Specific Proteolytic Activities Research Articles

Extraction of Crude Bromelain from Pineapple (<em>Ananas comosus</em> L.) Fruit Waste and it’s <em>in vitro</em> Protein Digestibility

Purpose: Crude bromelain is abundant in pineapple fruit waste and is possible to use in shrimp farming. The objectives of this present study were to investigate extraction conditions and to evaluate in vitro protein digestibility of the shrimp feed supplemented with the crude bromelain extract.Research Method: Crude bromelain was extracted from pineapple fruit waste with either distilled water or tap water and the pH was adjusted, ranging from 5 to 9. The different ripeness stages and weight ratios of waste to water were further investigated. The highest proteolytic and specific proteolytic activities of the extract were selected for suitable extraction conditions. The in vitro protein digestibility of soybean meal and shrimp feed pretreated with crude bromelain extract was evaluated to determine their feasibility to use in shrimp farming.Findings: The crude bromelain derived from the pH 7 preboiled tap water extract exhibited the highest values of both proteolytic activities. The weight ratio of fruit waste to water of 1:0.5 and a ripeness stage of more than 50% yellow peel were the conditions suitable to obtain the highest values of both activities. The in vitro protein digestibility of shrimp feed pretreated with crude bromelain was 68.68 ± 2.70 %, which indicated promotion capability for protein digestion in shrimp during shrimp farming.Research Limitations: A suitable crushing tool for pineapple peel was not available. Therefore, the fragments after crushing the peel were not consistent. Originality/Value: The crude bromelain can be extracted with tap water and used to supplement shrimp feed for the promotion of digestibility.

Local Colonic Administration of a Serine Protease Inhibitor Improves Post-Inflammatory Visceral Hypersensitivity in Rats

Dysregulation of the protease–antiprotease balance in the gastrointestinal tract has been suggested as a mechanism underlying visceral hypersensitivity in conditions such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). We aimed to study the potential therapeutic role of an intracolonically administered serine protease inhibitor for the treatment of abdominal pain in a post-inflammatory rat model for IBS. An enema containing 2,4,6-trinitrobenzene sulfonic acid (TNBS) was used to induce colitis in male Sprague–Dawley rats, whereas controls received a saline solution. Colonoscopies were performed to confirm colitis and follow-up mucosal healing. In the post-inflammatory phase, the serine protease inhibitor UAMC-00050 (0.1–5 mg/kg) or its vehicle alone (5% DMSO in H2O) was administered in the colon. Thirty minutes later, visceral mechanosensitivity to colorectal distensions was quantified by visceromotor responses (VMRs) and local effects on colonic compliance and inflammatory parameters were assessed. Specific proteolytic activities in fecal and colonic samples were measured using fluorogenic substrates. Pharmacokinetic parameters were evaluated using bioanalytical measurements with liquid chromatography–tandem mass spectrometry. Post-inflammatory rats had increased trypsin-like activity in colonic tissue and elevated elastase-like activity in fecal samples compared to controls. Treatment with UAMC-00050 decreased trypsin-like activity in colonic tissue of post-colitis animals. Pharmacokinetic experiments revealed that UAMC-00050 acted locally, being taken up in the bloodstream only minimally after administration. Local administration of UAMC-00050 normalized visceral hypersensitivity. These results support the role of serine proteases in the pathophysiology of visceral pain and the potential of locally administered serine protease inhibitors as clinically relevant therapeutics for the treatment of IBS patients with abdominal pain.

Secretion of Proteinases with Fibrinolytic Activity by Micromycetes of the Genus Aspergillus

Proteolytic activity of extracellular enzymes of 11 strains of different Aspergillus species was studied. Comparison of the enzymatic indices of strains grown on agar medium containing either casein or fibrin allowed the selection of the strain Aspergillus terreus 2 as a promising producer of fibrinolytic proteases. It was found that A. terreus 2 proteinases demonstrated maximum activity at pH 8.0. The highest values of fibrinolytic and total proteolytic activities expressed in UTyr (amount of micromoles of tyrosine released from fibrin or casein for 1 min) were 34.0 and 358.3, respectively. Maximum activities were detected when growing the producer on a medium containing only amine nitrogen sources (fish flour hydrolysate and peptone); however, the amount of extracellular protein and the specific fibrinolytic and total proteolytic activities were greater in the medium containing both mineral and amine nitrogen sources (fish flour hydrolysate and sodium nitrate) than in the medium containing only fish flour hydrolysate and peptone as nitrogen sources.

