Binding of Al by protein in plasma of patients on maintenance hemodialysis.

1985 ◽  
Vol 31 (8) ◽  
pp. 1314-1316 ◽  
Author(s):  
M Cochran ◽  
D Patterson ◽  
S Neoh ◽  
B Stevens ◽  
R Mazzachi
Keyword(s):  
Molecular Mass ◽  
Binding Protein ◽  
Equilibrium Dialysis ◽  
Gel Filtration ◽  
Human Albumin ◽  
Hemodialysis Patients ◽  
Single Peak ◽  
Molar Ratio ◽  
Maintenance Hemodialysis ◽  
Mass Fractions

Abstract Gel filtration of plasma from hemodialysis patients, with use of reagents and apparatus with carefully minimized background Al concentrations, reproducibly showed a single peak for Al, corresponding exactly to the elution position of transferrin. The Al/transferrin molar ratio in adjacent fractions was constant (mean 0.126, SE 0.006) in replicate experiments. In contrast, the association of Al with albumin varied. Using both equilibrium dialysis and gel-filtration techniques, in the presence and absence of calcium or phosphate, we could demonstrate no significant binding of Al by human albumin at Al concentrations of 1 to 12 mumol/L. We saw no Al peak in pooled, concentrated, low-molecular-mass fractions of plasma gel-filtered on Sephadex G-50. Evidently, transferrin is the sole Al-binding protein in plasma of hemodialysis patients.

Purification and characterization of prostate-specific antigen (PSA) complexed to alpha 1-antichymotrypsin: potential reference material for international standardization of PSA immunoassays

1995 ◽  
Vol 41 (9) ◽  
pp. 1273-1282 ◽  
Author(s):  
Z Chen ◽  
A Prestigiacomo ◽  
T A Stamey
Keyword(s):  
Molecular Mass ◽  
Prostate Specific Antigen ◽  
Specific Antigen ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Molar Ratio ◽  
Free Psa ◽  
Apparent Homogeneity ◽  
International Standardization

Abstract We describe for the first time a protocol to purify to apparent homogeneity an in vitro-prepared complex of prostate-specific antigen (PSA) and alpha 1-antichymotrypsin (ACT) by using a combination of gel filtration and ion-exchange chromatography. The purity of the PSA-ACT complex was confirmed by gel electrophoresis and Western blot. The PSA-ACT complex was stable in the pH range 6.0 to 7.8; it was also stable in various matrices, temperatures, and high concentrations of salt. Purification of the PSA-ACT complex was highly reproducible. An absorptivity of 0.99 L x g-1 x cm-1 at 280 nm was assigned to the PSA-ACT complex, based on amino acid analysis. Because PSA and ACT bind in a 1:1 molar ratio, we determined the molecular mass of the PSA-ACT complex as the mass encoded by the cDNA of ACT (plus 26% carbohydrate) plus the molecular mass of PSA (28,430 Da), which totals 89,280 Da. Using this material, we made two common calibrators, one of 100% PSA-ACT complex and one of 90% PSA-ACT complex plus 10% free PSA by volume (90:10 calibrator). Substitution of these calibrators for the manufacturers' calibrators in nine commercial immunoassays substantially reduced differences between immunoassays, especially for serum PSA values between 4 and 10 micrograms/L. The 90:10 calibrator is recommended as a universal calibrator for international standardization of PSA immunoassays.

scholarly journals Molecular Distinction of Circulating Pregnancy-Associated Plasma Protein A in Myocardial Infarction and Pregnancy

2005 ◽  
Vol 51 (1) ◽  
pp. 75-83 ◽  
Author(s):  
Qiu-Ping Qin ◽  
Saara Kokkala ◽  
Juha Lund ◽  
Natalia Tamm ◽  
Liisa-Maria Voipio-Pulkki ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Pregnant Women ◽  
Molecular Mass ◽  
Plasma Protein ◽  
Specific Antibody ◽  
Protein A ◽  
Gel Filtration ◽  
Single Peak ◽  
Serum Samples ◽  
A Subunit

