Isolation of bovine angiogenin using a placental ribonuclease inhibitor binding assay

Biochemistry ◽  
1988 ◽  
Vol 27 (17) ◽  
pp. 6282-6287 ◽  
Author(s):  
Michael D. Bond ◽  
Bert L. Vallee
Keyword(s):  
Binding Assay ◽  
Ribonuclease Inhibitor ◽  
Inhibitor Binding ◽  
Placental Ribonuclease Inhibitor

Competitive Inhibitor Binding Assay (CIBA) of ACE Inhibitors

2009 ◽  
Vol 220 (S714) ◽  
pp. 43-47
Author(s):  
C. GRÖNHAGEN-RISKA ◽  
I. TIKKANEN ◽  
F. FYHRQUIST
Keyword(s):  
Ace Inhibitors ◽  
Binding Assay ◽  
Competitive Inhibitor ◽  
Inhibitor Binding

Inhibitor binding assay for angiotensin-converting enzyme.

1984 ◽  
Vol 30 (5) ◽  
pp. 696-700 ◽  
Author(s):  
F Fyhrquist ◽  
I Tikkanen ◽  
C Grönhagen-Riska ◽  
L Hortling ◽  
M Hichens
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Binding Assay ◽  
Enzyme Inhibitor ◽  
Specific Binding ◽  
Biological Fluids ◽  
The Novel ◽  
Converting Enzyme ◽  
Inhibitor Binding ◽  
Standard Curve

Abstract We describe a new principle for the determination of enzymes, here applied to angiotensin-converting enzyme (ACE, EC 3.4.15.1) in human serum. The enzyme inhibitor binding assay is based on specific binding of labeled inhibitor to the active center of the enzyme. Serum (10-15 microL) is incubated with 125I-labeled ACE inhibitor (" 351A ," a p- hydroxybenzamidine derivative of N-(1-carboxy-3-phenylpropyl)-L-lysyl-L-proline) at pH 7.0 at 37 degrees C for 2 h in a non-equilibrated system. Inhibitor bound to ACE is separated by adsorption to coated charcoal, the radioactivity remaining in the supernate is counted, and the ACE value is calculated from a standard curve. Sensitivity for ACE in serum is 200 U/L, corresponding to 5.0 ng of ACE purified from human lung. The coefficient of variation was 3.9% within assay, and 6.4% between assays for normal ACE activities. Correlation with a comparison spectrophotometric method (Am J Med 59: 363-372, 1975) for ACE assay was excellent (r = 0.98) in 59 samples from healthy subjects and from patients with various diseases including active sarcoidosis. The novel assay principle presented here is simple and specific, and can be extended to use with various biological fluids and tissues, and to other enzymes as well.

scholarly journals Sensitivity of monomeric and dimeric forms of bovine seminal ribonuclease to human placental ribonuclease inhibitor

1992 ◽  
Vol 281 (2) ◽  
pp. 343-348 ◽  
Author(s):  
B S Murthy ◽  
R Sirdeshmukh
Keyword(s):  
Enzyme Activity ◽  
Gel Filtration ◽  
Enzyme Structure ◽  
Inhibitor Concentration ◽  
Ribonuclease Inhibitor ◽  
Inhibitor Study ◽  
Bovine Seminal Ribonuclease ◽  
Dimeric Form ◽  
Placental Ribonuclease Inhibitor ◽  
Assay Conditions

We have studied the inhibition of bovine pancreatic RNAase (RNAase A) and bovine seminal RNAase in its native dimeric form (RNAase BS-1) and in monomeric carboxymethylated form (MCM RNAase BS-1) by human placental RNAase inhibitor (RNAase inhibitor) in order to understand the effect of enzyme structure on its response to the inhibitor. Study of the inhibition as a function of inhibitor concentration revealed that RNAase A and MCM RNAase BS-1 were inhibited fully and the inhibitor-sensitivities of the two were comparable. But under identical inhibitor concentrations RNAase BS-1 was found to be virtually insensitive to the inhibitor; at higher (3-10-fold) inhibitor concentrations marginal inhibition of the native enzyme could be observed. When RNAase BS-1 was pretreated with 5 mM-dithiothreitol (DTT) and assayed, it exhibited greater inhibitor-sensitivity, presumably as a result of its partial monomerization on exposure to DTT. This DTT-mediated change in the response of RNAase BS-1 to the inhibitor did not, however, seem to occur either in the assay conditions (which included DTT) or even when the enzyme was pretreated with DTT in the presence of the substrate, suggesting an effect of the substrate on the enzyme behaviour towards the inhibitor. Independently, gel-filtration runs revealed that, although DTT treatment caused monomerization of RNase BS-1, this change did not take place when DTT treatment was carried out in the presence of the substrate. From our observations, we infer that differential inhibitor-sensitivity of the dimeric and monomeric forms of RNAase BS-1, the relative contents of the two forms and the influence of the substrate on them may be important determinants of the net enzyme activity in the presence of the inhibitor.

Inhibitor Binding Assay of rat Serum Angiotensin Converting Enzyme

1984 ◽  
Vol 67 (2) ◽  
pp. 237-241 ◽  
Author(s):  
Ilkka Tikkanen ◽  
Frej Fyhrquist ◽  
Terje Forslund
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Enzyme Assay ◽  
Binding Assay ◽  
Specific Binding ◽  
Great Sensitivity ◽  
Converting Enzyme ◽  
Inhibitor Binding ◽  
Rat Serum ◽  
Active Centre ◽  
Serum Angiotensin Converting Enzyme

1. Based on a specific binding of labelled inhibitor to the enzyme active centre, a new principle of enzyme assay, inhibitor binding assay (IBA), was developed and applied to measurement of rat serum angiotensin converting enzyme (ACE). 2. Serum diluted 1:50 was incubated with 125I-labelled ACE inhibitor, 351A, at pH 7.0, 37°C, for 2 h. Inhibitor bound to ACE was separated with coated charcoal and results were calculated from a standard curve. 3. The advantages offered by the novel inhibitor binding assay include simplicity, specificity, absence of interference by other enzymes or immunological cross-reactions, and great sensitivity enabling measurement of ACE in concentrations less than 0.1 units/ml. 4. This principle of enzyme assay will not only have potential new applications for research involving ACE but may also be extended to other enzymes.

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