Alkaline phosphatase intestine

Condensed (poly) phosphates may exert different effects on calcium utilization than the aforementioned effects of simple (ortho-) phosphates. Polyphosphates have a much greater affinity for calcium than do orthophosphates, and soluble calcium-polyphosphate complexes are readily formed in the gastric and intestinal environments. In addition, polyphosphates must be hydrolyzed by an intestinal alkaline phosphatase (27) prior to absorption. We have found polyphosphates to be incompletely (80.5%) hydrolyzed to orthophosphate during the digestive process in young adult males when calcium intake was low only 56% of a 1 g phosphorus supplement was absorbed from a polyphosphate sources as compared to 71% from an orthophosphate source (5). 

Enzyme labels are usually coupled to secondary antibodies or to (strept)avidin. The latter is used for detection of biotinylated primary or secondary antibodies in ABC methods (see Sect. 6.2.1). Enzyme labels routinely used in immunohisto-chemistry are horseradish peroxidase (HRP) and calf intestinal alkaline phosphatase (AP). Glucose oxidase from Aspergillus niger and E. coli (3-galactosidase are only rarely applied. 

The type of inhibition of chicken intestine alkaline phosphatase by forphenicine was not competitive, but uncompetitive with the stibstrate. Its derivative, forphenicinol, which contains a hydroxymethyl grorp instead of the formyl group in the forphenicine molecule, did not inhibit alkaline phosphatase but, it did bind to cells. Forphenicinol enhanced delayed-type lOT>ersensiti-vity (13,14) and the phagocytic activity of macrophages. 

FIGURE 2-21 The pH optima of some enzymes. Pepsin is a digestive enzyme secreted into gastric juice trypsin, a digestive enzyme that acts in the small intestine alkaline phosphatase of bone tissue, a hydrolytic enzyme thought to aid in bone mineralization. 

Enzymes. Xhol restriction enzyme, calf intestinal alkaline phosphatase, and T4 DNA ligase are available from commercial suppliers complete with reaction buffers. Follow the instructions for use, storage, and shelf-life... 

Dephosphorylate the vector DNA by adding 5 pL of 10X phosphatase buffer, 35 pL of water, and 0 02 U of calf intestinal alkaline phosphatase Incubate at 37°C for 30 min, then add another 0.02 U, and continue the incubation for a further 30 min. Extract with phenol/chloroform, followed by chloroform//isoamyl alcohol, and precipitate the DNA with ethanol as m step 2. 

Fig. 1. Hydrolysis of phenyl phosphate by calf intestinal alkaline phosphatase. The curves refer to the following substrate concentrations A, 25 pM B, 50 pM C, 100 pM D, 500 pM E, 750 pM F, 2.5 mM G, 25 mM and H, 75 mil/. Initial velocities are expressed as micromoles of product per milligram of enzyme per minute. From Morton (100).

Fig. 3. Hydrolysis of 4-methylumbelliferyl phosphate by calf intestinal alkaline phosphatase. Activities are recorded as turnovers per site per second at 20° and 1 = 0.02, using tris-acetic acid (< pH 8) or ammediol-HCl (> pH 8) buffers.

Fig. 5. Activation of calf intestinal alkaline phosphatase by Mg. Assays were performed at 38° in 0.05 M ethanolamine-HCl pH 9.9 with 2.5 mM phenyl phosphate. From Morton (90).

Digest 1 pg of the pLXSN retroviral plasmid with 1 pi of the restriction enzyme Hpal in 10 pi of 1 x NEBuffer 4 in a 1.5 ml Eppendorf tube at 37°C overnight. This produces a linear 5.9 kb backbone fragment with blunt ends. Add 1 pi of calf intestinal alkaline phosphatase to the tube which removes 5 and 3 phosphoryl groups. This prevents the plasmid from self ligation. 

Calf Intestinal Alkaline Phosphatase (CIAP) (20 IJ/pT) and its 10X reaction buffer (Promega). 

Immunoenzymatic staining methods utilize enzyme substrate reactions to convert colorless chromogens into colored end products. Of the enzymes used in these applications, only horseradish peroxidase and calf intestine alkaline phosphatase will be considered in some detail. Because of its low sensitivity, glucose oxidase (Aspergillus niger) is only rarely used today. 