Abstract Background: In the blood of pregnant women, pregnancy-associated plasma protein A (PAPP-A) is present as a covalent complex with the proform of eosinophil major basic protein (proMBP). Recently, increased serum concentrations of PAPP-A have been found in acute coronary syndromes (ACS). The aim of this study was to investigate whether the circulating PAPP-A in ACS is the same as that in pregnancy. Methods: We developed two time-resolved immunofluorometric assays based on a relative epitope map constructed by the use of 17 monoclonal antibodies. One assay, which measured total PAPP-A, used two PAPP-A subunit-specific antibodies. The other assay, which measured PAPP-A/proMBP complex, used one proMBP subunit-specific antibody and one PAPP-A subunit-specific antibody. Serum samples from four patients with myocardial infarction (MI), three pregnant women in their first trimester, and one in her third trimester were fractionated by gel filtration on a Superose™ 6 precision column. The two assays were used to analyze fractions obtained by gel filtration as well as serum samples serially collected from four other MI patients. Results: Pregnancy-related PAPP-A was eluted as a single peak with a molecular mass of ∼700 kDa, whereas ACS-related PAPP-A was also eluted as a single peak but with a molecular mass of ∼530 kDa. Pregnancy-related PAPP-A was detected equally by the two assays, whereas increased ACS-related PAPP-A was detected only by the assay for total PAPP-A. Conclusions: Our results provide the first evidence that circulating ACS-related PAPP-A is different from circulating pregnancy-related PAPP-A in that it is not complexed with proMBP. These findings provide a solid foundation for the design of immunoassays to accurately measure atherosclerosis-associated plasma protein A in the circulation.

scholarly journals Identification of Na+,Pi-binding protein in kidney and intestinal brush-border membranes

1988 ◽  
Vol 255 (1) ◽  
pp. 185-191 ◽  
Author(s):  
H Debiec ◽  
R Lorenc
Keyword(s):  
Molecular Mass ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Membrane Vesicles ◽  
High Specificity ◽  
Brush Border Membranes ◽  
Intestinal Brush Border

An Na+, Pi-binding protein has been extracted from kidney and intestinal brush-border membranes with an organic solvent and has been purified by Kieselghur and Sephadex LH-60 chromatography. The molecular mass of this protein has been estimated to be about 155 kDa as determined by gel-filtration chromatography on Sepharose 2B. Under denaturing conditions, polyacrylamide-gel electrophoresis revealed a monomer of molecular mass about 70 kDa. The protein has high specificity and high affinity for Pi [K0.5 (concentration at which half-maximal binding is observed) near 10 microM]. Na2+ binding also exhibits saturation behaviour, with a K0.5 near 7.5 mM. Pi binding is inhibited by known inhibitors of Pi transport in brush-border membrane vesicles. It appears that this protein could be involved in Na+/Pi co-transport across the renal and intestinal brush-border membranes.

Mid-term predictive value of calciprotein particles in maintenance hemodialysis patients based on a gel-filtration assay

2020 ◽  
Vol 303 ◽  
pp. 46-52 ◽  
Author(s):  
Yodo Gatate ◽  
Shintaro Nakano ◽  
Yosuke Mizuno ◽  
Toshihiro Muramatsu ◽  
Takaaki Senbonmatsu ◽  
...  
Keyword(s):  
Predictive Value ◽  
Gel Filtration ◽  
Hemodialysis Patients ◽  
Maintenance Hemodialysis ◽  
Calciprotein Particles ◽  
Maintenance Hemodialysis Patients

scholarly journals Isolation and characterization of riboflavin-binding protein from pregnant-rat serum

1980 ◽  
Vol 187 (2) ◽  
pp. 537-540 ◽  
Author(s):  
K Muniyappa ◽  
P R Adiga
Keyword(s):  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Disc Electrophoresis ◽  
Polyacrylamide Gel ◽  
Molar Ratio ◽  
Rat Serum ◽  
Pregnant Rat ◽  
Isolation And Characterization

A high-affinity riboflavin -binding protein was isolated and characterized for the first time from pregnant-rat sera by affinity chromatography on a lumiflavin-agarose column. The purified protein was homogeneous by the criteria of analytical polyacrylamide-gel disc electrophoresis, gel-filtration chromatography on Sephadex G-100 and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It had a molecular weight of 90000+/-5000 and interacted with [14C]riboflavin with a 1:1 molar ratio with a dissociation constant (Kd) of 0.42 micron.

scholarly journals Synthesis and secretion of a cobalamin-binding protein by HT 29 cell line

1991 ◽  
Vol 280 (2) ◽  
pp. 427-430 ◽  
Author(s):  
H Schohn ◽  
J L Guéant ◽  
M Girr ◽  
E Nexø ◽  
L Baricault ◽  
...  
Keyword(s):  
Cell Culture ◽  
Western Blot ◽  
Affinity Chromatography ◽  
Molecular Mass ◽  
Cell Line ◽  
Binding Protein ◽  
Gel Filtration ◽  
Secreted Protein ◽  
Sds Page ◽  
Ht 29

