Azocasein Assay Research Articles

Monoclonal antibodies against AsaP1, a major exotoxin of the fish pathogen Aeromonas salmonicida subsp. achromogenes, and their application in ELISA.

Two monoclonal antibodies (Mabs) binding to a toxic extracellular metallo-proteinase of Aeromonas salmonicida subsp. achromogenes, AsaP1, were produced. Both reacted with common epitopes of the native enzyme and recognized this 20 kDa antigen on Western blots. One of these Mabs had an inhibitory effect on the caseinase activity of the exotoxin. A Mab-based ELISA was set up and evaluated for serological detection of AsaP1 in bacterial culture filtrates. The exotoxin was identified serologically in the extracellular products of 11 of 26 atypical Aer. salmonicida isolates, including the type strain for subsp. achromogenes NCIMB 1110. The ELISA was approximately 100-fold more sensitive in detecting AsaP1 compared with an azocasein assay. The established serological test enables AsaP1 to be quantified reliably with a lower detection limit of about 0.12 ng ml-1 and has a potential use for the phenotypic differentiation of atypical Aer. salmonicida isolates.

Quorum sensing-dependent regulation and blockade of exoprotease production in Aeromonas hydrophila.

In Aeromonas hydrophila, the ahyI gene encodes a protein responsible for the synthesis of the quorum sensing signal N-butanoyl-L-homoserine lactone (C4-HSL). Inactivation of the ahyI gene on the A. hydrophila chromosome abolishes C4-HSL production. The exoprotease activity of A. hydrophila consists of both serine protease and metalloprotease activities; in the ahyI-negative strain, both are substantially reduced but can be restored by the addition of exogenous C4-HSL. In contrast, mutation of the LuxR homolog AhyR results in the loss of both exoprotease activities, which cannot be restored by exogenous C4-HSL. Furthermore, a substantial reduction in the production of exoprotease by the ahyI+ parent strain is obtained by the addition of N-acylhomoserine lactone analogs that have acyl side chains of 10, 12, or 14 carbons. The inclusion of N-(3-oxododecanoyl)-L-homoserine lactone or N-(3-oxotetradecanoyl)-L-homoserine lactone at 10 microM in overnight cultures of A. hydrophila abolishes exoprotease production in azocasein assays and reduces the activity of all the exoprotease species seen in zymograms.

Azocasein assay for alkaline protease in complex fermentation broth

An azocasein assay has been developed for determination of alkaline protease in fermentation broth from a complex substrate containing ca. 50 g/l protein which to a high degree interferes with azocasein. Methods described in the literature have been found inaccurate as results deviate ca. 50% from true activity, so one of the methods is modified in order to eliminate interference. Proportionality between the relative azocasein concentration and the deviation from true activity is found. An azocasein concentration of 350 mg azocasein per ml sample gives satisfactory results with an accuracy of ±5%. Application of a standard addition method also improves the accuracy, but is laborious and less precise.

Proteinases in the excretory/secretory products (ES) of adult Trichinella spiralis.

Adult Trichinella spiralis were maintained in vitro using defined media and the material excreted/secreted (ES) during this time examined for proteolytic enzyme (proteinase) activity using an azocasein assay and gelatin-substrate gels. Several discrete proteinases in the size range 14-100 kDa were observed with optimal activity at pH 7.5. The use of a class-differentiating panel of proteinase inhibitors indicated that serine proteinases were predominant although some inhibition was evident in the presence of cysteine and metalloproteinase inhibitors. Of a panel of potential natural protein substrates tested, ES proteinases only degraded fibrinogen and plasminogen and degradation was, in part, susceptible to the action of serine, cysteine and aspartyl proteinase inhibitors. In addition, antibody harvested from immune but not normal mice inhibited ES proteinase activity, an observation of relevance to the immunobiology of Trichinosis.

