scholarly journals Isoelectric focusing of human liver alkaline phosphatase

1970 ◽  
Vol 118 (2) ◽  
pp. 299-302 ◽  
Author(s):  
A. L. Latner ◽  
Mary E. Parsons ◽  
A. W. Skillen
Keyword(s):  
Alkaline Phosphatase ◽  
Density Gradient ◽  
Isoelectric Focusing ◽  
Isoelectric Point ◽  
Human Liver ◽  
Sucrose Density Gradient ◽  
Ph Gradient ◽  
Alkaline Phosphatases ◽  
Carrier Ampholytes

1. Isoelectric focusing of human liver alkaline phosphatase in a sucrose density gradient with LKB Ampholine as carrier ampholytes is described. 2. Problems due to the chelating properties of the ampholytes and the pH gradient were examined. 3. A reactivation procedure to counter these effects was devised that can probably be used for other alkaline phosphatases. 4. The isoelectric point of human liver alkaline phosphatase was found to be pH3.9.

Serum alkaline phosphatase isoenzymes in hepatobiliary disorders resolved by use of immobilized pH gradients.

1987 ◽  
Vol 33 (5) ◽  
pp. 653-657 ◽  
Author(s):  
P Sorroche ◽  
A Bianchi-Bosisio ◽  
P K Sinha ◽  
C Gelfi ◽  
P G Righetti
Keyword(s):  
Alkaline Phosphatase ◽  
Isoelectric Focusing ◽  
Serum Alkaline Phosphatase ◽  
Normal Serum ◽  
Alkaline Phosphatases ◽  
Ph Gradients ◽  
Carrier Ampholytes ◽  
Alkaline Phosphatase Isoenzymes ◽  
Serum Alkaline Phosphatase Isoenzymes ◽  
Acidic Components

Abstract This new method for fractionating alkaline phosphatase isoforms in hepatobiliary disorders is based on isoelectric focusing on a mixed-type polyacrylamide support containing an immobilized pH gradient with a superimposed carrier-ampholyte gradient. The high-Mr alkaline phosphatase forms typical of hepatobiliary disease (greater than 1 mega-dalton), which cannot migrate into the Immobiline gel, are disaggregated in zwitterionic detergents (the most effective being sulfobetaine 3-12)--20 g/L in the sample, 5 g/L in the gel--suggesting that they are still complexed with membrane fragments or that they tend to aggregate spontaneously in solution. These isoforms focus in the pI 5-6 range (while alkaline phosphatases in normal serum focus in the pI 4-5 interval) in immobilized pH gradients, but behave as strongly acidic components by agarose isoelectric focusing in the presence of carrier ampholytes, suggesting that they are strongly complexed with the latter. On treatment with neuraminidase, the low-pI isoforms in normal serum focus in the pI 5-6 range typical of the hepatobiliary isoforms, suggesting that the latter are poorly glycosylated. By a second-dimension run, in a porosity gradient, followed by activity staining, all alkaline phosphatase forms that have entered the Immobiline gel in the first dimension (normal forms and high-Mr species) exhibit the same Mr (ca. 140,000 Da), suggesting that no new chains are synthesized in hepatobiliary disorders.

Alkaline phosphatase. I. Kinetics and inhibition by levamisole of purified isoenzymes from humans.

1976 ◽  
Vol 22 (7) ◽  
pp. 972-976 ◽  
Author(s):  
H Van Belle
Keyword(s):  
Alkaline Phosphatase ◽  
Substrate Concentration ◽  
Human Liver ◽  
Alkaline Phosphatases ◽  
Mechanism Of Inhibition ◽  
Optimum Ph ◽  
Alkaline Phosphatase Isoenzymes

Abstract I studied the kinetics and sensitivity toward inhibition by levamisole and R 8231 of the most important human alkaline phosphatase isoenzymes. N-Ethylaminoethanol proved superior to the now widely used diethanolamine buffer, especially for the enzymes from the intestine and placenta, behaving as an uncompetitive activator. The optimum pH largely depends on the substrate concentration. The addition of Mg2+ has no effect on the activities. The meaning of Km-values for alkaline phosphatases is questioned. Isoenzymes from human liver, bone, kidney, and spleen are strongly inhibited by levamisole or R 8231 at concentrations that barely affect the enzymes from intestine or placenta. The inhibition is stereospecific, uncompetitive, and not changed by Mg2+. Inhibition is counteracted by increasing concentrations of N-ethylaminoethanol. The mechanism of inhibition is suggested to be formation of a complex with the phosphoenzyme.

