Method of determining proteolytic activity by using a conjugate of bovine serum albumin with peroxidase

The authors propose a method for determination of proteolytic activity, based on the hydrolysis of peroxidase-labeled molecules of bovine serum albumin immobilized on the surface of polystyrene microassay plates with the subsequent determination of peroxidase activity on the carrier or in the solution. The optimum conditions for the sorption of the labeled substrate have been established. The method permits the determination of bacillary alkaline protease at a concentration of 01. microgram/ml within 45 minutes. The determination of four proteases has demonstrated that this method shows good correlation with the routine one (r = 0.98), but is more sensitive and less time- and labor-consuming.     

Protease and phospholipase activities of Candida albicans isolated from vaginal secretions with different pH values

Even though vulvovaginal candidiasis and bacterial vaginosis are seldom simultaneously found, we have detected this association at an above average frequency. Thus, we set out to study the activity of proteinases and phospholipases, virulence factors of Candida albicans, to assess their role in the above mentioned association. Of a total of 70 Candida isolates were retrieved from samples of vaginal secretions analyzed at our Diagnostic Service, 65 were identified as C. albicans (a group of n=26 obtained from clinical samples of pH>4.5 and a group of n=39 from clinical samples of pH=or<4.5). The evaluation of phospholipases activity was performed on malt agar and Sabouraud dextrose agar with the addition of egg yolk as substrate. The proteolytic activity was detected on plates of agar base medium with the addition of bovine albumin serum as substrate as sole nitrogen source. Phospholipases activity was essentially the same in both groups of samples (p=0.2003). Proteolytic activity was detected in 61.5% of the isolates from the group with pH=or<4.5 and in 96.2% in the group with pH>4.5; being the former much higher than the latter (p=0.0001). Based on these results we postulate that the simultaneous occurrence of bacterial vaginosis and vulvovaginal candidiasis could be related to the proteolytic activity but unrelated to phospholipases activity.     

NanoDrop fluorometry adopted for microassays of proteasomal enzyme activities

NanoDrop spectrophotometry and NanoDrop fluorospectrometry are used almost exclusively to determine the concentrations of nucleic acids and proteins. We propose that NanoDrop fluorospectrometry can also be applied for measuring enzyme activities using fluorogenic substrates such as the proteolytic activities of the 26S proteasome. Because the NanoDrop ND-3300 device requires only 2 μl of sample, the amount of sample extract, substrate, and cofactors used for an enzyme assay can be significantly reduced. In this report, we present exemplary microassays for proteasomal activities (chymotrypsin-, trypsin-, and PGPH [peptidyl-glutamyl peptide hydrolase]-like sites) in extracts of isolated hemocytes from a marine crab, Cancer pagurus (Crustaceae).     

A microassay for proteolytic activity

A quantitative procedure for measuring proteolytic activity, utilizing azoalbumin as substrate, has been developed for use in microtiter plates. An enzyme-linked immunosorbent assay reader is used to measure absorbance. The procedure is sensitive, as well as being both rapid and economical. It is particularly convenient for measuring large numbers of samples, such as fractions from column chromatography.     

Preparation, properties, and uses of two fluorogenic substrates for the detection of 5'-(venom) and 3'-(spleen) nucleotide phosphodiesterases

A new method for ³H-labeling of native collagen and a specific microassay for collagenase activity are presented. Acid-soluble type I collagen derived from rat tail tendons was reacted with pyridoxal phosphate and then reduced with NaB³H₄ to yield [³H]collagen with a specific activity of more than 10 μCi/mg. With respect to rate of hydrolysis, trypsin susceptibility, and gelling properties this collagen compares favorably with biosynthetically labeled preparations. It was shown that chemical labeling procedures such as this, or N-acetylation with acetic anhydride, do not adversely affect properties of collagen which are important for its use as substrate in specific assays. The microassay employs 50-μl [³H]collagen gels (1 mg/ml) dispensed in microtest plates. At 36°C this assay combines rapid rate of hydrolysis with low trypsin susceptibility. As little as 1 ng of clostridial collagenase activity can be measured reproducibly. The high specific activity of the [³H]collagen allowed us to explore microassay conditions employing minute quantities of substrate in solution. These studies indicated that native type I collagen whether labeled or not, is cleaved in the helical region by trypsin at subdenaturation temperatures. It was concluded that, in order to remain specific, collagenase assays with collagen in solution as with collagen in fibrils must be performed at 10–12°C below the denaturation temperature, i.e., at 35–37°C with collagen gels and 27–29°C with collagen in solution.     

