Electrophoretic Separation on Agarose Thin Film of Isoenzymes of Alkaline Phosphatase from Human Serum and Tissue

1971 ◽  
Vol 17 (4) ◽  
pp. 290-295 ◽  
Author(s):  
J A Demetriou ◽  
J M Beattie
Keyword(s):  
Thin Film ◽  
Alkaline Phosphatase ◽  
Plasma Proteins ◽  
Enzymic Activity ◽  
Electrophoretic Method ◽  
Electrophoretic Mobilities ◽  
Alkaline Phosphatase Isoenzymes ◽  
Human Sera ◽  
Barbital Buffer ◽  
Isoenzyme Patterns

Abstract An electrophoretic method is presented for separating alkaline phosphatase isoenzymes on agarose gel by using barbital buffer. α-Naphthyl AS-MX phosphate was converted to the highly fluorescent α-naphthol AS-MX, to detect the zones of enzymic activity on the thin film. The fluorescent zones on the electropherograms were scanned with a recording fluorometer and the electrophoretic mobilities of the enzymes were compared with those of the plasma proteins. This method was used to assess the isoenzyme patterns in human sera and tissues (liver, bone, kidney, spleen, and placenta).

Cellulose Acetate Electrophoresis of Alkaline Phosphatase Isoenzymes in Human Serum and Tissue

1972 ◽  
Vol 18 (5) ◽  
pp. 417-421 ◽  
Author(s):  
H A Fritsche ◽  
H R Adams-Park
Keyword(s):  
Alkaline Phosphatase ◽  
Human Serum ◽  
Cellulose Acetate ◽  
High Sensitivity ◽  
Heat Inactivation ◽  
Cellulose Acetate Electrophoresis ◽  
Electrophoretic Method ◽  
Large Numbers ◽  
Alkaline Phosphatase Isoenzymes ◽  
Isoenzyme Patterns

Abstract We describe a new electrophoretic method for the characterization of human serum and tissue alkaline phosphatases on cellulose acetate plates. Enzymes are localized fluorometrically with the substrate α-naphthol AS-MX phosphate or colorimetrically by coupling the reaction product with Fast Blue RR. Both localization techniques are sensitive enough to demonstrate isoenzyme patterns in micro-scale samples of normal sera. Our electrophoretic studies indicate that sera of children and adults normally contain isoenzymes originating from both liver and bone. The high sensitivity of the method allows the use of normal sera as markers rather than tissue extracts, and isoenzyme patterns may be visually assessed after heat inactivation and chemical inhibition. The method is suitable for the electrophoretic fractionation of alkaline phosphatase in large numbers of sera, with equipment and technique familiar to many laboratories.

Improved agarose electrophoretic method for separating alkaline phosphatase isoenzymes in serum.

1988 ◽  
Vol 34 (9) ◽  
pp. 1857-1862 ◽  
Author(s):  
V O Van Hoof ◽  
L G Lepoutre ◽  
M F Hoylaerts ◽  
R Chevigné ◽  
M E De Broe
Keyword(s):  
Alkaline Phosphatase ◽  
Monoclonal Antibodies ◽  
Polyclonal Antibody ◽  
High Molecular Mass ◽  
High Reproducibility ◽  
Neuraminidase Treatment ◽  
Electrophoretic Method ◽  
Electrophoretic Mobilities ◽  
Before And After ◽  
Alkaline Phosphatase Isoenzymes

Abstract A modified agarose electrophoretic system for the separation of alkaline phosphatase (ALP, EC 3.1.3.1) isoenzymes is described. Bone, liver, high-molecular-mass, and intestinal ALP are separated with high reproducibility. The sensitivity of the agarose system is superior to cellulose acetate in detecting high-Mr ALP. Correlation is good between bone ALP fractions scanned before and after treatment with neuraminidase. Immunoglobulin-bound ALPs, the ALP-lipoprotein-X complex, and the additional ALP fraction observed in transient hyperphosphatasemia in children are detected by their peculiar electrophoretic mobility in the proposed system. Approximately 25% of the samples contained an additional fraction of intestinal-type ALP, as evidenced by neuraminidase treatment and use of polyclonal and monoclonal antibodies. Because the electrophoretic mobilities of this "intestinal variant" and of some immunoglobulin-bound ALP fractions are identical to those of bone and intestinal ALP, respectively, treatment of the samples with a polyclonal antibody that reacts with intestinal ALP is advised.

Electrophoretic Method for Assessing the Normal and Pathological Distribution of Alkaline Phosphatase Isoenzymes in Serum

1975 ◽  
Vol 21 (8) ◽  
pp. 1128-1135 ◽  
Author(s):  
Lilian M V Lee ◽  
Margaret A Kenny
Keyword(s):  
Alkaline Phosphatase ◽  
Distribution Patterns ◽  
Bone Diseases ◽  
Discontinuous System ◽  
Electrophoretic Method ◽  
Electrophoretic Mobilities ◽  
Alkaline Phosphatase Isoenzymes ◽  
Rf Values ◽  
Per Se ◽  
Tissue Of Origin

Abstract We describe a simple, reproducible, discontinuous system for polyacrylamide disc gel-electrophoresis, with which the alkaline phosphatase isoenzymes in human serum can be fractionated. No sample preparation is needed. The isoenzymes are classified according to their electrophoretic mobilities (RF values) and quantitated by peak area measurements from spectrophotometric scans. The four alkaline phosphatase isoenzymes usually present in normal sera, in order of descending mobilities (and designated according to principal tissue of origin) are: "fast" liver, "slow" liver, bone, and intestine. Sera of diseased patients show a greater variety of isoenzyme distribution patterns, but the most frequently observed patterns are the same as normal patterns. We conclude that the finding of "fast" liver only is not pathognomonic, as previously reported by others, and that information on relative distributions per se is not diagnostically useful, although information on specific increases in activity is useful. With this system, hepatobiliary disorders can be differentiated from other forms of liver and bone diseases.