Heat shock inhibits and activates different protein degradation pathways and proteinase activities in Neurospora crassa

In Neurospora crassa, heat shock treatment inhibits proteolytic activity. ATP-independent proteinases were analysed after polyacrylamide gel electrophoresis using renaturing gelatine gels. Proteinases of 24, 29, and 130 kDa were shown to be inhibited by heat shock and were further characterized as to their properties. A major part of the heat shock-induced inhibition is probably due to suppression of de novo synthesis of proteinases as deduced from experiments with cycloheximide. During several hours of recovery from heat shock, the inhibition of overall protein degradation and ATP-independent proteinases is reversed. Azocasein assays as well as pulse-chase experiments further showed that ATP-dependent protein degradation is only slightly affected by heat shock. Two ATP-binding proteinases of about 60 and 160 kDa even show an increased activity after heat shock. The degradation rate of heat shock proteins is inhibited by heat shock treatment, indicating that they are degraded by ATP-independent proteinases. Western blot analysis of a ∼40-kDa degradation product of HSP70 containing its amino terminal portion revealed a reduction in the amount of this peptide after heat shock.

Heat shock inhibits and activates different protein degradation pathways and proteinase activities in Neurospora crassa.

In Neurospora crassa, heat shock treatment inhibits proteolytic activity. ATP-independent proteinases were analysed after polyacrylamide gel electrophoresis using renaturing gelatine gels. Proteinases of 24, 29, and 130 kDa were shown to be inhibited by heat shock and were further characterized as to their properties. A major part of the heat shock-induced inhibition is probably due to suppression of de novo synthesis of proteinases as deduced from experiments with cycloheximide. During several hours of recovery from heat shock, the inhibition of overall protein degradation and ATP-independent proteinases is reversed. Azocasein assays as well as pulse-chase experiments further showed that ATP-dependent protein degradation is only slightly affected by heat shock. Two ATP-binding proteinases of about 60 and 160 kDa even show an increased activity after heat shock. The degradation rate of heat shock proteins is inhibited by heat shock treatment, indicating that they are degraded by ATP-independent proteinases. Western blot analysis of a approximately 40-kDa degradation product of HSP70 containing its amino terminal portion revealed a reduction in the amount of this peptide after heat shock.

Activity assays for characterizing the thrombolytic protein fibrolase

Characterization of the thrombolytic agent fibrolase was accomplished employing specific proteolytic and thrombolytic assays. This paper describes a method to measure enzyme proteolytic activity using the oxidized β-chain of insulin as a substrate. Advantages of this method include a short incubation time for substrate cleavage followed by an isocratic HPLC method with a retention time of approx. 5 min. Proleolytic activity can be rapidly and easily quantitated with this procedure. An azocasein assay was also used to quantitate proteolytic activity. This method was optimized with respect to substrate concentration and incubation time allowing for the rapid quantitation of fibrolase activity. A thrombolytic assay is described which employs fibrin plate clearance and has the advantage of rapid and accurate quantitation compared with previously described methods. It also allows for the standardization of fibrolase in plasmin-equivalent units.

A Sensitive Diffusion Plate Assay for Screening Inhibitors of Protease Activity in Plant Cell Fractions

Proteolytic activity was detected, using a sensitive radial diffusion plate assay, in the plasma membrane fractions of corn (Zea mays L.) roots and from roots of several other plant species. The proteases could be effectively inhibited in corn with phenylmethane sulfonyl fluoride or chymostatin. Protease activity of oat roots, however, was not significantly reduced by these inhibitors. The results of diffusion plate assay were confirmed with the less sensitive azocasein assay using crude cell homogenates. Chymostatin and phenylmethane sulfonyl fluoride were effective in preventing protease degradation of polypeptides as revealed by electrophoresis. The diffusion plate assay uses a permanent support for a 0.75 millimeter thick agarose slab containing 200 micrograms per milliliter casein. By staining the fixed and dried gel with Coomassie blue R-250, proteolytic activity was visualized as a cleared area around the sample well with a detection limit of about 0.3 nanograms trypsin. The diffusion plate assay should prove useful for screening inhibitors of proteases where limited amounts of material are available, such as with plant cell fractions or highly purified proteins.