Focusing of Proteins in a Horseshoe Microchannel

2008 ◽  
Author(s):  
Jaesool Shim ◽  
Prashanta Dutta ◽  
Cornelius F. Ivory
Keyword(s):  
Capillary Electrophoresis ◽  
Electric Field ◽  
Isoelectric Focusing ◽  
Ph Gradient ◽  
Complex Geometries ◽  
Electrokinetic Phenomena ◽  
Double Peaks ◽  
Zone Electrophoresis ◽  
Carrier Ampholytes ◽  
Ph Range

Ampholyte based isoelectric focusing (IEF) simulation was conducted to study dispersion of proteins in a horseshoe microchannel. Four model proteins (pls = 6.49, 7.1, 7.93 and 8.6) are focused in a 1 cm long horseshoe channel under an electric field of 300 V/cm. The pH gradient is formed in the presence of 25 biprotic carrier ampholytes (ΔpK = 3.0) within a pH range of 6 to 9. The proteins are focused at 380 sec in a nominal electric field of 300 V/cm. Our numerical results show that the band dispersions of a protein are large during the marching stage, but the dispersions are significantly reduced when the double peaks start to merge. This rearrangement of spreading band is very unique compared to linear electrokinetic phenomena (capillary electrophoresis, zone electrophoresis or electroosmosis) and is independent of channel position and channel shape. Hence, one can perform IEF in complex geometries without incorporating hyperturns.

Incorporation of human liver and placental alkaline phosphatases into liposomes and membranes is via phosphatidylinositol

1990 ◽  
Vol 68 (9) ◽  
pp. 1112-1118 ◽  
Author(s):  
Lee Kihn ◽  
Dorothy Rutkowski ◽  
Robert A. Stinson
Keyword(s):  
Alkaline Phosphatase ◽  
Phospholipase C ◽  
Human Liver ◽  
Red Cell ◽  
Osteosarcoma Cells ◽  
Alkaline Phosphatases ◽  
Membrane Anchor ◽  
Gradient Gel Electrophoresis ◽  
Triton X ◽  
Phosphatidylinositol Phospholipase C

As assessed by incorporation into liposomes and by adsorption to octyl-Sepharose, the integrity of the membrane anchor for the purified tetrameric forms of alkaline phosphatase from human liver and placenta was intact. Any treatment that resulted in a dimeric enzyme precluded incorporation and adsorption. An intact anchor also allowed incorporation into red cell ghosts. The addition of hydrophobic proteins inhibited incorporation into liposomes to varying degrees. Alkaline phosphatase was 100% releasable from liposomes and red cell ghosts by a phospholipase C specific for phosphatidylinositol. There was no appreciable difference in the rates of release of placental and liver alkaline phosphatases, although both were approximately 250 × slower in liposomes and 100 × slower in red cell ghosts than the enzyme's release from a suspension of cultured osteosarcoma cells. Both enzymes were released by phosphatidylinositol phospholipase C as dimers and would not reincorporate or adsorb to octyl-Sepharose. However, the enzyme incorporated, resolubilized by Triton X-100, and cleansed of the detergent by butanol treatment was tetrameric by gradient gel electrophoresis, was hydrophobic, and could reincorporate into fresh liposomes. A monoclonal antibody to liver alkaline phosphatase inhibited the enzyme's incorporation into liposomes, and abolished its release from liposomes and its conversion to dimers by phosphatidylinositol phospholipase C.Key words: alkaline phosphatase, liposome, phosphatidylinositol, membrane anchor.

