A micromethod for determination of proteolytle enzymes in the pH range of 2.8 to 4.8

Løwenstein, Henning; Ingild, A.

doi:10.1016/0003-2697(76)90028-2

Cited by 22 publications

(7 citation statements)

References 3 publications

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“…1). A similar relationship has been reported using the protein precipitation procedure (4,5,10,14). Below 39 ng, the standard curve deviates from linearity making quantitative interpretations of such low levels of proteolytic activity difficult even though a proteolytic clearing was evident around the well (Fig.…”

Section: Methodssupporting

confidence: 57%

“…4) indicates that complete inhibition was achieved by 0.1 M CuCl2, 10 mM PMSF, and 10 Ag/ml chymostatin. This compares with 5 pg/ml chymostatin and 1 mm PMSF based on the azocasein assay discussed below and indicates the diffusion plate assay can be used as a semiquantitative assay for protease inhibition.…”

Section: Methodsmentioning

confidence: 99%

“…After mixing, the absorbance was read at 440 nm. One unit of activity was defined as the amount of homogenate needed to cause an increase of 1 To compare the sensitivity of the Coomassie blue stained diffusion plates with that of previous methods that used protein precipitation to visualize the zones of hydrolysis (4,5,10,14), a diffusion plate was prepared assaying 0.3 ng to 2.5 ,ug purified trypsin (Fig. 1).…”

Section: Methodsmentioning

confidence: 99%

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

Gallagher

Carroll²,

Leonard³

1986

Plant Physiol.

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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 (2,12). This endogenous protease activity can also lead to artifacts during SDS-PAGE, such as a general smearing of the protein staining pattern on the gel, a loss of high mol wt polypeptides, and subtle shifts in electrophoretic mobility of specific polypeptides (1,8,12,18).Proteolytic enzymes are classified into two broad categories; the exopeptidases, or peptidases, which cleave peptide bonds at the N or C terminal positions producing very subtle modifications of the protein that are difficult to detect during protein purification, and the endopeptidases, or proteinases, which cleave internal peptide bonds producing dramatic effects on native protein structure and activity that are readily detected during protein purification (3,13). In this paper, we will use the term protease to refer to this later category of proteolytic enzyme.Based on catalytic mechanism, proteases can be separated into various classes which are usually distinguished by the use of specific inhibitors, rather than by activity with a particular substrate. Chemical inhibitors covalently modify a critical amino acid in the active site of the protease. For example, PMSF3 alkylates a reactive serine of the serine type of protease (13). However, chemical inhibitors are not always specific, and PMSF will also react with sulfhydryl proteases or with an important sulthydryl on the enzyme being purified. Competitive inhibitors compete at the active site of the protease and are usually very specific inhibitors (17). The microbial product, chymostatin, is an example of a competitive type of protease inhibitor. There are several approaches that can be taken to reduce protease activity during plant cell fractionation ( 12). One of the simplest approaches is to choose a tissue that has a low protease content. Because this is usually not possible, fractionation must be conducted under conditions which limit protease activity. This includes adjusting pH above or below the optimum for protease activity, lowering temperature to near 0WC, controlling the concentration of divalent cation activators of proteases, keeping the total time for fractionation as short as possible, removal of proteases from the homogenate by, for example, affinity chromatography, and/or adding an effective inhibitor of proteases. The addition of an inhibitor is by far the simplest and most effective way to control the activity of proteases. However, the effectiveness of a particular inhibitor in reducing protease activity will depend on the type of protease pr...

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Section: Methodssupporting

confidence: 57%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Gallagher

Carroll²,

Leonard³

1986

Plant Physiol.

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“…After the distance from the edge of the separating gel to the center of the clear lytic bands in each lane was measured, incubation of zymograms was terminated by separating the gels. Then the zymograms were fixed with 3% (v:v) acetic acid for 10 min, rinsed under tap water, dried, and stained with 0.1% (w:v) Amido Black 10B dye (Bio-Rad) in acetic isopropanol (25% [v:v] isopropanol and 10% [v:v] acetic acid in water) for permanent storage [35]. Polyacrylamide gels were stained with 0.05% Coomassie Brilliant Blue R-250 dye (Bio-Rad) in acetic isopropanol overnight without fixing and rinsing.…”

Section: Electrophoresis and Zymographymentioning

confidence: 99%

Production of Plasminogen Activators (PAs) in Bovine Cumulus-Oocyte Complexes during Maturation In Vitro: Effects of Epidermal Growth Factor on Production of PAs in Oocytes and Cumulus Cells1

