scholarly journals Isolation and immunochemical characterization of human cystatin C

2002 ◽  
Vol 2 (1-2) ◽  
pp. 12-15
Author(s):  
Lejla Begić ◽  
Selma Berbić
Keyword(s):  
Renal Failure ◽  
Chronic Renal Failure ◽  
Affinity Chromatography ◽  
Cystatin C ◽  
Gel Filtration ◽  
Physiological Role ◽  
Clinical Importance ◽  
Exchange Chromatography ◽  
Serum Samples ◽  
Human Cystatin C

Cystatin C is a natural inhibitor of the cysteine proteinases papain, and mammalian lysosomal cathepsins B, H, L and S. This protein is thought to serve an important physiological role as a local regulator of enzyme activity. The changes of levels of cystatin C in extracellular fluids have shown themselves having potential clinical importance. We have purified cystatin C from urine of patients with chronic renal failure by procedure using affinity chromatography on CM-papain Sepharose, gel filtration on Sephacryl S-200, and ion exchange chromatography on CM-cellulose. After isolation we obtained three inhibitory peaks (pI's from 7.8 to 9.2) which represent isoforms of the same protein. These isoforms are immunologically identical and differ in N-terminal sequence of the molecule. The form with pI 9.2 represents the intact inhibitor form, whereas the form with pI 7.8 is shortened for 8 amino-acid residues at N-terminal end. Purified cystatin C pI 9.2 was used for immunization of rabbits. Polyclonal antibodies, produced in rabbits, were isolated from rabbit sera by affinity chromatography on Protein A Sepharose. Enzyme immunoassay (ELISA) for cystatin C is developed on the basis of purified antibodies. Using ELISA test we determined amount of cystatin C in urine and serum samples of patients with chronic renal failure. The concentration of the inhibitor in the urine of these patients was approximately 100-fold more than in normal urine. In the serum from the same patients we found concentrations of cystatin C to be five times higher in comparison with the serum of healthy individuals.

Methods for studying the binding of aluminum by serum protein.

1985 ◽  
Vol 31 (12) ◽  
pp. 1969-1973 ◽  
Author(s):  
H Rahman ◽  
A W Skillen ◽  
S M Channon ◽  
M K Ward ◽  
D N Kerr
Keyword(s):  
Renal Failure ◽  
Chronic Renal Failure ◽  
Affinity Chromatography ◽  
Serum Protein ◽  
Reliable Method ◽  
Binding Protein ◽  
Gel Filtration ◽  
Normal Subjects ◽  
Quantitative Studies

Abstract We describe methods for studying the binding of Al by protein in serum: ultrafiltration, gel filtration, and immuno-affinity chromatography. For ultrafiltration we used an Amicon YM10 cellophane membrane with a nominal cutoff of 10 000 Da to separate ultrafiltrable and non-ultrafiltrable Al. For gel filtration we used Sephacryl S-300, and for immuno-affinity chromatography we used anti-transferrin coupled to CNBr-activated Sepharose to identify the Al-binding protein. For 30 normal subjects 54% of the total Al in serum was non-ultrafiltrable; for 30 patients with chronic renal failure being treated by hemodialysis 67% was non-ultrafiltrable. In both groups transferrin was identified as the major Al-binding protein in the serum. Results of gel-filtration studies should be interpreted with caution: some gel media adsorb "free" Al, which can be subsequently taken up by transferrin or desferrioxamine passing through the column. We find affinity chromatography to be a specific and reliable method, suitable for use in quantitative studies.

Isolation of specific protease inhibitors from Neurospora crassa

1976 ◽  
Vol 54 (8) ◽  
pp. 699-703 ◽  
Author(s):  
Peter H. Yu ◽  
Maria R. Kula ◽  
Hsin Tsai
Keyword(s):  
Neurospora Crassa ◽  
Protease Inhibitors ◽  
Gel Filtration ◽  
Physiological Role ◽  
Exchange Chromatography ◽  
Proteinase K ◽  
Tryptophan Synthase ◽  
Molecular Weights ◽  
Specific Protease

Four natural protease inhibitors have been partially purified by heat treatment, ion-exchange chromatography and gel filtration from Neurospora crassa. The inhibitory activity has been estimated by measuring the inhibition of proteolysis of casein as well as by the protection of Neurospora tryptophan synthase from proteolytic inactivation. The inhibitors are all oligopeptides and possess molecular weights in the range 5000 – 24 000 and appear to be very specific to Neurospora proteases. They may be classified into two types. The first are specific to Neurospora alkaline protease and the second to acidic protease. None of them exhibited any effect on other proteases including trypsin, chymotrypsin, papain, pepsin, thermolysin, subtilisin and proteinase K. The possible physiological role of these inhibitors is discussed.

scholarly journals Measurement of strontium in serum, urine, bone, and soft tissues by Zeeman atomic absorption spectrometry