Interspecies dynamics among bacteria associated with canine periodontal disease.

The etiology and pathogenic mechanisms associated with canine periodontal disease are less well understood than the disease in humans. In this study we have reconstructed defined consortia biofilms in vitro of microorganisms identified as prevalent in a same-breed cohort of dogs with or without periodontal disease. Frederiksenia canicola and Neisseria canis were selected as potential early colonizers of salivary pellicle, and Fusobacterium nucleatum and Porphyromonas gulae were included as high incidence canine oral bacteria. N. canis formed a biofilm substratum under aerobic conditions, but was unable to tolerate anaerobic conditions. Fr. canicola exhibited synergistic biofilm growth with Po.gulae under anaerobic conditions, but displayed an antagonistic relationship with Fu.nucleatum. However, strong co-adhesion between Fu.nucleatum and Po.gulae was able to overcome the inhibitory effects of Fr.canicola to facilitate three-species biofilm formation. Parvimonas micra, an anaerobic, asaccharolytic Gram-positive coccus found only under disease conditions in vivo, was able to form biofilms in conjunction with Fr.canicola and Po.gulae. Furthermore, the specific proteolytic activities of biofilms containing Fr.canicola and Po.gulae or Fu.nucleatum and Po.gulae were increased several-fold upon the addition of Pa.micra. This suggests that anaerobic cocci such as Pa.micra might provide a catalyst for progressive tissue destruction, inflammation and alveolar bone loss in canine periodontal disease, in keeping with the keystone-pathogen hypothesis.

Regulation of Proteasomal Catalytic Activity by Altering its Protein-Protein Interactions

The proteasome is a central protease of the main intracellular pathway responsible for regulated proteolysis that uses polyubiquitinylation for substrate tagging. The basic functional unit of proteasome called 20S or catalytic particle (CP) is a tube built from four stacked heptameric rings with 3 pairs of catalytic subunits equipped with specific proteolytic activities. The catalytic sites face inside of the tube and are additionally secured by the gates on the tubes’ alpha faces and blocking the access to the CP interior. To get to the active centers, substrates have to bypass the gate, which closed state is affected by binding ligands to the alpha face or catalytic sites. To gain capability to digest polyubiquitinylated substrates, the CP binds a regulatory particle (RP) or a 19S cap to least one site of the tube. The interface between CP and RP allosterically controls dynamics of substrates uptake, digest readiness, and efficiency and specificity of the active centers. The CP also wears other types of caps that allow it to digest efficiently particular populations of substrates and generate certain types of products. Also in these cases the interface between the protein complexes plays a crucial regulatory role. Here we designed a range of peptides, peptidomimetics and small molecule compounds that affect these protein – protein interactions to study selected mechanical aspects of proteasomal regulation. Using spectroscopic and microscopic tools we found that binding of certain ligands may affect status of the proteasomal gate, alpha face conformation, stability of the complexes or/and the CP dynamics. These structural changes alter the enzyme activity and specificity, which in turn influence intracellular metabolism. We attempted to correlate particular biological effects of the ligands with their mode of molecular action and their chemical properties.

Endogenous proteolytic enzymes – A study of their impact on cod (Gadus morhua) muscle proteins and textural properties in a fermented product

The aim of this study was to investigate endogenous proteolytic activities in a cod product and their impact on muscle proteins and textural properties during fermentation and storage. The result of specific proteolytic activities showed that cathepsins, especially cathepsin B, had the highest activities during fermentation and storage. SDS–PAGE indicated more degradation of myofibrillar proteins by cathepsin L than other proteases and that the hydrolysis by cathepsins was pronounced in the last stage of fermentation. Texture analysis showed that cathepsins had a negative impact on gel strength and this impact increased in the last stage of fermentation. However the product still had a firm texture. During storage (4°C) for one week, no significant changes were seen in the gel strength. In conclusion, cathepsins had more impact on muscle proteins and textural properties than other proteases during fermentation but had little impact on gel strength during storage at 4°C.

