Use of Chromogenic Substrate S-2251 for Determination of Plasminogen Activator in Rat Ovaries

1981 ◽  
Vol 46 (02) ◽  
pp. 507-510 ◽  
Author(s):  
H Shimada ◽  
T Mori ◽  
A Takada ◽  
Y Takada ◽  
Y Noda ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Enzymatic Reactions ◽  
Chromogenic Substrate ◽  
Assay Procedure ◽  
One Step ◽  
Reproducible Method ◽  
Plasmin Inhibitors ◽  
Endogenous Activity ◽  
Hydrolysis Of

SummaryA simple, specific and reproducible method for determination of plasminogen activator activity in rat ovaries has been developed by using the chromogenic substrate S-2251. The two steps of enzymatic reactions, i. e. activation of plasminogen and subsequent hydrolysis of the substrate was performed in one step incubation. A linear relationship was observed between the amount of chromogen produced and activator activity in the range of the optical density from 0.05 to 1.20 for 30 min’s incubation. Endogenous activity of non-specific proteases, plasmin or plasmin inhibitors which might be contained in rat ovaries turned out not to interfere with the specificity of a standardized assay procedure. Reproducibility was firmly established with coefficient of variation not exceeding 10%. Using this method, a marked increase followed by a drastic decrease in the activator activity was shown with rat ovaries around the time of ovulation after the injection of human chorionic gonadotropin.

scholarly journals A rapid direct assay for the determination of the separate activities of the three arylsulphatases of Aspergillus oryzae

1977 ◽  
Vol 166 (3) ◽  
pp. 411-413 ◽  
Author(s):  
G R J Burns ◽  
C H Wynn
Keyword(s):  
Phenolic Compounds ◽  
Aspergillus Oryzae ◽  
Substrate Specificities ◽  
Direct Assay ◽  
Assay Procedure ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of ◽  
Potential Use

1. The three arylsulphatases of Aspergillus oryzae exhibit pronounced kinetic differences and substrate specificities. Arylsulphatase I hydrolyses all substrates tested, whereas arylsulphatase III will not hydrolyse tyrosine O-sulphate or phenolphthalein disulphate. Arylsulphatase II does not hydrolyse p-nitrophenyl sulphate or phenolphthalein disulphate at appreciable rates in the absence of added phenolic compounds. Phenols such as tyramine increase the rate of hydrolysis of these substances by this enzyme 1000-fold. At pH 6.9 arylsulphatase I exhibits an apparent Km of 0.1 mM for p-nitrophenyl sulphate, whereas the Km of arylsulphatase III for this substrate is 1 mM. 2. These differences were utilized to develop an assay procedure which can be used to determine the separate activities of the three enzymes present in mixtures. This assay has potential use as a means of examining the relative activities of the three enzymes in investigations of the differences in the mechanisms regulating their synthesis.

scholarly journals Fluorogenic Substrates for the Determination of Plasminogen Activators and Plasmin

1977 ◽  
Author(s):  
W. Nieuwenhuizen ◽  
G. Wijngaards ◽  
E. Groeneveld
Keyword(s):  
Plasminogen Activator ◽  
Enzyme Histochemistry ◽  
Biological Fluids ◽  
Kinetic Studies ◽  
Plasminogen Activators ◽  
Fluorogenic Substrates ◽  
Highly Sensitive ◽  
Tissue Plasminogen ◽  
Hydrolysis Of

Plasminogen activator activities in biological fluids are low and specific for plasminogen. Highly sensitive and accurate assays are needed for direct quantification and for kinetic studies. We therefore, synthesized tripeptide amides i. e., t. BOC. val. gly. arg β-NA (l) and val. gly. arg β-NA (II) from which the fluorescent group β-naphtylamine (β-NA) is released upon enzymatic hydrolysis.Kinetic parameters found for the hydrolysis of these substrates by plasmin, urokinase and tissue plasminogen activator are:*in nmol/CU/min; **in pmol/CTA/minStrikingly, tissue activator is not active on substrate II, allowing discrimination between this activator and other proteases.The sensitivity of the assay is at least ten times higher than that observed for other synthetic substrates available. Moreover, substrates I and II can be used in enzyme histochemistry.