An HT 29 cell line derived from human colonic carcinoma was shown to synthesize and release a cobalamin-binding protein. The cobalamin-binding protein was classified as transcobalamin (TC). By gel filtration on Sephacryl S200 HR, we observed that the secreted protein bound to cobalamin had the same size as plasma transcobalamin. Like transcobalamin, the cobalamin-binding protein bound cobalamin but not cobinamide. Purification of the cobalamin-binding protein was performed by heparin-Sepharose affinity chromatography and by Sephacryl S200 gel filtration. The molecular mass of the purified protein was estimated at 44 kDa by SDS/PAGE. The isoelectric point was determined to be 6.4. The purified cobalamin-binding protein reacted with an antiserum produced against human transcobalamin. A 44 kDa band was also identified by SDS/PAGE of an immunoprecipitated homogenate from HT 29 cells labelled with [35S]methionine and in a Western blot of cell homogenates. The secretion of the cobalamin-binding protein was maximal between 10 and 12 days of cell culture and was inhibited by cycloheximide.

scholarly journals Distribution of manganese in rat pancreas and identification of its primary binding protein as pro-carboxypeptidase B

1991 ◽  
Vol 278 (3) ◽  
pp. 857-862 ◽  
Author(s):  
H Kodama ◽  
N Shimojo ◽  
K T Suzuki
Keyword(s):  
Binding Protein ◽  
Gel Filtration ◽  
Chemical Characterization ◽  
Molar Ratio ◽  
Emission Spectrometry ◽  
Repeated Injection ◽  
Plasma Atomic Emission Spectrometry ◽  
Carboxypeptidase B ◽  
Rat Pancreas

Distribution of manganese (Mn) and its binding to specific proteins were examined in rat pancreas. A MnCl2 solution was injected subcutaneously into Wistar rats daily at a single dose of 15 mg of Mn/kg body weight for 10 days and the animals were killed 1 day after the last injection. The concentration of Mn in the pancreas increased considerably from 1.4 +/- 0.2 (control) to 13.3 +/- 3.7 micrograms/g wet tissue by the repeated injection of Mn. The distribution of Mn in the soluble fraction of the pancreas (170,000 g supernatant) was determined on a gel-filtration column (Asahipak GST-520) using an h.p.l.c.-inductively coupled argon plasma atomic-emission spectrometry (i.c.p.) technique. The metal was eluted as a single peak in the high-molecular-mass protein fraction, where Mn had been observed as a small peak in the control profile, suggesting that the administered Mn was bound to the same Mn-binding component as that in the control. On the basis of enzymic and chemical characterization of the protein, it was identified as a zymogen of carboxypeptidase B (pro-carboxypeptidase B, pro-CPB). The elution profiles of the protein by h.p.l.c.-i.c.p. indicated that Mn and zinc (Zn) were bound to the zymogen with a molar ratio of 1:4 in normal rat pancreas. Mn bound to the zymogen was easily replaced by Zn in vitro, suggesting that Mn was bound to the Zn-binding site and that the binding affinity to Zn was higher than that to Mn. The present results indicate that pro-CPB is the primary Mn-binding protein in the pancreas of control and also Mn-administered rats.

scholarly journals Isolation, purification and characterization of an iron-binding protein from the horseshoe crab (Limulus polyphemus)

1988 ◽  
Vol 252 (1) ◽  
pp. 151-157 ◽  
Author(s):  
R Topham ◽  
B Cooper ◽  
S Tesh ◽  
G Godette ◽  
C Bonaventura ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Horseshoe Crab ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Limulus Polyphemus ◽  
Evolutionary Development ◽  
Iron Binding ◽  
Oxygen Carriers ◽  
Iron Binding Protein

The presence of an iron-binding protein in the haemolymph of the horseshoe crab, Limulus polyphemus, was detected by gel filtration of 59Fe-labelled haemolymph. Lysis of amoebocytes did not change the amount of iron-binding protein in haemolymph samples. The protein was purified to homogeneity by ion-exchange chromatography. The molecular mass of the purified protein was estimated to be 282,000 +/- 10,000 Da by gel filtration and analytical ultracentrifugation. SDS/polyacrylamide-gel electrophoresis demonstrated that the protein is composed of ten subunits having a molecular mass of 28,000 +/- 2,000 Da. The purified, unlabelled protein efficiently sequestered 59Fe in the absence of haemolymph indicating that no other haemolymph factors are required for the incorporation of iron into the protein. No 59Fe was removed from the purified protein with EDTA or 2,2′-bipyridyl. Partial removal of 59Fe was achieved by dialysis with nitrilotriacetic acid or desferal. Analysis of the iron-loaded protein indicated that each subunit has the capacity to bind two iron atoms with high affinity. The isolation of an iron-binding protein from L. polyphemus supports the proposal that such proteins are an ancient evolutionary development not necessarily linked to the appearance of iron proteins which serve as oxygen carriers.