Hybrid isoelectric focusing: Adsorption of proteins onto immobilized pH gradient matrices and desorption by carrier ampholytes

1987 ◽  
Vol 8 (1) ◽  
pp. 52-62 ◽  
Author(s):  
Klaus Altland ◽  
Arnold von Eckardstein ◽  
Angelika Banzhoff ◽  
Michael Wagner ◽  
Ute Rossmann ◽  
...  
Keyword(s):  
Isoelectric Focusing ◽  
Ph Gradient ◽  
Carrier Ampholytes ◽  
Hybrid Isoelectric Focusing

Presence of γ-glutamyltransferase in the renal microvascular compartment

1981 ◽  
Vol 59 (6) ◽  
pp. 383-386 ◽  
Author(s):  
P. D. Dass ◽  
R. P. Misra ◽  
T. C. Welbourne
Keyword(s):  
Alkaline Phosphatase ◽  
Phosphatase Activity ◽  
Density Gradient ◽  
Alkaline Phosphatase Activity ◽  
Brush Border ◽  
Sucrose Density Gradient ◽  
Isolated Glomeruli ◽  
Crude Homogenate ◽  
Brush Border Enzyme ◽  
Enzyme Alkaline Phosphatase

The association between the brush border enzyme alkaline phosphatase and γ-glutamyltransferase was determined by sucrose density gradient analysis of crude kidney homogenates, isolated glomeruli, and isolated microvessels. As previously established there is an overlap of these enzyme activities in the crude homogenate corresponding to a density of 1.17 g∙cm−3. In contrast, isolated glomeruli sedimented with a peak of 1.25 g∙cm−3 and exhibited γ-glutamyltransferase activity but little alkaline phosphatase activity; homogenizing isolated glomeruli shifted the fragments to a density coincident with that observed for the crude homogenate γ-glutamyltransferase peak. A second population of capillaries, isolated microvessels, were homogenized and analyzed on the sucrose density gradient. These fragments sedimented over the same range as crude homogenate γ-glutamyltransferase peak but were devoid of alkaline phosphatase activity and yet exhibited remarkable γ-glutamyltransferase activity. The results indicate homogenization of renal cortex results in a heterogenous collection of particles from both tubular and microvascular locations exhibiting γ-glutamyltransferase activity which overlap with the brush border alkaline phosphatase containing membranes. However, isolation of microvessels and glomeruli prior to homogenization allows separation of γ-glutamyltransferase from alkaline phosphatase activity; between 10 and 20% of the total homogenate γ-glutamyltransferase activity is estimated to be associated with the microvascular compartment.

scholarly journals Crystallization and characterization of human liver kynurenine–glyoxylate aminotransferase. Identity with alanine–glyoxylate aminotransferase and serine–pyruvate aminotransferase

1980 ◽  
Vol 189 (3) ◽  
pp. 581-590 ◽  
Author(s):  
Etsuo Okuno ◽  
Yohsuke Minatogawa ◽  
Masayuki Nakamura ◽  
Naoki Kamoda ◽  
Junko Nakanishi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Density Gradient ◽  
Human Liver ◽  
Analytical Ultracentrifugation ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Glyoxylate Aminotransferase