Digestive proteolytic activity in Apodiphus amygdali Germar (Hemiptera: Pentatomidae): effect of endogenous inhibitors

The digestive proteolytic profile of <em>Apodiphus amygdali</em> was determined by using several substrates and specific inhibitors. Analysis of optimal pH and temperature showed the highest enzymatic activity at the pH range of 6-7 and temperature of 40°C when azocasein was used as a substrate. By using a negative control, the presence of several specific proteases were determined including tryspin-like, chymotrypsinlike, elastase, cathepsin B, cathepsin L, amino- and carboxypeptidases in the midgut content of <em>A. amygdali</em>, with the highest and the lowest activities of cathepsin L and carboxypeptidase, respectively. pH dependency of specific proteases revealed optimal pHs of 9, 8 and 9 for trypsin-, chymotrypsin-like, 6 for cathepsins and 5-6 for carboxy- and aminopeptidases, respectively. Specific inhibitors, including phenylmethylsulfonyl fluoride, Na-p-tosyl-L-lysine chloromethyl ketone, Ntosyl- L-phenylalanine chloromethyl ketone, L-trans-epoxysuccinylleucylamido-(4-guanidino)-butane, phenanthroline and ethylendiamidetetraacetic acid, significantly decreased proteolytic activity, indicating the presence of different proteases in the midgut of <em>A. amygdali</em>. Extracted inhibitors from the midgut demonstrated significant inhibition of specific proteolytic activities of <em>A. amygdali</em> except for cathepsin B and aminopeptidase. The results indicated that determination of digestive proteolytic activity could be helpful to clarify digestion process in insects. Moreover, understanding the nature of digestive proteases might be used to develop several inhibitors for providing resistant crop varieties against pests.

Effects of a Lectin from Polygonum Persicaria L. on Pieris Brassicae L. (Lepidoptera: Pieridae)

Abstract In the current study, the entomotoxic effects of a lectin, extracted from Polygonum persicaria, were determined on Pieris brassicae larvae. The purified molecule significantly decreased larval survival, weight, and mass protein. Different concentrations of P. persicaria agglutinin (PPA) (0.5, 1 and 2 mg/ml) significantly decreased nutritional indices including: the efficiency of conversion of ingested food (ECI), the efficiency of conversion of digested food (ECD), and approximate digestibility (AD) in a dose-dependent manner. There was a significant decrease in larval α-amylase activity after larval fed on PPA. The decreased activity was found in both the biochemical assessment and gel electrophoresis; sharpness of one isozyme decreased and another completely disappeared. Meanwhile, PPA significantly decreased activities of glucosidases mainly β-glucosidase. Fed of P. brassicae larvae on different concentrations of PPA, significantly decreased activity of TAG-lipase in both the biochemical assessment and gel electrophoresis. It was found that alkaline phosphatase (ALP) and acid phosphatase (ACP) were also affected by PPA but concentrations of 1 and 2 mg/ml showed statistical differences compared to the control. General and specific proteolytic activities statistically decreased after larvae fed on different concentrations of PPA. Results of our study could bring attention to this molecule, showing it to be a safe and efficient control of the pest worldwide.