Hydroxyl Radical-Induced Characteristic Chemiluminescent Spectra from Plasma of Hemodialysis Patients

1992 ◽  
Vol 38 (1) ◽  
pp. 48-55 ◽  
Author(s):  
Shinichi Agatsuma ◽  
Toshiyuki Nagoshi ◽  
Masaki Kobayashi ◽  
Masashi Usa ◽  
Haruo Watanabe ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Emission Spectrum ◽  
Molecular Mass ◽  
Hydroxyl Radicals ◽  
Gel Filtration ◽  
Hemodialysis Patients ◽  
Clinical Samples ◽  
Gel Chromatography ◽  
Characteristic Emission ◽  
Forced Oxidation

Abstract Plasma from hemodialysis patients evoked weak photon emissions (chemiluminescence) in a characteristic emission spectrum with a peak at 430 nm, attributed to attack by hydroxyl radicals generated from the iron-catalyzed breakdown of hydrogen peroxide (Fenton reaction), whereas plasma from normal healthy subjects showed a rather weak red chemiluminescence peak at around 680 nm, similar to that resulting from attack by hydroxyl radicals. However, the addition of hydrogen peroxide in the absence of divalent irons induced almost the same red chemiluminescent emission spectrum in both plasmas. The HPLC-gel-filtration chromatography carried out with both plasmas revealed that a primary emitter evoking a peak emission at 430 nm was located in the fraction of lower-molecular-mass substances in fractionated plasma from hemodialysis patients. In contrast, the elution peaks evoking red chemiluminescence with the addition of hydrogen peroxide were mainly observed for the higher-molecular-mass fraction, as determined by gel chromatography of both plasmas. Therefore, the observation of a chemiluminescence peak at 430 nm, induced by the generation of hydroxyl radicals, correlated well with chemiluminescent emissions in plasma samples from patients with chronic renal failure. Spectral analyses of clinical samples that show weak chemiluminescence by forced oxidation by such an active oxygen may provide a new and more sensitive method for diagnosing metabolic disorders.

scholarly journals Interactions of small molecules with phospholipid bilayers. Binding to egg phosphatidylcholine of some organic anions (bromosulphophthalein, oestrone sulphate, haem and bilirubin) that bind to ligandin and aminoazo-dye-binding protein A

1979 ◽  
Vol 180 (2) ◽  
pp. 327-337 ◽  
Author(s):  
E Tipping ◽  
B Ketterer ◽  
L Christodoulides
Keyword(s):  
Intracellular Transport ◽  
Binding Protein ◽  
Equilibrium Dialysis ◽  
Protein A ◽  
Molar Ratio ◽  
Oestrone Sulphate ◽  
Dye Binding ◽  
Order Of Magnitude ◽  
Saturation Effects ◽  
Limiting Values

1. To assess the possible involvement of ligandin and aminoazo-dye-binding protein A in intracellular transport it is necessary to know how their ligands, most of which are molecules with hydrophobic moieties, interact with cellular membranes. To obtain such information we examined the interactions of bromosulphophthalein, oestrone sulphate, haem and bilirubin with aqueous dispersions of egg phosphatidylcholine and egg phosphatidylchone/cholesterol (1:1, molar ratio) by equilibrium dialysis and spectrophotometry. 2. In all four cases, saturation effects were observed. Values of Vmax (v = mol of compound bound/mol of lipid phosphorus) at 25 degrees C were: for bromosulphophthalein, approximately 0.1; for oestrone sulphate, approximately 0.25; for haem, approximately 0.25 (all at pH 7.4); and for bilirubin 0.1–0.2 (at pH 8.2). 3. Limiting values of v/c (c = unbound concentration) as v leads to 0 at 25 degrees C and pH 7.4 are: for bromosulphophthalein, 6.25 × 10(4) litre-mol-1; for oestrone sulphate, 7.8 × 10(2) litre-mol-1; for haem, 4.5 × 10(5) litre-mol-1; and for bilirubin, approximately 1.2 × 10(4) litre-mol-1. For haem the result depends on the assumption that only the monomeric form binds to the lipid. 4. The binding of each compound was decreased by cholesterol; bromosulphophthalein and oestrone sulphate were affected more than haem and bilirubin. 5. Bromosulphophthalein at saturating concentration decreased the limiting values of v/c of the other three compounds by approximately one order of magnitude. 6. By assuming that the interactions with egg phosphatidylcholine resemble those with the phospholipid components of mammalian intracellular membranes the binding data for phosphyatidylcholine, together with data for binding to the intracellular proteins ligandin and aminoazo-dye-binding protein A, enable the subcellular distributions of the four compounds to be estimated. For the rat hepatocyte up to 92, 51, 98 and 47% of the total bromosulphophthalein, oestrone sulphate, haem and bilirubin respectively may be membrane-bound.

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