scholarly journals QUANTITATION OF TISSUE-BOUND RENAL AMINOPEPTIDASE BY A MICRODENSITOMETRIC TECHNIQUE

1966 ◽  
Vol 14 (1) ◽  
pp. 53-63 ◽  
Author(s):  
K. FELGENHAUER ◽  
G. G. GLENNER
Keyword(s):  
Tissue Section ◽  
Diazonium Salt ◽  
Rat Kidney ◽  
Significant Proportion ◽  
Enzymic Activity ◽  
Assay Method ◽  
Assay Procedure ◽  
Order Of Magnitude ◽  
Proximal Tubuli ◽  
Ph Level

Relationships between biochemical and histochemical assay systems can be evaluated by appropriate techniques. With rat kidney as the test object the enzyme localized in the tissue section hydrolyzing l-leucyl-β-naphthylamide (LNA) was found to be identical with a purified particle-bound, cobalt-activated aminopolypeptidase previously characterized biochemically. A soluble sulfhydryl-dependent aminopeptidase, present in the rat kidney and also known to hydrolyze LNA, was not demonstrated in the histochemical system, although the diazonium salt employed incidentally caused the insolubilization in the tissue section of a significant proportion of previously extractable, particle-bound enzyme. Linearity between the enzymic activity present and the measurable enzymic activity was demonstrated to exist at the substrate concentration, pH level, section thickness and diazonium salt concentrations used in the assay procedure, but did not exist when these factors were varied beyond certain well defined limits. In addition, the effect of diazonium salt on both the inhibition and insolubilization of enzyme in the tissue section could be quantitated. Based on this evaluation of the enzymes hydrolyzing LNA in the rat kidney, a microdensitometric assay method employing a constant flow incubating chamber was developed to characterize and quantitate the LNA-hydrolyzing enzyme in the proximal tubuli. This study defines many of the parameters necessary for the future investigation by quantitative and qualitative methods of histochemical systems capable of providing resolutions of a higher order of magnitude and the retention of a greater proportion of extractable enzyme within the tissue section.

scholarly journals Determination of high-affinity oestrogen-receptor sites in uterine supernatant preparations

1970 ◽  
Vol 120 (4) ◽  
pp. 831-836 ◽  
Author(s):  
J. Méšter ◽  
D. M. Robertson ◽  
Patricia Feherty ◽  
A. E. Kellie
Keyword(s):  
Dissociation Constant ◽  
Oestrogen Receptor ◽  
Scatchard Plot ◽  
Assay Method ◽  
Physiological Conditions ◽  
High Affinity ◽  
Receptor Sites ◽  
Assay Procedure ◽  
Order Of Magnitude

An assay method was developed for the determination of high-affinity oestradiol receptors in uterine supernatant preparations. When only high-affinity sites are present in such preparations, or when they predominate, the analysis of the equilibrium between oestradiol and receptor sites based on the Scatchard (1949) plot may be used to determine the dissociation constant of the equilibrium and the molar concentration of the high-affinity sites. When both high-affinity and low-affinity sites are present the Scatchard plot is no longer linear and cannot be used directly to determine high-affinity sites. Determination of the reverse velocity constants of the reaction between high-affinity (k−1) and low-affinity (k−2) receptor sites and [3H]oestradiol has shown that these constants differ by at least one order of magnitude. Advantage has been taken of this difference to introduce an additional step into the assay procedure that eliminates oestradiol bound to low-affinity sites and permits the determination of high-affinity sites in different species and under a variety of physiological conditions.

Studies of calcium-45 desaturation from kidney slices in flow-through chambers

1978 ◽  
Vol 234 (1) ◽  
pp. R34-R38
Author(s):  
T. Uchikawa ◽  
A. B. Borle
Keyword(s):  
Kinetic Parameters ◽  
Rat Kidney ◽  
Kidney Cells ◽  
Tissue Slices ◽  
Kidney Slices ◽  
System A ◽  
Order Of Magnitude ◽  
Exponential Equations ◽  
Flow Through ◽  
Best Fit

This paper describes a method to measure calcium fluxes and calcium exchangeable pools in tissue slices by continuous perifusion in flow-through chambers. 45Ca desaturation from rat kidney slices can be analyzed as in an open three-compartment catenary system. A set of equations is given to calculate all the relevant kinetic parameters from the triple exponential equations which best fit the desaturation curves. The results show that the kinetic parameters obtained in kidney slices by this new method are in the same order of magnitude as those previously observed in cultured monkey kidney cells.