Increase in plasminogen activator in the involuting uterus of the postpartum rat

1985 ◽  
Vol 104 (2) ◽  
pp. 295-298 ◽  
Author(s):  
H. Shimada ◽  
H. Okamura ◽  
L. L. Espey ◽  
T. Mori
Keyword(s):  
Plasminogen Activator ◽  
Indirect Method ◽  
Post Partum ◽  
Control Level ◽  
Collagenolytic Activity ◽  
Tissue Remodelling ◽  
Rat Uterus ◽  
Wet Weight ◽  
Plasmin Inhibitors ◽  
Hydrolysis Of

ABSTRACT Plasminogen activator (PA) activity in the rat uterus was measured at fixed intervals post partum in order to determine whether this serine protease increases during the acute remodelling of tissue which occurs in the involuting uterus. Plasminogen activator activity was measured by an indirect method based on the hydrolysis of the chromogenic substrate S-2251 by PA-generated plasmin. At the time of parturition the control level of PA activity was 0·033 ± 0·018 (s.d.) μmol/4 mg uterine wet weight per 30 min. This activity increased fourfold to a peak of 0·131 ±0·036 at 3 days post partum, and then it declined steadily towards the control level during the next 7 days. Concomitantly, uterine weight decreased to 25% of the control weight by 3 days post partum, and it continued to decrease until day 15. In the 30 days post partum during which PA activity was monitored there was no significant change in plasmin inhibitors in the uterine extracts. The results suggest a correlation between PA activity and the process of tissue remodelling which occurs during involution of the rat uterus. This increase in PA might serve to activate a latent collagenase since the measured peak in PA activity happens to coincide with a reported increase in collagenolytic activity in the involuting rat uterus. J. Endocr. (1985) 104, 295–298

Isolation and partial characterization of a peptidase with trypsin-like activity from bovine adenohypophysis

1989 ◽  
Vol 54 (8) ◽  
pp. 2276-2286
Author(s):  
Tsezengijn Dash ◽  
Tomislav Barth ◽  
Jiřina Slaninová ◽  
Jana Barthová ◽  
Hana P. Mašková ◽  
...  
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Partial Characterization ◽  
Chromogenic Substrate ◽  
Fluorogenic Substrate ◽  
Basic Amino Acids ◽  
Optimum Ph ◽  
Reproducible Method ◽  
Hydrolysis Of

A reproducible method has been developed for the isolation of the adenohypophyseal enzyme with a trypsin-like activity. The enzyme is able to hydrolyze Nα-benzoyl-L-arginine-p-nitroanilide, a fluorogenic substrate CBzl-Arg-Arg-β-naphthyl amide and some peptides with one or two accumulated basic amino acids in the chain. The optimum pH for hydrolysis of the chromogenic substrate was within the range 6.0-7.0 (Km = 0.66 mmol l-1), in the case of the fluorogenic substrate the range was between 7.0 and 7.5 (Km = 1.2 μmol l-1). The enzyme is activated by cysteine and dithiothreitol and inhibited by SH-poisons. The molecular weight of the enzyme, determined by means of two independent methods, was approximately 25 kDA.

Effect of mastitis on plasminogen activator activity of milk somatic cells

1992 ◽  
Vol 59 (4) ◽  
pp. 461-467 ◽  
Author(s):  
Teffi Zachos ◽  
Ioannis Politis ◽  
Ronald C. Gorewit ◽  
David M. Barbano
Keyword(s):  
Cell Count ◽  
Plasminogen Activator ◽  
Somatic Cell Count ◽  
Physiological Function ◽  
Somatic Cells ◽  
Fold Increase ◽  
Chromogenic Substrate ◽  
Stage Of Lactation ◽  
Milk Somatic Cells ◽  
Hydrolysis Of

SummaryThis study was conducted to examine the effects of mastitis and stage of lactation on plasminogen activator (PA) activity in milk somatic cells. An assay System, which measures the plasmin-mediated hydrolysis of the chromogenic substrate D-valyl-leucyl-lysine p−nitroanilide, was used to assess PA activity present within milk somatic cells. Milk cell associated PA activity was increased (P < 0·05) by 50% in the presence of fibrin fragments. This suggests that milk somatic cells contain tissue PA which, unlike urokinase PA, is preferentially activated in the presence of fibrin fragments. An increase of the milk somatic cell count from < 5 × 104 to > 106 cells/ml resulted in an 8-fold increase in PA activity per cell. Elevated levels of PA activity were associated with milk somatic cells isolated from mastitic quarters obtained from cows in early (<4 months in lactation) or late lactation (>8 months in lactation). We conclude that PA activity is increased during severe mastitic inflammation. Although the physiological function of this enzyme is as yet unclear, we propose that it may be involved in the conversion of plasminogen to plasmin, contributing to the higher levels of plasmin occurring in milk isolated from mastitic quarters.