Micropropagation of Hohenbergia penduliflora (A. Rich.) Mez. for sustainable production of plant proteases

Hohenbergia penduliflora (A. Rich.) Mez. inhabits the protected ecological area of Cunagua Highland, Ciego de Avila, Cuba. The availability of this plant for experimental purposes is exceedingly limited. Tissue cultured plants of this specie, would be useful for propagation purposes. Experiments were carried out to optimize the micropropagation process from the disinfection of fruits to ex vitro hardening of regenerated plantlets. The best results were obtained when seeds were disinfected with 2 % (v:v) sodium hypochlorite for 20 min and placed in vitro for 45 days for seed germination. Tissue cultured shoots (1 cm) with vertical wounds in the basal region (5 mm long) were placed in a medium containing Murashige and Skoog (MS) salts, 100 mg l−1 myo-inositol, 0.1 mg l−1 thiamine-HCl, 30 g l−1 sucrose, 8.8 μM 6-benzyladenine (BA) and 1.6 μM naphthalene acetic acid (NAA). Shoots were proliferated for 45 days to obtain 8.21 new shoots per explant; they were subsequently divided and rooted on a medium containing 1.6 μM NAA for 30 days. For ex vitro hardening, plastic trays containing 82 cm3 of filter-cake-sugarcane ashes were used; 100 % survival rate was recorded. After 6 months of hardening, plants were established ex vitro and ready for protease extraction. Comparisons between protein contents, proteolytic activities and specific proteolytic activities of extracts from stems of macro- and micropropagated plants were acquired. Tissue cultured stems showed statistically lower figures which is why Ethrel was tested here to increase proteolytic activity in micropropagated plant stems. After Ethrel applications, protein contents, proteolytic activities and specific proteolytic activities of extracts from stems were the three main indicators recorded. However, other biochemical effects of Ethrel were also evaluated, such as, levels of chlorophyll pigments, malondialdehyde and other aldehydes; and superoxide dismutase, and guaiacol peroxidase activities. Rising concentrations of Ethrel (0, 1.5, 3.0, 4.5 and 6.0 mg l−1) decreased protein contents at 72 h but increased proteolytic and specific proteolytic activities of stem extracts. Ethrel was effective in increasing proteolytic activity in in vitro culture-derived plant stems, at a level higher than in field-grown plant stems. Moreover, Ethrel increased superoxide dismutase and guaiacol peroxidase-specific activities in leaves; and decreased chlorophyll pigments. Ethrel did not affect levels of malondialdehyde and other aldehydes.

Subcellular Distribution and Dynamics of Active Proteasome Complexes Unraveled by a Workflow Combining in Vivo Complex Cross-Linking and Quantitative Proteomics

Through protein degradation, the proteasome plays fundamental roles in different cell compartments. Although the composition of the 20S catalytic core particle (CP) has been well documented, little is known about the composition and dynamics of the regulatory complexes that play a crucial role in its activity, or about how they associate with the CP in different cell compartments, different cell lines, and in response to external stimuli. Because of difficulties performing acceptable cell fractionation while maintaining complex integrity, it has been challenging to characterize proteasome complexes by proteomic approaches. Here, we report an integrated protocol, combining a cross-linking procedure on intact cells with cell fractionation, proteasome immuno-purification, and robust label-free quantitative proteomic analysis by mass spectrometry to determine the distribution and dynamics of cellular proteasome complexes in leukemic cells. Activity profiles of proteasomes were correlated fully with the composition of protein complexes and stoichiometry. Moreover, our results suggest that, at the subcellular level, proteasome function is regulated by dynamic interactions between the 20S CP and its regulatory proteins-which modulate proteasome activity, stability, localization, or substrate uptake-rather than by profound changes in 20S CP composition. Proteasome plasticity was observed both in the 20S CP and in its network of interactions following IFNγ stimulation. The fractionation protocol also revealed specific proteolytic activities and structural features of low-abundance microsomal proteasomes from U937 and KG1a cells. These could be linked to their important roles in the endoplasmic reticulum associated degradation pathway in leukemic cells.