Radioimmunoassay for 4-hydroxyoestrone in human urine

1981 ◽  
Vol 97 (2) ◽  
pp. 251-257 ◽  
Author(s):  
Günter Emons ◽  
Claudia Mente ◽  
Rudolf Knuppen ◽  
Peter Ball
Keyword(s):  
Human Urine ◽  
Gas Chromatography Mass Spectrometry ◽  
Clear Cut ◽  
High Affinity ◽  
Assay Procedure ◽  
Female Children ◽  
Bovine Serum Albumin Conjugate ◽  
Order Of Magnitude ◽  
Excretion Rates

Abstract. Under the protection of ascorbic acid a 4-hydroxyoestrone-bovine serum albumin conjugate was prepared containing intact 4-hydroxyoestrone as determined by gas chromatography-mass spectrometry. Using this antigen, antibodies with high affinity and specificity for 4-hydroxyoestrone were raised in rabbits. An assay procedure for the determination of 4-hydroxyoestrone in human urine and the assessment of its reliability are described. The following urinary excretion rates were found: male children 0.29 μg/24 h, female children 0.35 μg/24 h, men (20–40 years) 1.6 μg/24 h, men (>50 years) 1.8 μg/24 h, women, follic. 2.0 μg/24 h, pre-ov. 5.3 μg/24 h, luteal 2.4 μg/24 h, women, pregnant, first trim. 30.0 μg/24 h, second trim. 64.0 μg/24 h, third trim. 48.0 μg/24 h, women, post-men. 1.5 μg/24 h. Thus the amounts of 4-hydroxyoestrone excreted in human urine are about 1/3 to 1/10 of those of 2-hydroxyoestrone. During the menstrual cycle the excretion rates of 4-hydroxyoestrone are in the same order of magnitude as those of oestradiol and show a clear-cut pre-ovulatory peak.

Clinical studies of the use of a fluorogenic substrate assay method for the determination of plasminogen

1979 ◽  
Author(s):  
Douglas Triplett ◽  
Cathy Harms ◽  
Lori Hermelin ◽  
Rolf Huseby ◽  
Gary Mitchell ◽  
...  
Keyword(s):  
Clinical Studies ◽  
Assay Method ◽  
Fluorogenic Substrate ◽  
Normal Range ◽  
Thrombosis Research ◽  
Normal Ranges ◽  
Assay Procedure ◽  
Medical Population ◽  
Pulmonary Bypass

Utilization of a recently reported method for the determination of plasminogen by fluorometric technique (Ref. 3, Pochron, et. al., Thrombosis Research, Vol. 13, pg. 733, 1978) was compared with acceptable methodologies (e.g. the caseinolytic and radial–immunodiffusion assays). Excellent results were seen with the normal range of 2.4–3.8 CTA U/ml of plasminogen by the synthetic substrate assay as compared to 8.7–14.3 mg/dl by the radialimmunodiffusion assay. Normal ranges for the caseinolvtic assay were seen to be 2.2–4.0 CTA U/ml. Patients’ studies were performed on a general medical population but centered primarily on “pre” and “post” operative coronary-pulmonary bypass patients. In all cases studied, a drop in Plasminogen level was noted in this group of patients following surgery (20-50%). Other clinical data will be presented indicating that the determination of plasminogen by the fluorometric assay procedure is sensitive and accurate for clinical situations.

Clinical Studies of the Use of a Fluorogenic Substrate Assay Method for the Determination of Plasminogen

1979 ◽  
Author(s):  
Douglas A. Triplett ◽  
Cathy Harms ◽  
Lori Hermelin ◽  
Rolf M. Huseby ◽  
Gary A. Mitchell ◽  
...  
Keyword(s):  
Radial Immunodiffusion ◽  
Assay Method ◽  
Fluorogenic Substrate ◽  
Normal Range ◽  
Thrombosis Research ◽  
Normal Ranges ◽  
Assay Procedure ◽  
Medical Population ◽  
Pulmonary Bypass

Utilization of a recently reported method for the determination of plasminogen by fluorometrit technique (Ref. 3, Pochron, et. al., Thrombosis Research, Vol. 13, pg. 733, 1978) was compared with acceptable methodologies (e.g. the caseinolvtic and radial-immunodiffusion assays). Excellent results were seen with the normal range of 2.4–3.8 CTA U/ml of plasminogen by the synthetic substrate assay as compared to 8.7-14.3 mg/dl by the radial immunodiffusion assay. Normal ranges for the caseinolvtic assay were seen to be 2.2-4.0 CTA U/ml. Patients’ studies were performed on a general medical population but centered primarily on “pre” and “post” operative coronarv-pulmonary bypass patients. In all cases studied, a drop in Plasminogen level was noted in this group of patients following surgery (20–50%). Other clinical data will be presented indicating that the determination of plasminogen by the fluorometric assay procedure is sensitive and accurate for clinical situations.

