Purification of an alkaline phosphatase-lipoprotein-X complex.

1980 ◽  
Vol 26 (5) ◽  
pp. 604-608
Author(s):  
R N Weijers ◽  
H Kruijswijk ◽  
J M Baak
Keyword(s):  
Alkaline Phosphatase ◽  
Affinity Chromatography ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Relative Molecular Mass ◽  
Agarose Gel ◽  
Extrahepatic Cholestasis ◽  
Abnormal Position ◽  
Alkaline Phosphatase Isoenzyme ◽  
Isoenzyme Patterns

Abstract An alkaline phosphatase isoenzyme was observed in an abnormal position in the agar-agarose gel pattern of sera from several patients suffering from intrahepatic and extrahepatic cholestasis. We purified this isoenzyme, by gel filtration and affinity chromatography, from the serum of a patient suffering from extrahepatic cholestasis. Analysis demonstrated an alkaline phosphatase-lipoprotein-X complex with a relative molecular mass of at least 669,000. We discuss the interpretation of alkaline phosphatase isoenzyme patterns produced by different techniques.

Purification of an alkaline phosphatase-lipoprotein-X complex.

1980 ◽  
Vol 26 (5) ◽  
pp. 604-608 ◽  
Author(s):  
R N Weijers ◽  
H Kruijswijk ◽  
J M Baak
Keyword(s):  
Alkaline Phosphatase ◽  
Affinity Chromatography ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Relative Molecular Mass ◽  
Agarose Gel ◽  
Extrahepatic Cholestasis ◽  
Abnormal Position ◽  
Alkaline Phosphatase Isoenzyme ◽  
Isoenzyme Patterns

Abstract An alkaline phosphatase isoenzyme was observed in an abnormal position in the agar-agarose gel pattern of sera from several patients suffering from intrahepatic and extrahepatic cholestasis. We purified this isoenzyme, by gel filtration and affinity chromatography, from the serum of a patient suffering from extrahepatic cholestasis. Analysis demonstrated an alkaline phosphatase-lipoprotein-X complex with a relative molecular mass of at least 669,000. We discuss the interpretation of alkaline phosphatase isoenzyme patterns produced by different techniques.

Atypical alkaline phosphatase isoenzyme in serum from a patient with Hodgkin's disease.

1984 ◽  
Vol 30 (5) ◽  
pp. 800-802 ◽  
Author(s):  
J P Beilby ◽  
P Garcia-Webb ◽  
C I Bhagat ◽  
A Prins
Keyword(s):  
Alkaline Phosphatase ◽  
Molecular Mass ◽  
Hodgkin's Disease ◽  
Serum Alkaline Phosphatase ◽  
Hodgkin’S Disease ◽  
Heat Inactivation ◽  
Relative Molecular Mass ◽  
Agarose Gel Electrophoresis ◽  
Post Translational Modification ◽  
Alkaline Phosphatase Isoenzyme

Abstract An alkaline phosphatase isoenzyme that did not move from the origin in agarose gel electrophoresis was detected in serum from a 51-year-old woman with Hodgkin's disease. Inhibitor and heat-inactivation studies of the patient's serum alkaline phosphatase showed properties resembling those of both liver and bone isoenzymes. No immunoglobulin or high-molecular-mass complexes with the alkaline phosphatase isoenzyme were detected. The relative molecular mass (Mr) of the atypical alkaline phosphatase isoenzyme was 182 000, that of the liver alkaline phosphatase isoenzyme control 170 000. Treatment of both of these isoenzymes with neuraminidase gave a product with an Mr of 140 000. We propose that a post-translational modification increased the carbohydrate content of the liver alkaline phosphatase isoenzyme, thus changing the charge characteristics of the enzyme and decreasing its electrophoretic mobility. We believe this to be the first report of a post-translational modification in a heat-sensitive isoenzyme of alkaline phosphatase.

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.

scholarly journals Separation and purification of the alkaline phosphatase and a phosphodiesterase from Halobacterium cutirubrum

1979 ◽  
Vol 181 (2) ◽  
pp. 347-353 ◽  
Author(s):  
P S Fitt ◽  
P Baddoo
Keyword(s):  
Alkaline Phosphatase ◽  
Gel Filtration ◽  
Adsorption Chromatography ◽  
Agarose Gel ◽  
Good Recovery ◽  
Separation And Purification ◽  
Phosphodiesterase Activity ◽  
No Adsorption ◽  
Molecular Weights ◽  
Crude Extracts

1. Halobacterium cutirubrum alkaline phosphatase is associated in crude extracts with a phosphodiesterase. 2. The enzymes were stabilized in buffers containing both (NH4)2SO4 and 10 mM-Mn2+. 3. Adsorption chromatography on Sepharose 6B/agarose-gel columns in the presence of 1.4M-(NH4)2SO4 gave a phosphatase-free phosphodiesterase and the alkaline phosphatase associated with some phosphodiesterase activity. 4. Further chromatography of the separated enzymes gave a good recovery of greater than 600-fold purified phosphodiesterase and greater than 3000-fold purified alkaline phosphatase. 5. The requirements of these enzymes and their relationship to each other was examined. 6. A detailed study showed that the alkaline phosphatase was adsorbed at least partially to agarose and dextran columns at all (NH4)2SO4 concentrations from 0.25 to 2M. 7. In contrast, no adsorption of the enzyme or protein standards was evident in 2.5M-KCl/l M-NaCl or 0.25 M-KCl/0.1 M-NaCl, in agreement with previous studies by Louis, Peterkin & Fitt [(1971) Biochem. J. 121, 635-641], thus confirming the validity of gel filtration in 2.5 M-KCl/1 M-NaCl as a method for determining the approximate molecular weights of extremehalophile proteins.