Evaluation of a D-amino-acid-containing fluorescence resonance energy transfer peptide library for profiling prokaryotic proteases

Bacterial proteases play an important role in a broad spectrum of processes, including colonization, proliferation, and virulence. In this respect, bacterial proteases are potential biomarkers for bacterial diagnosis and targets for novel therapeutic protease inhibitors. To investigate these potential functions, the authors designed and used a protease substrate fluorescence resonance energy transfer (FRET) library comprising 115 short d- and l-amino-acid-containing fluorogenic substrates as a tool to generate proteolytic profiles for a wide range of bacteria. Bacterial specificity of the d-amino acid substrates was confirmed using enzymes isolated from both eukaryotic and prokaryotic organisms. Interestingly, bacterial proteases that are known to be involved in housekeeping and nutrition, but not in virulence, were able to degrade substrates in which a d-amino acid was present. Using our FRET peptide library and culture supernatants from a total of 60 different bacterial species revealed novel, bacteria-specific, proteolytic profiles, although in-species variation was observed for Pseudomonas aeruginosa, Porphyromonas gingivalis, and Staphylococcus aureus. Overall, the specific characteristic of our substrate peptide library makes it a rapid tool to high-throughput screen for novel substrates to detect bacterial proteolytic activity.     

Protease-producing psychrotrophic bacteria isolated from Antarctica

The extracellular protease production capacity of 840 bacterial strains isolated during the austral summers of 1989/90 and 1991/92 from different sources of the Antarctic ecosystem was analysed in skim-milk agar plates. Thirty-four psychrotrophic strains were selected, classified at genus level and tested from proteolytic activity by the azocasein method from the cell-free supernatant of submerged cultures. Thirty-two of the selected strains were Gram-negative bacteria and Pseudomonas was the predominant genus. Three Pseudomonas maltophilia strains showed the highest levels of proteolytic activity at 20°C. No correlation was observed between the proteolytic activity estimated by the ring of hydrolysis in skim-milk agar plates and the activity measured by the azocasein method. The results suggest that these psychrotrophic strains are potentially useful for developing a biotechnological process to produce proteases with high activity at moderate temperatures.     

Characterization of the structure and function of Escherichia coli DegQ as a representative of the DegQ-like proteases of bacterial HtrA family proteins

HtrA family proteins play a central role in protein quality control in the bacterial periplasmic space. DegQ-like proteases, a group of bacterial HtrA proteins, are characterized by a short LA loop as compared with DegP-like proteases, and are found in many bacterial species. As a representative of the DegQ-like proteases, we report that Escherichia coli DegQ exists in?vivo primarily as a trimer (substrate-free) or dodecamer (substrate-containing). Biochemical analysis of DegQ dodecamers revealed that the major copurified protein substrate is OmpA. Importantly, wild-type DegQ exhibited a much lower proteolytic activity, and thus higher chaperone-like activity, than DegP. Furthermore, using cryo-electron microscopy we determined high-resolution structures of DegQ 12- and 24-mers in the presence of substrate, thus revealing the structural mechanism by which DegQ moderates its proteolytic activity.     

Nonanoic acid, other alkanoic acids, and related compounds as antifeedants in Hylobius abietis pine weevils

A medium‐length, straight‐chain alkanoic acid, nonanoic acid, is known from laboratory microassays to be an antifeedant in adults of the large pine weevil, Hylobius abietis (L.) (Coleoptera: Curculionidae). Our hypothesis was that we could find new, less volatile alkanoic acids or related compounds suitable for field application and with improved long‐term duration. Alkanoic acids of varying chain lengths (C6–C13) were tested for antifeedant activity in H. abietis adults. Microassay choice tests showed that straight‐chain (C6–C11) alkanoic acids were active. However, high activities were restricted to the (C6–C10) acids, with the C9 (nonanoic acid) at 4 µmol cm^?2 being the most active one. In a no‐choice test on pine twigs, the antifeedant effect of C10 acid was lower than that of the C8 and C9 acids. In microassays, less volatile methyl‐branched alkanoic acids exhibited lower antifeedant activities than did the corresponding straight‐chain ones. However, the most active of the methyl‐branched acids, 2‐methyldecanoic acid, had an activity similar to that of nonanoic acid. Compounds related to nonanoic acid were either active (1‐nonanol), weakly active (nonanoic anhydride), or inactive (nonanal, sodium nonanoate). The anhydride was highly active in the microassay, but less active on twigs. The antifeedant effects of the straight chain (C8–C10) alkanoic acids against pine weevil feeding were tested in the field. In contrast to the results from the twig tests, the less volatile C10 acid was more active in the field for the protection of transplants on fresh clear cuts over a 3‐month period than both the C8 and C9 acids. Phytotoxic effects of the alkanoic acids were observed both in the field and in laboratory studies. If a protective layer of paraffin was applied to the stem prior to application of the alkanoic acids, these undesired side effects were reduced.     