Adaptive differences in digestive enzyme activity in the crab Neohelice granulata in relation to sex and habitat

Neohelice granulata (Dana, 1851) is a semi-terrestrial burrowing crab that inhabits estuaries and saltmarshes feeding on grasses or sediment depending on the microhabitat they occupy (vegetated saltmarsh or bare mudflat). Specific cellulolytic, amylolytic, and proteolytic enzyme activities were analyzed in midgut gland homogenates of males and females from each microhabitat fed in the laboratory with Spartina densiflora leaves and sediment, respectively, in order to detect sex, food and microhabitat related differences. The presence of β-1,4-glucosidase, endo-β-1,4-glucanase, α amylase, trypsin and chymotrypsin were confirmed. Specific cellulolytic activity was higher in crabs fed on leaves than in those fed on sediment or in no fed controls and variable differences between sexes were observed. Specific amylase activity of crabs fed on leaves was the lowest recorded. Trypsin and chymotrypsin specific activities were higher in saltmarsh crabs fed on leaves than in mudflat crabs fed on sediment. Different mechanisms of enzyme regulation to explain the observed differences among groups were suggested. Additionally, differences between sexes suggest different metabolic needs related to gonad maturation. It is concluded that N. granulata has the ability to adapt digestive enzyme production to support its physiological and metabolic needs based on the different food sources available at each microhabitat.

Proteolytic Activity Matrix Analysis (PrAMA) for simultaneous determination of multiple protease activities.

Matrix metalloproteinases (MMPs) and A Disintegrin and Metalloproteinases (ADAMs) are two related protease families that play key roles in matrix remodeling and growth factor ligand shedding. Directly ascertaining the proteolytic activities of particular MMPs and ADAMs in physiological environments in a non-invasive, real-time, multiplex manner remains a challenge. This work describes Proteolytic Activity Matrix Analysis (PrAMA), an integrated experimental measurement and mathematical analysis framework for simultaneously determining the activities of particular enzymes in complex mixtures of MMPs and ADAMs. The PrAMA method interprets dynamic signals from panels of moderately specific FRET-based polypeptide protease substrates to deduce a profile of specific MMP and ADAM proteolytic activities. Deconvolution of signals from complex mixtures of proteases is accomplished using prior data on individual MMP/ADAM cleavage signatures for the substrate panel measured with purified enzymes. We first validate PrAMA inference using a compendium of roughly 4000 measurements involving known mixtures of purified enzymes and substrates, and then demonstrate application to the live-cell response of wildtype, ADAM10-/-, and ADAM17-/- fibroblasts to phorbol ester and ionomycin stimulation. Results indicate PrAMA can distinguish closely related enzymes from each other with high accuracy, even in the presence of unknown background proteolytic activity. PrAMA offers a valuable tool for applications ranging from live-cell in vitro assays to high-throughput inhibitor screening with complex enzyme mixtures. Moreover, our approach may extend to other families of proteases, such as caspases and cathepsins, that also can lack highly-specific substrates.

Digestive proteolytic and amylolytic activities of Helicoverpa armigera in response to feeding on different soybean cultivars

Digestive proteolytic and amylolytic activities of the larvae of Helicoverpa armigera (Hübner) fed either on artificial diet or on different soybean cultivars (356, M4, M7, M9, Clark, Sahar, JK, BP, Williams, L17, Zane, Gorgan3 and DPX) and response of the larvae to feeding on some soybean-based protease inhibitors were studied. The highest general and specific proteolytic activities were in artificial-diet-fed larvae. Although the highest general proteolytic activity was in the larvae fed on L17, M4 and Sahar cultivars, the lowest tryptic activity was on L17 and Sahar, which may be due to the presence of some serine protease inhibitors in these two cultivars, resulting in hyperproduction of chymotrypsin- and elastase-like enzymes in response to the inhibition of these enzymes. The highest amylolytic activity was on M4, and the lowest was on Williams and DPX. General proteolytic activity of SKTI-fed larvae was the highest compared with SBBI- and STI-fed larvae. The findings demonstrated that the cultivars L17 and Sahar were partially resistant to this pest, probably because of some secondary chemicals or proteinaceous protease inhibitors of these cultivars.