Park

Choi

Song

et al. 1999

Biology of Reproduction

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We examined whether plasminogen activators (PAs) are produced by bovine cumulus-oocyte complexes (COCs) during maturation in vitro. The effects of epidermal growth factor (EGF) on production of PAs in oocytes and cumulus cells were also examined. When COCs were cultured for 24 h with 30 ng/ml EGF, three plasminogen-dependent lytic zones (58.5 +/- 3.5 kDa, 79.0 +/- 3.0 kDa, and 113.5 +/- 6.5 kDa) were observed. Addition of amiloride, a competitive inhibitor of urokinase-type PA (uPA), to the zymogram eliminated the activity of the 58.5 +/- 3.5-kDa zone, suggesting that this band is a uPA. However, since the activity of the remaining two bands was not eliminated, it was suggested that the 79.0 +/- 3.0-kDa band is a tissue-type PA (tPA) and the 113.5 +/- 6.5-kDa band is possibly a tPA-PA inhibitor (tPA-PAI) complex. In COCs before culture, however, no activity of PAs was detected. At 6 h of culture, the same level of uPA activity was detected in COCs cultured both in the absence and in the presence of EGF. The uPA activity was increased at 12 h of culture but without further increase at 24 h of culture, with higher activity in the presence than in the absence of EGF. The activity of tPA and tPA-PAI was first detected at 24 h of culture in the absence of EGF. In the presence of EGF, however, some activity of tPA-PAI was detected at 12 h of culture. At 24 h of culture, the activity of all PAs was detected in cumulus cells, but only uPA activity was detected in oocytes, with higher activity in the presence than in the absence of EGF. The uPA activity in oocytes was not detected when they were cultured without cumulus cells in either the presence or absence of EGF, although cumulus expansion was stimulated by EGF, exhibiting a time-course similar to that observed in PA production. These results suggest that uPA, tPA, and tPA-PAI are all produced by bovine COCs, but only uPA by oocytes, during maturation in vitro. However, cumulus cells play an essential role or roles in the production of uPA by oocytes, and EGF enhances the roles of cumulus cells.

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“…To terminate the incubation, zymographs were removed from the polyacrylamide gels and fixed with 3.0% acetic acid for 15 min. Zymographs were dried and stained for permanent storage [16], and polyacrylamide gels were stained with 0.05% Coomassie Brilliant Blue dye (Bio-Rad) in 65:25:10 water: isopropanol: acetic acid and destained with 80:10:10 water: isopropanol:acetic acid. Zymographs containing amiloride (Sigma), the competitive inhibitor of urokinase [17], were prepared with the following modifications: equal volumes of 2.2% agarose and 2.2% nonfat dry milk were combined, and 1 ml containing 200 pg human plasminogen and either PBS or 100 mM amiloride was added to 9 ml of the melt.…”

Section: Electrophoresis and Zymographymentioning

confidence: 99%

Partial Characterization of the Plasminogen Activator Produced by Ovine Embryos in Vitro1

Bartlett

Menino²

1993

Biology of Reproduction

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The type of plasminogen activator (PA) produced by Day 7, 9, and 11 ovine embryos in vitro was partially characterized. PA activity in the conditioned medium did not change (p > 0.05) up to equivalent gestational Day (EGD) 14 but increased to peak levels on EGD 15 (p < 0.05). Zymographic analysis of conditioned medium and embryos revealed one plasminogen-dependent lytic zone (48-51 kDa) within EGD 8-15 and a second plasminogen-dependent lytic zone that appeared after EGD 13 (79-83 kDa). Addition of the urokinase competitive inhibitor, amiloride, to the zymograph inhibited plasminogen-dependent lytic activity; and forms of both high and low molecular mass were immunoprecipitated with antiserum to bovine urokinase-type PA. PA activity in conditioned medium was completely suppressed by amiloride treatment (p < 0.05) but was increased (p < 0.05) after incubation with antibodies to human PA inhibitor (PAI)-2. Treatment with antibodies to human PAI-1 did not increase (p > 0.05) PA activity in conditioned medium. These results suggest that Day 7, 9, and 11 ovine embryos produce a urokinase-type PA (49.9 +/- 0.5 kDa) and possibly a PAI-2-like protein that associates with the PA and forms a PA-PAI complex of high molecular mass (81.4 +/- 1.2 kDa).

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A micromethod for determination of proteolytle enzymes in the pH range of 2.8 to 4.8

Cited by 22 publications

References 3 publications

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

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

Production of Plasminogen Activators (PAs) in Bovine Cumulus-Oocyte Complexes during Maturation In Vitro: Effects of Epidermal Growth Factor on Production of PAs in Oocytes and Cumulus Cells1

Partial Characterization of the Plasminogen Activator Produced by Ovine Embryos in Vitro1

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