Electrophoretic Patterns of Alkaline Phosphatase Isoenzymes in Human Sera with Abnormally High Activity, and an Unusual Band Observed in Sera of Patients with Pancreatic Cancer

1975 ◽  
Vol 21 (8) ◽  
pp. 1067-1071 ◽  
Author(s):  
Chung-Ja Mo Cha ◽  
Balduino Mastrofrancesco ◽  
Sungman Cha ◽  
Henry T Randall
Keyword(s):  
Alkaline Phosphatase ◽  
Heat Stability ◽  
Diffuse Band ◽  
Electrophoretic Patterns ◽  
Cancer Of The Pancreas ◽  
Cellulose Acetate Membranes ◽  
Intestinal Origin ◽  
Alkaline Phosphatase Isoenzymes ◽  
Human Sera ◽  
Moving Band

Abstract Alkaline phosphatase isoenzymes in sera were resolved by electrophoresis on cellulose acetate membranes into seven different bands (L1, B, Pl, L2, I1, I2, and Pa, in decreasing order of electrophoretic mobility). The slowest-moving band (Pa) was observed in the sera of 16 patients—15 with cancer of the pancreas and one with hemochromatosis. Sera of 50 other patients with malignant or benign diseases did not show the Pa band. The Pa band is more heat labile than is the liver isoenzyme (L1). Its behavior toward inhibitors (L-phenylalanine and L-homoarginine) is similar to that of L1. Sera containing the Pa band exhibit a diffuse band in the region where isoenzymes of intestinal origin migrate; however, its heat stability and stereospecific inhibition are different from those of intestinal isoenzymes in sera that show no Pa band.

Alkaline phosphatase isoenzymes resolved by electrophoresis on lectin-containing agarose gel.

1986 ◽  
Vol 32 (8) ◽  
pp. 1570-1573 ◽  
Author(s):  
W E Schreiber ◽  
L Whitta
Keyword(s):  
Heat Treatment ◽  
Alkaline Phosphatase ◽  
Cellulose Acetate ◽  
Wheat Germ ◽  
Agarose Gel ◽  
Electrophoretic Method ◽  
Activity Staining ◽  
Agarose Gels ◽  
Alkaline Phosphatase Isoenzymes ◽  
Wheat Germ Lectin

Abstract With this electrophoretic method the liver, biliary, and bone isoenzymes of alkaline phosphatase are clearly separated on agarose gels. Wheat-germ lectin, incorporated in the gel matrix, binds the bone isoenzyme selectively, forming a precipitate near the origin. Neither liver nor biliary isoenzyme is affected. Activity staining with an indigogenic dye substrate reveals the liver isoenzyme migrating nearest the anode, followed by the biliary and bone isoenzymes. Results are generally similar to those of electrophoresis on cellulose acetate. However, the lectin-agarose gels better resolve the liver and bone isoenzymes, and heat treatment of samples is not required before electrophoresis.

Separation of Isoenzymes of Alkaline Phosphatase by Substrate—Gel Imprint after Electrophoresis on Cellulose Acetate

1972 ◽  
Vol 18 (7) ◽  
pp. 662-666 ◽  
Author(s):  
Douglas P Rhone ◽  
Florence M Mizuno
Keyword(s):  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Cellulose Acetate ◽  
Serum Proteins ◽  
Diazonium Salt ◽  
Acid Phosphate ◽  
Electrophoretic Mobilities ◽  
Human Sera ◽  
Chemical Inhibition

Abstract Isoenzymes of alkaline phosphatase, separated by electrophoresis on cellulose acetate, were developed by a substrate—gel imprint. The gel was prepared in an alkaline buffer and contained β-naphthyl acid phosphate, which was hydrolyzed to β-naphthyl and phosphorus. The β-naphthyl was coupled to the diazonium salt, Fast Violet B, and an insoluble red compound was formed on a cellulose acetate strip to mark the sites of enzyme activity. The electrophoretic mobilities of the isoenzymes were compared with those of serum proteins. Supporting data included thermostability and chemical inhibition (urea and phenylalanine) studies. This method was used to evaluate patterns in tissues and human sera.

An improved electrophoretic method for alkaline phosphatase isoenzymes determination

1975 ◽  
Vol 8 (1-6) ◽  
pp. 415-418
Author(s):  
Samuel Y. Chu ◽  
V.E. Turkington ◽  
C. Vea ◽  
P. Cheung
Keyword(s):  
Alkaline Phosphatase ◽  
Electrophoretic Method ◽  
Alkaline Phosphatase Isoenzymes

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.

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