Plasma alkaline phosphatase isozymes: Isolation and characterization of isozymes (original) (raw)
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Clinica Chimica Acta, 1974
Using a new electrophoretic method employing cellulose acetate plates we have found that up to four high molecular weight isoenzymes of alkaline phosphatase may be present in the sera of cancer patients. 134 of 201 sera contained a "pre-liver" isoenzyme which migrated with serum alpha-l globulins. Twenty-two of the 134 sera contained the pre-liver isoenzyme with 1 or 2 of 3 "slower" isoenzymes which migrated with the beta and gamma globulins. Chromatographic studies showed that the 4 unusual isoenzymes elute from Sephadex G-200 with high molecular weight serum proteins. The pre-liver isoenzyme showed isoenzyme inhibition properties that are similar to those of an isoenzyme extracted from bile, while the 3 slower isoenzymes gave inhibition data that is similar to the intestinal isoenzyme. The high molecular weight isoenzymes are completely inhibited by heating at 65". The four unusual isoenzymes of alkaline phosphatase were present most frequently in the sera of patients who had clinical and laboratory evidence of metastatic and obstructive liver disease.
Separation of Human Serum-Alkaline-Phosphatase Isoenzymes by Polyacrylamide Gel Electrophoresis
The Lancet, 1969
Electrophoresis on polyacrylamide gel slabs was found to separate human serum-alkaline-phosphatase into four distinct isoenzymes. The first was found primarily in the sera of patients with liver disorders and migrated just ahead of a more diffuse isoenzyme that was present in serum from patients with bone disorders. A third isoenzyme, identical with that of intestinal origin, was readily distinguishable from these two and was found in only two sera, both from patients with Laennec's cirrhosis. A fourth isoenzyme remained at the origin during electrophoresis, was present most often in patients with liver disease, but was of unknown origin and significance. With electrophoresis on polyacrylamide gel slabs, the presence of liver or bone disorders was predicted in more than 90% of one hundred and eleven patients with elevated serumalkaline-phosphatase.
Specific assays for human alkaline phosphatase isozymes
Clinica Chimica Acta, 1987
Specific assays for human intestinal and liver alkaline phosphatases were developed by use of isozyme specific monoclonal antibodies bound to paper discs. The assays are fast, specific and convenient to use as demonstrated by determinations of alkaline phosphatase isozymes in sera and tissues. In sera from forty healthy individuals the activity of the tissue unspecific alkaline phosphatase was determined to 32 k 12 IU/l (mean k SD). The activity of the intestinal alkaline phosphatase was found to be tenfold lower, 3.5 f 6.3 II-J/l (mean + SD), and of the placental alkaline phosphatase another tenfold lower, 0.3 f 0.2 IU/l (mean & SD), than that of the tissue unspecific alkaline phosphatase. Several normal tissues contained all three isozymes, the intestinal mucosa, for example, which besides intestinal alkaline phosphatase also expresses trace amounts of placental and liver-bone-kidney alkaline phosphatase. Seminomas, known to express eutopically placental-like alkaline phosphatase were demonstrated to contain increased levels of both intestinal and liver-bone-kidney alkaline phosphatases as compared to the normal testis.
Comparison of human alkaline phosphatase isoenzymes. Structural evidence for three protein classes
Biochemical Journal, 1979
The structural relationships among human alkaline phosphatase isoenzymes from placenta, bone, kidney, liver and intestine were investigated by using three criteria. 1. Immunochemical characterization by using monospecific antisera prepared against either the placental isoenzyme or the liver isoenzyme distinguishes two antigenic groups: bone, kidney and liver isoenzymes cross-react with anti-(liver isoenzyme) serum, and the intestinal and placental isoenzymes cross-react with the anti-(placental isoenzyme) antiserum. 2. High-resolution two-dimensional electrophoresis of the 32P-labelled denatured subunits of each enzyme distinguishes three groups of alkaline phosphatase: (a) the liver, bone and kidney isoenzymes, each with a unique isoelectric point in the native form, can be converted into a single form by treatment with neuraminidase; (b) the placental isoenzyme, whose position also shifts after removal of sialic acid; and (c) the intestinal isoenzyme, which is distinct from all ot...