Kynurenine–glyoxylate aminotransferase, alanine–glyoxylate aminotransferase and serine–pyruvate aminotransferase were co-purified and crystallized as yellow cubes from human liver particulate fraction. The crystalline enzyme was homogeneous by the criteria of electrophoresis, isoelectric focusing, gel filtration, sucrose-density-gradient centrifugation and analytical ultracentrifugation. The molecular weight of the enzyme was calculated as approx. 90000, 89000 and 99000 by the use of gel filtration, analytical ultracentrifugation and sucrose-density-gradient centrifugation respectively, with two identical subunits. The enzyme has a s20,w value of 5.23S, an isoelectric point of 8.3 and a pH optimum between 9.0 and 9.5. The enzyme solution showed absorption maxima at 280 and 420nm. The enzyme catalysed transamination between several l-amino acids and pyruvate or glyoxylate. The order of effectiveness of amino acids was alanine>serine>glutamine>glutamate>methionine>kynurenine = phenylalanine = asparagine>valine>histidine>lysine>leucine>isoleucine>arginine>tyrosine = threonine>aspartate, with glyoxylate as amino acceptor. The enzyme was active with glyoxylate, oxaloacetate, hydroxypyruvate, pyruvate, 4-methylthio-2-oxobutyrate and 2-oxobutyrate, but showed little activity with phenylpyruvate, 2-oxoglutarate and 2-oxoadipate, with kynurenine as amino donor. Kynurenine–glyoxylate aminotransferase activity was competitively inhibited by the addition of l-alanine or l-serine. From these results we conclude that kynurenine–glyoxylate aminotransferase, alanine–glyoxylate aminotransferase and serine–pyruvate aminotransferase activities of human liver are catalysed by a single protein. Kinetic parameters for the kynurenine–glyoxylate aminotransferase, alanine–glyoxylate aminotransferase, serine–pyruvate aminotransferase and alanine–hydroxypyruvate aminotransferase reactions of the enzyme are presented.

The Microheterogeneity of Human Haptoglobin and Its Complex with Hemoglobin

1973 ◽  
Vol 51 (5) ◽  
pp. 597-605 ◽  
Author(s):  
H. J. Yang ◽  
M. Przybylska
Keyword(s):  
Sialic Acid ◽  
Density Gradient ◽  
Isoelectric Focusing ◽  
Acid Content ◽  
Sucrose Density Gradient ◽  
Polyacrylamide Gel ◽  
Ascites Fluid ◽  
Sialic Acid Content ◽  
Symmetrical Distribution ◽  
Complex Components

Human haptoglobin, type 1S-1S, isolated from plasma and from ascites fluid was subjected to isoelectric focusing in polyacrylamide gel and its complex with horse cyanomethemoglobin was analyzed by isoelectric focusing in a column using a sucrose density gradient. Both methods revealed microheterogeneity. Similar patterns were obtained consisting of five to eight strong bands with a symmetrical distribution giving the highest yield in the center. An investigation of the nature of these bands was carried out and it was found that the sialic acid content varied in the different components. Experiments were undertaken to show that the multiple bands were not due to the binding of Ampholine to the protein and that this heterogeneity preexisted in haptoglobin prior to its isolation. No observable difference in heterogeneity was observed between samples of haptoglobin isolated by different methods and for complexes obtained by adding hemoglobin to haptoglobin and haptoglobin to hemoglobin.The unfractionated complex and the separated complex components were crystallized.

Separation and identification of alkaline phosphatase isoenzymes and isoforms in serum of healthy persons by isoelectric focusing.

1987 ◽  
Vol 33 (12) ◽  
pp. 2171-2177 ◽  
Author(s):  
J Griffiths ◽  
J Black
Keyword(s):  
Alkaline Phosphatase ◽  
Isoelectric Focusing ◽  
Diazonium Salt ◽  
Independent Gene ◽  
Gel Film ◽  
Isoelectric Points ◽  
Carrier Ampholytes ◽  
Alkaline Phosphatase Isoenzymes ◽  
Age Range ◽  
Healthy Persons

Abstract We have developed an isoelectric focusing procedure for resolving alkaline phosphatase (EC 3.1.3.1) isoenzymes and isoforms in serum. We use a thin-layer agarose gel film containing synthetic carrier ampholytes and a "separator" to flatten the pH gradient in the region of the isoenzyme and isoform isoelectric points. Sharp, highly resolved zones of enzyme activity are obtained by limiting diffusion; for this we rapidly couple the released product, 1-naphthol, to a diazonium salt, which forms a colored precipitate at the site of activity. We have resolved and identified 12 zones of alkaline phosphatase activity in the serum of ostensibly healthy persons within a wide age range. Theoretically, three basic isoenzymes are produced from independent gene loci: intestinal, placental, and nonspecific tissue alkaline phosphatase. The other zones of activity may be isoforms.

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