Induction of a proteinase by heat-shock in yeast

In Saccharomyces cerevisiae heat-shock induces an increase in proteinase activity. The induction is probably due to newly synthesized enzyme molecules, since the increase in proteinase activity can be inhibited by cycloheximide. Degradation of endogenous proteins is enhanced by EDTA, while the azocasein assay is not affected by MnCl2, MgCl2, or EDTA. The proteinase has a pH optimum of 8, and phenylmethylsulfonyl fluoride (PMSF) as well as chymostatin are strong inhibitors. We infer that the induced proteinase is probably identical with proteinase B of yeast.

Intracellular segregation and degradation of bovine serum albumin and lactate dehydrogenase microinjected into 3T3-L1 cells

n.d. properties of the four cathepsins are recorded in Table 1. Cathepsin S from rabbit spleen was similar to the previously described bovine cathepsin S except that it exhibited a different response in the two azocasein assays. As reported previously (Etherington, 1980), cathepsin N caused little degradation of azocasein. However, in the presence of 3M- urea, which is now used routinely in the assay for cathepsin L, cathepsin N exhibited substantial activity. Furthermore cathepsin N exhibited only a weak gelatinase activity unless 3M-urea was also included. Cathepsins N and S were com- pletely resolved from cathepsins B and L by IEF. IEF gels were prepared and the enzymes located by applying an agarose-substrate overlay. In each overlay examined the differences in specificity were confirmed from the intensities of the digestion zones. IEF gels were also used in an imniunodiffusion assay with an antiserum raised against rabbit cathepsin L (Etherington et al., 1984). A precipitin arc was formed only at the position of cathepsin L and not the positions of cathepsin B, N or S. This is the first time that all four cathepsins have been identified in a single tissue. It is concluded that they are different enzymes, but their respective contributions to intracellular proteolysis are still unclear.

Plasminogen activator content of neoplastic and benign human prostate tissues; fibrin augmentation of an activator activity.

The plasminogen activator content of the extracts of excise prostate cancers (25 specimens) was determined with an azocasein assay and found to be on the average 1.7 times higher than that of extracts of excised prostate benign hyperplasias (29 specimens). Both groups contained the same average percentage of human urokinase type activator (approximately 45%) as determined by the inhibition of activity when anti-human urokinase antibody was included in the assay system. The two types of activators were partially purified and found to have distinctly different properties. The most striking difference was the large augmentation of activity o the non-urokinase enzyme in fibrinolysis. The implications of an enhanced fibrinolysis relative to azocaseinolysis (or other) is discussed, particularly with respect to its importance in the quantitation and characterization of activators by different investigators. Highly purified urokinase-like activator was found to be similar to commercial urokinase preparation with respect to molecular weight, isoelectric point, inhibition by the antibody, and inhibition by placenta inhibitor.

29] Human plasminogen

Publisher Summary Human plasminogen is known to possess multiple isoelectric forms, which can be isolated by isoelectric-focusing techniques. Glu-plasminogen 1 possesses multiple forms of pI values of 6.2, 6.3, 6.4, and 6.6. Glu-plasminogen possesses two isoelectric forms with pI values of 6.4 and 6.6. On the other hand, Lys77-plasminogen 1 contains three major isoelectric forms of pI 6.7, 7.2, and 7.5; whereas LysrT-plasminogen 2 contains three major forms of pI values 7.5, 7.8, and 8.1. Assays of plasminogen first require its conversion to plasmin by common activators. The conversion of human plasminogen to human plasmin is accomplished as a result of cleavage of the Arg-Val­-sl peptide bond. The enzyme is then assayed based on its ability to hydrolyze a-N-tosyl-Larginine methyl ester (TAME), utilizing a recording pH-stat to titrate the amount of acid, a-N-tosyl-L-arginine, liberated with time. Assays that rely on the spectrophotometric determination of the plasmin-catalyzed release of p -nitroanilide from the substrate H-D-valyl-L-leucyl-L-lysine-pnitroanilide dihydrochloride and on the determination of the plasmin-catalyzed release of p-nitrophenolate from N-Cbz-L-lysine-p - nitrophenyl ester 3° have also been fruitfully employed. Other assay methods for plasmin are available, such as the casein, azocasein, and fibrin plate assay.