Characterization of a cysteine protease from wheat Triticum aestivum (cv. Giza 164)

Enzymes, especially proteases, have become an important and indispensable part of the processes used by the modern food and feed industry to produce a large and diversified range of products for human and animal consumption. A cysteine protease, used extensively in the food industry, was purified from germinated wheat Triticum aestivum (cv. Giza 164) grains through a simple reproducible method consisting of extraction, ion exchange chromatography and gel filtration. The molecular weight of the enzyme was estimated to be 61000+/-1200-62000+/-1500 by SDS-PAGE and gel filtration. The cysteine protease had an isoelectric point and pH optimum at 4.4 and 4.0, respectively. The enzyme exhibited more activity toward azocasein than the other examined substrates with K(m) 2.8+/-0.15 mg azocasein/ml. In addition, it had a temperature optimum of 50 degrees C and based on a heat stability study 55% of its initial activity remained after preincubation of the enzyme at 50 degrees C for 30 min prior to substrate addition. All the examined metal cations inhibited the enzyme except Co(2+), Mg(2+), Mn(2+) and Li(+). The proteolytic activity of the enzyme was inhibited by thiol-specific inhibitors, whereas iodoacetate and p-hydroxymercuribenzoate caused a competitive inhibition with Ki values 6+/-0.3 mM and 21+/-1.2 microM, respectively. Soybean trypsin inhibitor had no effect on the enzyme. The enzyme activity remained almost constant for 150 days of storage at -20 degrees C. The properties of this enzyme, temperature and pH optima, substrate specificity, stability and sensitivity to inhibitors or activators, meet the prerequisites needed for food industries.     

DNA-bound proteases. Partial characterization of proteolytic activities in commercial sources of DNA.

The presence of variable amounts of protease in commercial sources of calf thymus DNA has been established by means of three independent methods; electrophoresis, substrate labelling and microfluorometry. The microfluorometric method, which is particularly useful for initial velocity measurements, has been used to characterize this proteolytic activity, either bound to DNA or solubilized by salt extraction. The proteolytic activity had a maximum at neutral pH and was not affected by NaCl concentration up to 0.5 M. Studies of specificity with natural substrates showed a higher V for protamine and lower Km for histones. The proteolytic activity was resolved by gel filtration chromatography in two major molecular weight species, differing in thermal stability and pH activity profile.     

Thermally modified azocasein--a new insoluble substrate for the determination of proteolytic activity

An insoluble chromogenic substrate for the determination of proteolytic activity was prepared by heating azocasein in a thin layer at 200 degrees C for 4 h in a hot-air thermostat. The activity of an extracellular bacterial proteinase produced by Bacillus sp. was determined with this new substrate.     

The fungal chimerolectin MOA inhibits protein and DNA synthesis in NIH/3T3 cells and may induce BAX-mediated apoptosis

The Marasmius oreades mushroom agglutinin (MOA) is a blood group B-specific lectin carrying an active proteolytic domain. Its enzymatic activity has recently been shown to be critical for toxicity of MOA toward the fungivorous soil nematode Caenorhabditis elegans. Here we present evidence that MOA also induces cytotoxicity in a cellular model system (murine NIH/3T3 cells), by inhibiting protein synthesis, and that cytotoxicity correlates, at least in part, with proteolytic activity. A peptide-array screen identified the apoptosis mediator BAX as a potential proteolytic substrate and further suggests a variety of bacterial and fungal peptides as potential substrates. These findings are in line with the suggestion that MOA and related proteases may play a role for host defense.     

Microassay of acetylcholinesterase activity in small portions of single mosquito homogenates

1. A simple, rapid microassay method is described for measuring acetylcholinesterase (AChE) activity accurately and precisely in small portions of single mosquito homogenates. 2. Up to 30 microassay replicates were possible for individual insects. 3. Microassay data on individual mosquitoes were compared with conventional enzyme assay data acquired using pools of the same homogenates. 4. Under the optimum reaction conditions established, an average Vmax of 7.1 nmol/l/min/mosquito and an average Km of 1.3 x 10(-4) M were observed with acetylthiocholine iodide as substrate. 5. Variability in AChE activity within a sample population of Anopheles albimanus was observed using measurements from individual insects. 6. Such information is fundamental to comparative studies of pesticide physiology (in particular, the resistance phenomenon) in the individual mosquitoes in a population pool; this technique forms the basis for a recently developed resistance microassay.     

Toxoplasma gondii: Microassay to differentiate toxoplasma inhibiting factor and interleukin 2

Using a sensitive, economical, and reproducible microassay, the relationship of toxoplasma inhibiting factor to interleukin 2 has been examined. The assay developed took advantage of the observation that (1) Toxoplasma gondii tachyzoites replicated efficiently in the murine monocytic cell line, RAW 264; (2) treatment of RAW 264 cells with toxoplasma inhibiting factor prevented intracellular replication of the parasite to an extent similar to that observed with identical treatment of freshly isolated murine peritoneal exudate cells; and (3) [³H]uracil incorporation was an efficacious means to quantify replication (or inhibition of replication) of tachyzoites within the cell line. Although toxoplasma inhibiting factor and interleukin 2 were both present in the same lectin -and antigen-stimulated splenocyte supernatant fluids, results from microassays strongly suggested that the molecules were two distinct entities.     