scholarly journals Improved Surface Display of Human Hyal1 and Identification of Testosterone Propionate and Chicoric Acid as New Inhibitors

2020 ◽  
Vol 13 (4) ◽  
pp. 54 ◽  
Author(s):  
Isabelle Lengers ◽  
Fabian Herrmann ◽  
Marc Le Borgne ◽  
Joachim Jose
Keyword(s):  
Enzyme Activity ◽  
Single Cell ◽  
Testosterone Propionate ◽  
Surface Display ◽  
Assay Method ◽  
Activity Assay ◽  
Enzyme Activity Assay ◽  
Chicoric Acid ◽  
Ic50 Value ◽  
Order Of Magnitude

Degradation of high molecular weight hyaluronic acid (HA) in humans is mainly catalyzed by hyaluronidase Hyal1. This enzyme is involved in many pathophysiological processes and therefore appears an interesting target for drug discovery. Until now, only a few inhibitors of human Hyal1 are known due to obstacles in obtaining active enzymes for inhibitor screening. The aim of the present work was to provide a convenient enzyme activity assay and show its feasibility by the identification of new inhibitors. By autodisplay, Escherichia coli F470 can present active Hyal1 on its surface. In this study, the inducible expression of Hyal1 on the cell surface of E. coli under the control of a rhamnose-dependent promoter (Prha) was performed and optimized. Enzyme activity per single cell was increased by a factor of 100 compared to the constitutive Hyal1 surface display, as described before. An activity of 6.8 × 10−4 mU per single cell was obtained under optimal reaction conditions. By this modified activity assay, two new inhibitors of human Hyal1 were identified. Chicoric acid, a natural compound belonging to the phenylpropanoids, showed an IC50 value of 171 µM. The steroid derivative testosterone propionate showed and IC50 value of 124 ± 1.1 µM. Both values were in the same order of magnitude as the IC50 value of glycyrrhizic acid (177 µM), one of the best known inhibitors of human Hyal1 known so far. In conclusion, we established a new enzyme activity assay for human Hyal1 and identified new inhibitors with this new assay method.

scholarly journals Assay of Catechol-O-Methyltransferase Activity in Human Erythrocytes Using Norepinephrine as a Natural Substrate

2002 ◽  
Vol 39 (6) ◽  
pp. 589-594 ◽  
Author(s):  
Mayumi Masuda ◽  
Makoto Tsunoda ◽  
Yukie Yusa ◽  
Shizuo Yamada ◽  
Kazuhiro Imai
Keyword(s):  
High Performance ◽  
Physiological State ◽  
Human Erythrocytes ◽  
Assay Method ◽  
Catecholamine Metabolism ◽  
Natural Substrate ◽  
Spontaneously Hypertensive ◽  
Assay Procedure ◽  
Comt Activity ◽  
Liquid Chromatography Separation

Background Catechol- O-methyltransferase (COMT) catalyses the inactivation of catecholamines. It is widely distributed in most tissues in soluble (S-COMT) and membrane-bound (MB-COMT) forms. Recently, we used a new assay for COMT activity and demonstrated that COMT plays an important role in blood pressure regulation in spontaneously hypertensive rats. In order to investigate whether this is true for human hypertension, we have evaluated the erythrocyte COMT assay in humans. Method The assay procedure included the use of norepinephrine (NE) as a natural substrate and the quantification of the reaction product, normetanephrine, followed by high-performance liquid chromatography separation and fluorescence or chemiluminescence detection. Results After evaluation of the method, the optimum conditions were obtained for the assay of human erythrocyte COMT. The S- and MB-COMT activities obtained were 50·6 (24·5) and 329·8 (179·4) fmol/min/mg protein, respectively [mean (standard deviation); n = 54]. The Km values for NE were 91·3 (14·1) and 11·7 (1·1) μmol/L for S- and MB-COMT, respectively ( n=6). Conclusion The established assay method used to assess S- and MB-COMT activities in human erythrocytes could be useful to elucidate catecholamine metabolism in the normal physiological state as well as in the pathology of certain diseases.