1997 ◽  
Vol 43 (1) ◽  
pp. 121-128 ◽  
Author(s):  
Patrick C D’Haese ◽  
Glen F Van Landeghem ◽  
Ludwig V Lamberts ◽  
Vera A Bekaert ◽  
Iris Schrooten ◽  
...  
Keyword(s):  
Renal Failure ◽  
Chronic Renal Failure ◽  
Atomic Absorption Spectrometry ◽  
Atomic Absorption ◽  
Soft Tissues ◽  
Absorption Spectrometry ◽  
Serum Samples ◽  
Tissue Samples ◽  
Triton X

Abstract To study the possible accumulation of Sr in chronic renal failure patients, methods were developed for the determination of the element in serum, urine, bone, and soft tissues by using Zeeman atomic absorption spectrometry. Serum samples were diluted 1:4 with a Triton X-100–HNO3 mixture, whereas urine samples were diluted 1:20 with HNO3. Bone samples were digested with concentrated HNO3 in stoppered polytetrafluoroethylene (Teflon®) tubes, whereas soft tissues were dissolved in a tetramethylammonium hydroxide solution in water. For serum and urine we used matrix-matched calibration curves, whereas bone and tissue samples were measured against aqueous calibrators. Atomization was performed from the wall of pyrolytically coated graphite tubes for all of the matrices under study. Both inter- and intraassay CVs were <6% (n = 12, n = 10, respectively), and the recovery of added analyte was close to 100% for all of the biological matrices under study. Detection limits were 1.2 μg/L (serum), 0.3 μg/L (urine), 0.4 μg/g (bone), and 2.2 ng/g (soft tissues), whereas the sensitivity determined by the slope of the calibration curve, i.e., the amount of Sr producing a 0.0044 integrated absorbance change in signal, was 2.4 pg, 2.4 pg, 3.9 pg, and 2.6 pg for these matrices respectively. We conclude that the present methods are precise and accurate and easily applicable for both routine use and research investigations. They will allow us to study the metabolism of the element in chronic renal failure patients and shed some light on the association that was recently noted between increased bone Sr concentrations and the development of osteomalacia in these individuals.

False-Positive Titers of Thyroid Autoantibodies in Patients Undergoing Hemodialysis?

1991 ◽  
Vol 37 (12) ◽  
pp. 2153-2154 ◽  
Author(s):  
Ljerka Lukinac ◽  
Zvonko Kusic ◽  
Petar Kes
Keyword(s):  
Renal Failure ◽  
Chronic Renal Failure ◽  
False Positive ◽  
Serum Samples ◽  
Standard Procedures ◽  
Before And After ◽  
Thyroid Function Testing ◽  
Total Triiodothyronine ◽  
The Difference ◽  
Function Testing

Abstract Concentrations of thyroid-related hormones in serum of patients with chronic renal failure are known to be abnormal (1, 2). In our study on thyroid-function testing of patients undergoing hemodialysis, we determined, in addition to concentrations of free and total triiodothyronine, free and total thyroxin, “reverse” triiodothyronine, and thyroxin-binding globulin, the titers of thyroglobulin and microsomal autoantibodies (TGA and TMA, respectively). The study was provoked by the appearance of an uncommon agglutination pattern in the control wells of some samples from patients with chronic renal failure during the standard procedures of detecting TGA and TMA with hemagglutination methods (Thymune-T and Thymune-M assays from Welcome, London, U.K.). For these samples we were not certain whether positive titers for TGA and (or) TMA represented false-positive or true-positive values. Therefore, we assayed the absorbed serum samples and samples after addition of excess nonspecific immunoglobulin. Furthermore, we wanted to determine the difference in TGA and TMA titers of serum samples before and after hemodialysis.

scholarly journals Mouse Uterine Alkaline Phosphatase: Improved Purification by Affinity Chromatography and Further Characterization of the Enzyme

1980 ◽  
Vol 33 (3) ◽  
pp. 279 ◽  
Author(s):  
RN Murdoch ◽  
Louise E Buxton ◽  
DJ Kay
Keyword(s):  
Alkaline Phosphatase ◽  
Affinity Chromatography ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Anion Exchange Chromatography ◽  
Pregnant Mouse ◽  
Exchange Chromatography ◽  
Enzyme Preparations

An improved procedure for the purification of alkaline phosphatase from about 10 g of day 7 pregnant mouse uterine tissue is described. Following homogenization, the procedure involved solubilization and extraction with 0�8% (v/v) Triton X-lOO and 20% (v/v) n-butanol, ammonium sulfate precipitation, concanavalin A-Sepharose 4B affinity chromatography, DEAE-cellulose anion-exchange chromatography and Sephacryl S200 gel filtration. On subjecting 2162-fold purified enzyme preparations to polyacrylamide-gel electrophoresis, a single band of protein coincident with the zone of enzyme activity and having an apparent molecular weight of 205 OOO� lOOOO was identified. Affinity chromatography yielded the largest increase in purity of any step in the procedure and established the glycoprotein nature of the uterine enzyme.

