed during days 12-20 of pregnancy, induced a further increase in protease activity in the gastrocnemius (42%), diaphragm (47%), and heart (25%). In rats with diabetes lasting from day 5 to 20 of gestation there were only slight further increases in protease activity. In contrast, maternal diabetes did not induce changes in myofibrillar protease activity of fetal leg or heart muscles compared with the corresponding muscles of fetuses of nondiabetic rats. The increased protease activity on the maternal side occurred at low insulin levels and/or reduced insulin sensitivity, whereas the absence of changes in protease activity on the fetal side was associated with sustained or even increased presence of insulin. This suggests that the adaptive changes in protease activity, in both pregnancy and superimposed diabetes, are related to insulin availability and its effectiveness.