Role of central and peripheral aminopeptidase activities in the control of blood pressure: a working hypothesis

Although there is a large body of knowledge on protein synthesis, the available data on protein catabolism, although quite substantial, are still inadequate. This is due to the marked differences in the activity of proteolytic enzymes, compounded by different substrate specificities and multiple environmental factors. Understanding enzyme behavior under physiological and pathological conditions requires the identification of specific proteolytic activities, such as aminopeptidases, as able to degrade certain peptidergic hormones or neuropeptides. Another requirement is the isolation, purification and characterization of the enzymes involved. In addition, systematic studies are needed to determine each enzyme's subcellular location, tissue distribution, and the influence of environmental factors such as diurnal rhythm, age, gender, diet, cholesterol, or steroids. Central and peripheral aminopeptidases may play a role in the control of blood pressure by coordinating the effect of the different peptides of the renin-angiotensin system cascade, acting through the AT(1), AT(2), and AT(4) receptors. Our review of the available data suggests the hypothesis that cholesterol or steroids, particularly testosterone, significantly influence aminopeptidase activities, their substrate availability and consequently their functions. These observations may have relevant clinical implications for a better understanding of the pathophysiology of cardiovascular diseases, and thus for their treatment with aminopeptidase inhibitors.

Intermediate-type 20 S Proteasomes in HeLa Cells: “Asymmetric” Subunit Composition, Diversity and Adaptation

The 20 S proteasomes are cylinder-shaped heteromeric dimers with a subunit configuration of α7, β7, β7, α7. Replacement of the three active site-containing standard β-subunits (β1, β2, β5) by immuno-β-subunits (β1i, β2i, β5i) results in formation of 20 S immuno-proteasomes, while only partial replacement leads to intermediate-type proteasomes. Synthesis of immuno-subunits can be induced by interferon-γ, which causes a complete transformation of three subtypes of standard proteasomes into three subtypes of intermediate-type proteasomes in HeLa cells, a process that results in a change in the proteolytic activities of the enzymes. HeLa cells producing the proteasome β1-subunit tagged with the Fc region-binding ZZ domain of protein A were grown in the presence of interferon-γ. From these cells, we have purified 20 S proteasomes by using IgG-affinity resin and analysed them by 2D PAGE. Our study showed that subunit replacement can be confined to one half of the proteasome cylinder, resulting in the formation of intermediate-type proteasomes with ”asymmetric” subunit composition. Analysis of proteasomes purified from the cytoplasm, nucleoplasm, and microsomes of HeLa S3 cells reveals that all three compartments are furnished with intermediate-type proteasomes of different subtype and subunit composition, exhibiting different specific proteolytic activities.

Digestive enzymes during development of Ceratitis capitata (Diptera:Tephritidae) and effects of SBTI on its digestive serine proteinase targets

The digestive system of Ceratitis capitata was characterized during its larval development and in the insect stage. Disaccharidases against maltose and sucrose were more evident in the 2nd and 3rd day of larval development and in the adult stage, respectively. Glycosil-hydrolyses with higher specific α-galactosidasic and β-galactosidasic activities were detected in the 2nd and 3rd day of the larval stage, respectively. Specific proteolytic activities against azocasein showed an increase in the 4th and 5th day of the larval stage and in the adult stage. Specific hemoglobin activities were constant between 2nd and 6th day of the larval stage. The larvae used mainly serine proteinases, such as trypsin/chymotrypsin, and the adult insects only chymotrypsin-like enzymes in their digestive process. Two serine proteinases were separated from zymogram between the 4th and 5th day of larval development and in the adult stage. Effect of soybean trypsin inhibitor (SBTI, a serine proteinase inhibitor) on development of C. capitata was examined by bioassay. C. capitata was susceptible to SBTI which affected larval mass at ED 50 3.01%.