A sensitive and practical bioassay for thyrotropin using cultured FRTL-5 cells: assessment of bioactivity for serum TSH in patients with chronic renal failure

1989 ◽  
Vol 121 (2) ◽  
pp. 191-196 ◽  
Author(s):  
Masateru Horimoto ◽  
Mitsushige Nishikawa ◽  
Norio Yoshikawa ◽  
Mitsuo Inada
Keyword(s):  
Renal Failure ◽  
Chronic Renal Failure ◽  
Regression Line ◽  
Normal Subjects ◽  
Analysis Of Covariance ◽  
Serum Samples ◽  
Solution Ph ◽  
Phosphate Buffered Saline ◽  
Serum Tsh ◽  
Hcl Solution

Abstract. A sensitive bioassay for TSH employing a practical extraction method was developed, and the bioactivities in patients with chronic renal failure receiving hemodialysis were compared with those in normal subjects. Serum samples were obtained from 12 normal subjects and 12 patients with chronic renal failure receiving hemodialysis. TSH was extracted from the serum using anti-human TSH monoclonal antibody coated tubes, followed by elution with 2.0 mol/l guanidine-HCl solution (pH 3.2). After the eluate had been dialyzed against phosphate buffered saline (pH 7.4) and again against TRIS-HCl solution (pH 7.4) and then lyophilized, it was reconstituted with hypotonic Hanks' solution. Bioassay for TSH was performed by measuring the levels of cAMP released into the medium from cultured FRTL-5 cells incubated with the extract. The mean immunoreactive recovery rates of TSH from the serum in normal subjects and patients with chronic renal failure were about 42% (± 6) and 40% (± 2), respectively. The present bioassay was sufficiently sensitive to detect a serum TSH level of 1.0 mU/l when 3.0 ml of serum was used. Extracts from standard sera at concentrations ranging from 1.0 to 10 mU/l added to the culture medium caused significant linear increases in cAMP production. Based on analysis of covariance the regression line between the immunoreactivities and bioactivities of serum TSH in patients with chronic renal failure (y = 0.90x + 0.3, r = 0.92) was not significantly different from that in normal subjects (y = 1.04x + 0.1, r = 0.93). These results suggest that the present bioassay for TSH is sensitive and practical, and that the bioactivity of TSH in patients with chronic renal failure is similar to that in normal subjects.

scholarly journals Current Trends in Protein Purification : A Review

2020 ◽  
pp. 279-310
Author(s):  
Angela Boxi ◽  
Isha Parikh ◽  
Radhika B S ◽  
Shryli K S
Keyword(s):  
High Pressure ◽  
Liquid Chromatography ◽  
Ion Exchange ◽  
Affinity Chromatography ◽  
Protein Purification ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Current Trends ◽  
Comparative Information

The present review is based on papers published between 1990 and 2020 and gives Comparative information about the most common protein purification techniques Gel-Filtration, Chromatography, Ion-Exchange Chromatography, Electrophoresis, Affinity Chromatography, and Dialysis, High-Pressure Liquid Chromatography. and their applications.

scholarly journals Evaluation Activity of Alanine Aminopeptidase (AAP) in Patients with Renal Dialysis, Purification and Isolation of Isoenzymes

2019 ◽  
pp. 265-272
Author(s):  
Taghreed Mohammad ◽  
Sawsan Mahdi ◽  
Tammara Al-Kareim
Keyword(s):  
Renal Failure ◽  
Chronic Renal Failure ◽  
Renal Dialysis ◽  
Exchange Chromatography ◽  
Aminopeptidase Activity ◽  
Control Group ◽  
Efficacy Rate ◽  
Loss Of Function ◽  
Evaluation Activity ◽  
Alanine Aminopeptidase

Chronic renal failure is the progressive loss of function of kidney and patient requires a long treatment in the form of renal replacement therapy. Alanine aminopeptidase activity has been assayed in (70) patient’s sera samples of both sexes, aged (25-70) years. The sample were divided in to two groups, the first group(G1) is of (35) samples before dialysis. The second group(G2) is of (35) samples after dialysis, as well as (30) sample of healthy as control group(C). The result showed a significant increase (p> 0.001) in general in the enzyme Alanine aminopeptidase in the serum of chronic renal failure patients (116.0±3.4) U/L while the efficacy rate is (105.5±3.4) U/L in healthy persons. Goal of the research was partial purified of enzyme from sera patients with chronic renal failure. by using two techniques: The first gel filtration chromatography packed with spadix G50 and giving 3.45% yield and 0.964-fold purification. Two isoenzymes were obtained by using ion exchange chromatography and the purity degree of isoenzymes were 0.687(1.08%) and 2.539(1.04%) fold respectively. Also, the results showed a single band for isoenzymes following steps.

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