A microassay for the peptidase activity of enzymes utilizing ribonuclease S peptide as a substrate

A microassay for the peptidase activity of proteins obtained in minute amounts was devised. The method uses ribonuclease S peptide as a substrate. The substrate when cleaved is unable to reconstitute an active ribonuclease S complex. Therefore the loss in activity of the reconstituted complex is a measure of the peptidase activity. The method was previously tested with known peptidases such as clastase (9), chymotrypsin (8), and trypsin. In this work the peptidase activity of a protein related to a sperm-decapitating factor (1) is evidenced.     

Purification and characterization of a novel broad-spectrum bacteriocin from Bacillus licheniformis MKU3

A bacterial strain Bacillus licheniformis MKU3, isolated from slaughterhouse sediments showed a strong antimicrobial activity. The antimicrobial substance produced by this strain was found to be a protein that inhibited a broad range of bacterial strains, such as Bacillus sp., Staphylococcus sp., Streptococcus sp., and Listeria monocytogenes. The antimicrobial peptide was purified to homogeneity by cut off membrane filtration followed by gel filtration chromatography. The purified protein with low molecular mass (< 8 kDa) was resolved as single band on Tricine SDS-PAGE. This protein was stable at 100°C for 10 min, but lost its activity at 121°C in 15 min. It was resistant to the proteolytic action of trypsin, proteinase K, and pronase E and stable within a wide range of pH (3.0∼11.0). This protein exhibited lytic activity on selected indicator strain Kurthia gibsonii GCS6.     

A microassay for ATPase

A newly developed microtechnique for quantitating activity of myosin ATPase (EC 3.6.1.32) is more sensitive and less time-consuming than existing spectrophotometric methods. Measurement of ATPase activity using the new method can be accomplished in a final volume of 0.25 ml, allowing the assay to be conducted in individual wells of 96-well microplates commonly used for the enzyme-linked immunosorbent assay (ELISA). The microassay is performed by adding purified myosin to microplate wells followed by addition of ATP to initiate the enzymatic reaction. The reaction is subsequently terminated by addition of an acidic solution containing malachite green and ammonium molybdate. The level of inorganic phosphate produced by enzymatic hydrolysis of ATP is measured by scanning the microplates using a microELISA plate reader. An entire 96-well microplate can be scanned in less than 2 min, and data from the microassay can be transferred directly to a microprocessor for statistical analysis. The microassay is capable of detecting between 0.2 and 3 nmol of inorganic phosphate in a reaction volume of 50 microliter, and the ATPase activity of as little as 10 ng of rat cardiac myosin can be measured. The increased sensitivity compared with that of other spectrophotometric assays and ease of performing the microassay enable a detailed analysis of the enzymatic properties of cardiac myosin to be conducted on large numbers of small tissue specimens. Several kinetic properties of rat cardiac myosin were determined using this technique.     

A convenient automated method for the determination of proteolytic enzymes

An automated method for the assay of proteolytic enzymes is described. The insoluble dye-protein complex, Remazolbrilliant Blue-hide powder, is used as the substrate and is readily digested by trypsin, elastase, subtilisin, thermolysin, chymotrypsin, and to a lesser extent pronase, pepsin, and bacterial collagenase. The proteolytic activity of crude microbial culture preparations is expressed in terms of an equivalent concentration of crystalline trypsin, which itself can be readily determined within the concentration range 0.25–5.0 μg/ml.     

A microwell assay for anchorage independent cell growth

We describe modifications of the conventional assay for anchorage independent growth of fibroblasts that enable the assay to be carried out in microwell plates, as opposed to the conventional Petri dishes. The microwell assay is a good discriminator of final EGF concentrations in the range 10-100 picograms/ml, and can be used to detect absolute amounts of EGF below 2 pg. Addition of TGF beta to EGF enhances colony formation in the microassay in the usual manner. We describe the use of this microassay to identify and map local production of transforming growth factor activity by the component pieces of individual chick embryos. Transforming activity was identified in all the stages tested (Hamburger and Hamilton stages 13-23). Highest levels were found near the mid-line of the embryo. No clear differences in the cranio-caudal axis have so far been identified. This technique will enable the spatial and temporal distribution of transforming activity throughout vertebrate embryos to be completely mapped. It seems likely that this mapping process will help elucidate the normal role of transforming growth factors in embryos.