Understanding Critical Illness Myopathy: Approaching the Pathomechanism

Myopathies occurring in critically ill patients have gained increasing interest during the past years. For the patient, they represent a crucial factor for prolonged intensive care unit treatment and secondary complications. Critical illness myopathies (CIMs) seem to be related to various pathogenic factors. Among those, the septic inflammatory response syndrome seems to play a major role. It has been suggested that, similar to sepsis-related multiorgan failure, CIM might be considered a failure of the organ muscle. Muscle function might be impaired by proposed "myotoxic" humoral factors. These could be endogenously produced during the innate immune response to sepsis. This article follows up recent evidence for such active fractions in the blood serum of CIM patients. To explain muscle weakness in CIM, serum fractions acutely modified membrane excitability and subcellular Ca2+ regulation in an animal model. From the differential serum effects, early-phase CIM seems to involve a reduction in the overall force generation in muscle but also a compensation by the membrane, increasing the excitability. Different animal models will help to elucidate the underlying mechanisms accounting for the specific proteolytic activities found in different forms of CIM. CIM represents a systemic rather than a local disorder. Humoral factors might initiate the local reaction of skeletal muscle clinically seen as muscle weakness, altered excitability, and proteolysis of contractile proteins. Establishing the interactions in the excitation-contraction cascade in CIM is a challenging task, not only to clarify its pathomechanism but also to deduce clinical interventions.

Polymorphonuclear proteolytic activity and milk composition change.

Relationships between Quarter Milk Cell Count (QMCC) and Tank Milk Cell Count (TMCC) with milk biochemical and technological parameters in milk and dairy products were investigated. All parameters measured were affected by the increase of TMCC and QMCC between 0 and 600 000 cells/mL. The variable effect of lactation stage which is different for different authors, is discussed. The three mechanisms, measured during the inflammation of the udder, implicated in the modification of milk quality are described (a decrease in synthesis, a decrease in the milk barrier permeability and an increase in proteolytic activities). The direct effect of plasmin in caseinolysis is well known; the specific role of the increase of somatic cells (especially PMN) in the modification of milk quality is described. Several specific proteolytic activities of PMN are described and the impact of these activities on caseinolysis is evaluated. Two hypothetical mechanisms of caseinolysis by PMN are suggested and a synthetic scheme of the role of plasmin, bacteria and somatic cells in caseinolysis is discussed.

Green Fluorescent Protein Rendered Susceptible to Proteolysis: Positions for Protease-Sensitive Insertions

The green fluorescent protein (GFP) is highly resistant to proteolysis and remains uncleaved after prolonged incubation with trypsin or pronase despite several putative tryptic and chymotryptic sites in exposed loops. We have rendered GFP sensitive to proteolysis by inserting five amino acids, IEGRS, in loops at position 157, 172, or 189. Excitation and emission maxima of the three insertion mutants were similar to those of wild type, but quantum yields of mutants Ω172 and Ω189 were lower, indicating increased freedom of the fluorophore. Trypsin cleaved the native (folded) form of each mutant at a unique site defined by the insert. Pronase also yields similar digestion patterns in these variants, but further proteolysis was also observed, suggesting that the primary cleavage relaxes GFP structure and reveals previously inaccessible sites. Fluorescence of Ω189 changed little upon digestion with trypsin but decreased progressively by as much as 40% upon digestion with increasing amounts of pronase. Fluorescence of other variants was not affected significantly by the proteases, further confirming the remarkable stabilities of GFP variants. These constructs define a new conformation-sensitive site around residue 189 of GFP and show that GFP may be useful for design of protease-susceptible molecules for monitoring of specific proteolytic activities in vivo.

Structure and functions of the interaction domains of C1r and C1s: keystones of the architecture of the C1 complex

C1r and C1s, the proteases responsible for activation and proteolytic activity of the C1 complex of complement, share similar overall structural organizations featuring five nonenzymic protein modules (two CUB modules surrounding a single EGF module, and a pair of CCP modules) followed by a serine protease domain. Besides highly specific proteolytic activities, both proteases exhibit interaction properties associated with their N-terminal regions. These properties include the ability to bind Ca2+ ions with high affinity, to associate with each other within a Ca2+-dependent C1s–C1r–C1r–C1s tetramer, and to interact with C1q upon C1 assembly. Precise functional mapping of these regions has been achieved recently, allowing identification of the domains responsible for these interactions, and providing a comprehensive picture of their structure and function. The objective of this article is to provide a detailed and up-to-date overview of the information available on these domains, which are keystones of the assembly of C1, and appear to play an essential role at the interface between the recognition function of C1 and its proteolytic activity.