Evaluation of an Automated Dual-Channel Hydroxylamine Assay Procedure for Formulated Penicillin Products. II. Intact Solid Dosage Formulations

1974 ◽  
Vol 57 (6) ◽  
pp. 1325-1337
Author(s):  
Jonathan R Lane
Keyword(s):  
Drug Administration ◽  
Automated System ◽  
Assay Method ◽  
Manual Method ◽  
Solid Dosage ◽  
Dual Channel ◽  
Assay Procedure ◽  
Wide Range ◽  
Ampicillin Trihydrate

Abstract The dual-channel hydroxylamine method of Stevenson, Bechtel, and Coursen for the potency determination of formulated penicillins has been evaluated for use as an official Food and Drug Administration (FDA) alternative assay method for solid dosage formulations. A modified Technicon SOLIDprep Sampler I was used to assay a wide range of penicillin capsules and compressed tablets. In some instances, as many as 10 tablets/sample cup were tested. Results by this method and the official FDA manual method are compared. Some of the problems encountered in the analysis of ampicillin trihydrate capsule formulations and with compressed, film-coated tablets are also described. Based on the performance of the automated system and the data obtained, recommendations have been made to make the dual-channel hydroxylamine procedure an official FDA alternative assay technique for penicillin formulations.

scholarly journals THE ENZYMATIC HYDROLYSIS OF AMINO ACID β-NAPHTHYLAMIDES II. PARTIAL PURIFICATION AND PROPERTIES OF A PARTICLE-BOUND COBALT-ACTIVATED RAT KIDNEY AMINOPEPTIDASE

1966 ◽  
Vol 14 (5) ◽  
pp. 401-413 ◽  
Author(s):  
K. FELGENHAUER ◽  
G. G. GLENNER
Keyword(s):  
Amino Acid ◽  
Rat Kidney ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Disc Electrophoresis ◽  
Partial Purification ◽  
Soluble Enzyme ◽  
Solvent Fractionation ◽  
Biochemical Systems ◽  
Hydrolysis Of

In order to determine the relationship and characteristics of the soluble and tissue-bound (so-called "lyo" and "desmo") components of enzymes in histochemical and biochemical systems, a biochemical investigation of the hydrolysis of l-leucyl β-naphthylamide in rat kidney was undertaken. By ultracentrifugation, autolysis, salt and solvent fractionation procedures and DEAE-cellulose column chromatography primary soluble and tissuue-bound enzymes were separated and partially purified. The soluble enzyme was found to be inhibited by p-chloromercuribenzoate and reactivated by cysteine. The tissue-bound enzyme was found to be inhibited by o-phenanthroline and reactivated by Co++, and hydrolyzed peptides and amino acid β-naphthylamides at rates differing markedly from those of the soluble enzyme and commercial leucine aminopeptidase. Disc electrophoresis of the tissue-bound enzyme preparation demonstrated two protein bands, both revealing enzymic activity with almost identical hydrolytic characteristics, i.e., isozymes. This evidence established the identity of the tissue-bound Co++-activated enzyme with that found histochemically in the brush border of the proximal tubuli, and indicated its functional capacity as that of an aminopolypeptidase. It was also concluded that in the system investigated the "lyo" and "desmo" components represented, in reality, distinct enzymes with unique characteristics.

scholarly journals Cellular activation of mesangial gelatinase A by cytochalasin D is accompanied by enhanced mRNA expression of both gelatinase A and its membrane-associated gelatinase A activator (MT-MMP)

1996 ◽  
Vol 313 (3) ◽  
pp. 879-884 ◽  
Author(s):  
Menachem AILENBERG ◽  
Mel SILVERMAN
Keyword(s):  
Mesangial Cells ◽  
Rat Kidney ◽  
State Level ◽  
Enzymic Activity ◽  
Cytochalasin D ◽  
Gelatinase A ◽  
Cellular Activation ◽  
Reverse Transcription Pcr ◽  
Extracellular Compartment ◽  
Membrane Type

Activation of gelatinase A represents a crucial regulatory step in the control of its enzymic activity. Rat kidney mesangial cells secrete predominantly latent gelatinase A that can be activated following treatment with cytochalasin D. In the present paper we provide new evidence, using reverse transcription-PCR, that treatment of rat mesangial cells with cytochalasin D enhances the steady-state level of mRNA of the membrane-type matrix metalloproteinase (MT-MMP), as well as of gelatinase A, with no change in the level of tissue inhibitor of metalloproteinases-2 (TIMP-2) mRNA. Since the TIMP-2 protein level is reduced in conditioned medium from cytochalasin D-treated cells, the results of the present study are consistent with a model in which the action of cytochalasin D is to cause extracellular gelatinase A and TIMP-2 to be sequestered at the plasma membrane, forming a heterotrimeric complex with MT-MMP. In this manner, TIMP-2 may assume a bifunctional role causing: (i) inhibition of gelatinase A in the extracellular compartment; and (ii) guiding gelatinase A to activation through a membrane association with MT-MMP.

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