Urine contains an inhibitor for turbidimetric determinations of protein.

1982 ◽  
Vol 28 (11) ◽  
pp. 2294-2296
Author(s):  
J Nakamura ◽  
M Yakata
Keyword(s):  
Molecular Mass ◽  
Gamma Globulin ◽  
Gel Filtration ◽  
Relative Molecular Mass ◽  
Sulfosalicylic Acid ◽  
Turbidimetric Method ◽  
Analytical Recovery ◽  
Dye Binding ◽  
Turbidimetric Assay ◽  
Contrast Measurement

Abstract Our examination of urine components separated by gel filtration revealed the presence of an inhibitor that decreases the analytical recovery of protein in a turbidimetric assay involving sulfosalicylic acid as reagent (Proc. Soc. Exp. Biol. Med. 92: 748, 1956). The apparent relative molecular mass of this inhibitor was in the range 160 000-240 000. A study with purified proteins showed a similar inhibition by gamma-globulin, glycoprotein, and beta-lipoprotein in the assay of albumin by the same turbidimetric method. In contrast, measurement of protein by a dye binding method was not affected by these materials. The low values for apparent urinary protein given by the turbidimetric method as compared with those by the dye-binding method are at least partly ascribable to the inhibitor.

scholarly journals Cardiac Troponin T Circulates in the Free, Intact Form in Patients with Kidney Failure

2006 ◽  
Vol 52 (3) ◽  
pp. 414-420 ◽  
Author(s):  
Michael N Fahie-Wilson ◽  
David J Carmichael ◽  
Michael P Delaney ◽  
Paul E Stevens ◽  
Elizabeth M Hall ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Kidney Failure ◽  
Cardiac Troponin ◽  
Troponin T ◽  
Gel Filtration ◽  
Serum Prolactin ◽  
Relative Molecular Mass ◽  
Cardiac Troponin T ◽  
Coronary Syndrome ◽  
Esrd Patients

Abstract Background: The clinical significance of the increased concentrations of cardiac troponins observed in patients with end stage renal disease (ESRD) in the absence of an acute coronary syndrome (ACS) is controversial. One proposed explanation is that immunoreactive fragments of cardiac troponin T (cTnT) accumulate in ESRD. We used gel-filtration chromatography (GFC) to ascertain whether fragments of cTnT, which could cross-react in the commercial diagnostic immunoassay (Roche Diagnostics), were the cause of the increased cTnT in the serum of patients with ESRD. Methods: We subjected sera from ESRD patients (n = 21) receiving dialysis and having increased cTnT concentrations to size-separation GFC. We detected cTnT in the chromatography fractions by use of the same antibodies used in the commercial assay for serum cTnT. Results: In all patients, cTnT immunoreactivity eluted as a major, homogeneous peak in an identical position between the peaks of serum prolactin [relative molecular mass (Mr) 23 000] and albumin (Mr 67 000): the elution pattern of cTnT in samples obtained from ACS patients was identical to that of the ESRD patients. There was no evidence that low–molecular-mass (Mr <23 000) cTnT fragments were the cause of the increased cTnT in the patients studied. Conclusions: The form of cTnT observed in the serum of patients with kidney failure and immunoreactive in the diagnostic assay is predominantly the free intact form, as in patients with ACS. Our data are consistent with the view that circulating cTnT in renal failure reflects cardiac pathology.

Plasma alkaline phosphodiesterase I in intrahepatic cholestasis induced by α-naphthylisothiocyanate in rats

1988 ◽  
Vol 75 (1) ◽  
pp. 13-20 ◽  
Author(s):  
Julie A. Quayle ◽  
Alison Capstick ◽  
Anthony I. Morris ◽  
David Billington
Keyword(s):  
Alkaline Phosphatase ◽  
Bile Acid ◽  
Bile Duct ◽  
Intrahepatic Cholestasis ◽  
Bile Duct Ligation ◽  
Gel Filtration ◽  
Extrahepatic Cholestasis ◽  
Rat Serum ◽  
Duct Ligation ◽  
Dose Dependent

1. Administration of α-naphthylisothiocyanate (ANIT) to rats produced dose-dependent increases in plasma bile acid and bilirubin concentrations. Similar increases in plasma bile acid and bilirubin concentrations were evident in bile duct ligated rats, indicating that the severity of cholestasis is almost identical in both models. 2. Plasma alkaline phosphodiesterase I was increased by only 50–80% while alkaline phosphatase was increased more than threefold after ANIT administration. This is in contrast to an earlier study [S. R. Simpson, K. Rahman & D. Billington (1984) Clinical Science 67, 647–652] where, after bile duct ligation, serum alkaline phosphodiesterase I was elevated sixfold before any increase in alkaline phosphatase activity became apparent. Thus, plasma alkaline phosphodiesterase I does not offer as sensitive a marker of intrahepatic cholestasis (induced by ANIT) as it does of extrahepatic cholestasis (induced by bile duct ligation). 3. Hepatic alkaline phosphodiesterase I was unaffected by ANIT pretreatment while hepatic alkaline phosphatase was increased up to seven times. It is suggested that raised plasma alkaline phosphodiesterase I is due to regurgitation of the biliary enzyme rather than overspill of the enzyme from liver into blood. 4. Gel filtration showed that 24 h and 96 h after ANIT administration, rat serum contained a high molecular weight form of alkaline phosphodiesterase I, suggesting a different isoenzyme profile.

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