Journal of Chromatography A, 1993
Two commercially available electrophoretic methods for the separation of the human serum enzyme alkaline phosphatase (ALP, EC 3.1.3.1) were evaluated: the Isopal system (Beckman, Brea, CA, USA) was compared with the Iso-PAL system (Sebia, Issy-les-Moulineux, France). Both use agarose as supporting medium to separate ALP into its clinically relevant isoenxymes: bone, liver, high-M, (or "fast liver"), intestinal and placental ALP. With both methods, additional fractions for bone, liver and intestinal ALP were found, true isoforms that were called "variant" fractions. The migration pattern differed considerably between the systems, owing to the use of different detergents. Bone and liver ALP were partially separated with both methods. However, when bone ALP exceeded 50% of the total ALP activity, sample treatment was necessary, either with neuraminidase (Beckman) or by applying the sample on a second gel containing wheat-germ lectin to precipitate bone ALP (Sebia). The withinand between-gel reproducibilities of both systems were comparable and remained between 2 and 6% for normal isoenzyme activities. Both systems correlated well, except for high M, ALP. The Sebia system was more sensitive for detecting intestinal ALP, whereas higher liver and bone variant ALP activities were detected with the Beckman system. It is concluded that both methods are convenient for routine use in the clinical laboratory.
Alkaline phosphatase and its isoenzymes in the tissues and sera of normal dogs
Veterinary Research Communications, 1986
The total alkaline phosphatase (AP) activity and the pattern of its isoenzymes were studied in the tissues and sera of normal adult dogs. Small intestine mucosa showed the greatest total AP activity followed by kidney, bone, pancreas, liver, lung, skeletal muscle and heart muscle. After separation by agarose gel electrophoresis, each tissue showed only one isoenzyme except lung which showed two. The tissue isoenzymes, in decreasing order of migration distance towards the anode, were as follows: fast lung isoenzyme, liver or slow lung isoenzyme, the group consisting of skeletal muscle, bone, small intestine and pancreas isoenzymes and, finally, the kidney isoenzyme. Two isoenzymes occurred in serum. The major band corresponded to liver and the slow lung isoenzyme, while the minor band was considered to be the corticosteroid-induced isoenzyme, previously thought to be absent from normal serum. The AP isoenzyme patterns in lung and skeletal muscle and the presence of an isoenzyme migrating an identical distance to the corticosteroid-induced isoenzyme do not appear to have been reported before in normal dogs.
Substrate specificity of alkaline phosphatase from human polymorphonuclear leukocytes
Clinica Chimica Acta, 1986
The ability of alkaline phosphatase in purified preparations from human neutrophils and liver to utilize ATP or inorganic pyrophosphate as substrate depended upon the Mg2+ concentration. With pyrophosphate present (1.0 mmol/l), activity peaked at Mg2+ concentrations of 0.25 to 0.50 mmol/l and fell sharply above this. By contrast, p-nitrophenylphosphatase activity was activated with Mg2' concentration up to 0.75 mmol/l but above this was constant to 5.0 mmol/l. Hydrolysis was abolished by r_-levamisole, a specific inhibitor of alkaline phosphatase. Testing butanol extracts of neutropbils from 50 healthy subjects showed good correlation of enzyme activity with ~-nitrophenylphosphate and ADP (r = 0.90), and between p-nitrophenylphosphate and pyridoxaf phosphate (r = 0.96) as substrate, consistent with hydrolysis of all three phosphoesters by one enzyme. Inhibition studies yielded no evidence of a specific pyridoxal phosphatase. Alkaline phosphatase from human neutrophils has the same broad substrate specificity as other molecular forms of the human enzyme and, like other forms, has little or no activity towards phosphoesters complexed with Mg2+.
2013
Abstract: Measuring serum alkaline phosphatase (ALP) isoenzymes activity is very important for evaluation of two groups of conditions including hepatobiliary and bone diseases. The present study aimed to compare phenylalanine inhibition and heat inactivation techniques in the quantification of liver ALP isoenzyme activity. Fasting serum from 50 healthy adults were used to evaluate total ALP and liver isoenzyme activities. Comparison of Intra- and inter-assay precisions for normal and cholestatic liver samples using phenylalanine inhibition (PI) and heat inactivation (HI) methods were done. Total alkaline phosphatase activity was 91.2 ±24.7 in 50 normal serum samples using IFCC standard method. Intra-assay coefficients of variations (CV) were 3.17% and 4.22 % for normal and 2.98 % and 3.75 % for cholestatic liver samples using HI and PI methods, respectively. The inter-assay CV were 3.64 % and 4.45 % for normal and 3.23 % and 4.06 % for cholestatic liver samples using HI and PI metho...
Distribution and properties of rat intestinal alkaline phosphatase isoenzymes
Experimental animals, 2001
The ALP activities and properties of rat intestine cut into 20 segments were examined, and we were able to demonstrate that the ALP activity of upper intestine is high compared to that of lower intestine. This result coincided with those of other reports. However, we newly clarified that there is an ALP isoenzyme found in the lower intestine which can be inhibited by L-homoarginine. The molecular weight of the ALP isoenzyme was 136 kDa. In addition, it was clarified that there are several isoenzymes from upper to lower intestine. This study demonstrates that there exist isoenzymes, which are inhibited by L-HArg, in the intestine which are similar to the isoenzymes in the liver, bone and kidney.