Direct Assay For Factor XII In Plasma

1981 ◽  
Author(s):  
T Zuffi ◽  
R Jordan
Keyword(s):  
Trypsin Inhibitor ◽  
Dextran Sulfate ◽  
Factor Xii ◽  
Chromogenic Substrate ◽  
Plasma Samples ◽  
Contact Activation ◽  
Assay Procedure ◽  
Sulfate Activation ◽  
Kinetics Of ◽  
Hydrolysis Of

A chromogenic substrate assay for FXII in plasma samples has been developed. The method involves 0°C activation of the contact factors with dextran sulfate, and a direct selective measurement of the FXIIa activity generated by hydrolysis of the synthetic substrate S2302 in the presence of the kallikrein inhibitor, soybean trypsin inhibitor. Using these procedures, the kinetics of both FXII and pre- kallikrein activation could be studied and it was found that the relative rates of activation of these two zymogens did not necessarily coincide.When this dextran sulfate treatment of FXII or HMWK-deficient plasmas was tried, insignificant kallikrein or FXIIa activity was generated, demonstrating that contact activation was not occurring. These studies have shown that a dextran sulfate activation can be used as an assay procedure for FXII in plasma and can distinguish normal from certain deficient plasmas.

Inhibition of Enzymatic Reactions. A Rapid Method to Determine the Index pI50

2002 ◽  
Vol 57 (5-6) ◽  
pp. 496-499 ◽  
Author(s):  
David Kulhavý ◽  
Alexander Čegan ◽  
Karel Komers ◽  
Jaromír Mindl
Keyword(s):  
Rapid Method ◽  
Mixed Type ◽  
Enzymatic Reaction ◽  
Reaction Rates ◽  
Fast Method ◽  
Enzymatic Reactions ◽  
Initial Substrate ◽  
The One ◽  
Hydrolysis Of

The activity of every substance I inhibiting an enzymatic reaction can be approximately evaluated by the index pI50. This paper describes a simple and fast method of estimate and/or determination of this index. The method is based on the linearity of the dependence of the ratio of reaction rates of uninhibited and inhibited reaction vs. concentration of the inhibitor at constant initial substrate and enzyme concentrations for fully competitive, noncompetitive, uncompetitive and mixed type of inhibition by the one inhibitor. The validity of the method is demonstrated by four inhibitors of hydrolysis of acetylthiocholine by butyrylcholine esterase.

Chromogenic Substrate Methods For The Determination Of FVIIIc, Endotoxin And Plasminogen Activator

1981 ◽  
Author(s):  
E Eriksson ◽  
S Rosén ◽  
M Knös ◽  
P Friberger
Keyword(s):  
Heat Treatment ◽  
Cerebrospinal Fluid ◽  
Enzymatic Activity ◽  
Plasminogen Activator ◽  
Chromogenic Substrate ◽  
Good Reproducibility ◽  
Reaction Conditions ◽  
Assay Conditions ◽  
Selection Of

FVIII clotting activity in plasma and concentrates can be assayed with good reproducibility in the range 20-120% (C.V. approximately 3%) using purified FIXfl and FX in excess. By incubating the sample with above factors, Ca2+ and phospholipid, FX is activated in proportion to the amount of FVIIIc. Generated FXa is measured with the substrate S-2222. FVIIIc in the range below 20% can be determined by modifying the reaction conditions.Endotoxin in water solutions can be accurately determined down to 1 pg/ml by incubating the sample with an excess of Limulus lysate. The generated enzymatic activity which is linear in proportion to the amount of endotoxin, is then measured with the substrate S-2423 (Ac-Ile-Glu-Gly-Arg-pNA). Accurate endotoxin determinations in plasma and cerebrospinal fluid are also possible after destroying interfering inhibitors by heat treatment.Plasminogen activator activities can be measured via plasminogen and the plasmin substrate S-2251 or directly by substrate S-2288 (H-D-Ile-Pro-Arg-pNA). Interfering activities can be minimized by strategic selection of various protease inhibitors.In all the methods the assay conditions, reagents and procedure have been optimized.

Quantitative Determination of Plasminogen

1970 ◽  
Vol 23 (02) ◽  
pp. 191-201 ◽  
Author(s):  
H. D Bruhn ◽  
L Müller ◽  
F Duckert
Keyword(s):  
Quantitative Determination ◽  
Plasminogen Activator ◽  
Assay System ◽  
Two Stage ◽  
System Activation ◽  
Frozen Plasma

SummaryA modification of the caseinolytic assay for plasminogen is described. This assay system is characterized by the following features :1. Urokinase is used as activator achieving a complete activation of the plasminogen whereas with streptokinase caseinolytically inactive plasminogen-activator complexes are formed.2. All incubation times are reduced to the minimum which is still compatible with accuracy.3. Results are expressed in percent of a standard of ten normal plasmas.4. In this two-stage assay-system (activation of plasminogen to plasmin, digestion of casein by plasmin) both stages proceed simultaneously in the same system, thus the plasmin formed is stabilized “in statu nascendi” by the casein.5. Several conditions (stability of plasminogen in frozen plasma, use of anticoagulants, reproducibility) are defined.

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