Horseradish peroxidase and calf intestine alkaline phosphatase meet most of these criteria, and the following will list their properties in more detail. 

Calf intestine alkaline phosphatase (molecular weight 100 kDa) removes (by hydrolysis) and transfers phosphate groups from organic esters by breaking the P 0 bond an intermediate enzyme-substrate bond is briefly formed. The chief metal activators for AP are Mg++, Mn++ and Ca++. 

AP had not been used extensively in immunohistochemistry until publication of the unlabeled alkaline phosphatase-antialkaline phosphatase (APAAP) procedure (2, 3). The soluble immune complexes utilized in this procedure have molecular weights of approximately 560 kDa. The major advantage of the APAAP procedure compared to the earlier peroxidase techniques was the lack of interference posed by endogenous peroxidase activity. Because of the potential distraction of endogenous peroxidase activity, the alkaline phosphatase techniques were particularly recommended for use on blood and bone marrow smears. Endogenous alkaline phosphatase activity from bone, kidney, liver and some white cells can be inhibited by the addition of 1 mM levamisole to the substrate solution (4), although 5 mM has been found to be more effective (5). Intestinal alkaline phosphatases are not adequately inhibited by levamisole. 

Levamisole + chromogen except intestinal alkaline phosphatase Alkaline phosphatase label... 

Unquenched endogenous alkaline phosphatase activity may be seen in leucocytes, kidney, liver, bone, ovary bladder, salivary glands, placenta and gastro-intestinal tissue. Add levamisole to the alkaline phosphatase chromogen reagent or use another enzyme label such as horseradish peroxidase. Intestinal alkaline phosphatase is not quenched by the addition of levamisole. Pretreat the tissue with 0.03 N HCI. 115-121... 

NO STAINING SEEN. GO TO NEXT STEP. Block with levamisole (Intestinal alkaline phosphatase may be... 

B. Lorenz and H. C. Schroder (2001). Mammalian intestinal alkaline phosphatase acts as highly active exopolyphosphatase. Biochim. Biophys. Acta, 1547, 254-261. 

Lieverse, A.G., van Essen, G.G., Beukeveld, G.J.J., Gazendam, J., Dom-peling, E.C., ten Kate, L.P., van Belle, S.A., Weits, J. Familial increased serum intestinal alkaline phosphatase a new variant associated with Gilbert s syndrome. J. Clin. Pathol. 1990 43 125-128... 

AttoPhos, 2 -(2-benzothiazoIyl)-6 -hydroxybenzothiazole phosphate, is a weakly fluorescent alkaline phosphate substrate marketed by JBL Scientific (San Luis Obispo, CA) which is converted into the highly fluorescent product AttoFluor, 2 -(2-benzothiazolyl)-6 -hydroxybenzothiazole. Calf intestinal alkaline phosphatase was obtained from Life Technologies (Gaithersburg, MD). Boric acid and p-nitrophenyl phosphate were purchased from Sigma (St. Louis, MO) and chloroform, MgCl2 and diethanolamine from Fisher Scientific (Ottawa, ON). 

When considering the application of urinary enzymes to monitor subtle renal dysfunction and/ or to clarify mechanisms of nephrotoxicity, only a limited number of enzymes have been generally accepted as valuable urinary biomarkers. These include lactic dehydrogenase, N-acetyl-P-l>glucosarriitiidase (NAG), alanine aminopeptidase (AAP), intestinal alkaline phosphatase, glutathione-S-transferase, gamma-glutamyl transferase and fructose-l,6,-biphosphatase. 

Intestinal alkaline phosphatase and human tissue non-specific alkaline phosphatase are two urinary isoenzymes that have ehcited interest as potential segment specific markers of the human nephron [190]. Both are members of the closely related group of alkaline phosphatases. Intestinal alkaline phosphatase is the intestinal isoenzyme that is locahzed on the brush border of human intestinal epithelial cells. It is also present in normal human kidney, where it is exclusively expressed on the brush border of tubulo-epithelial cells of the S3-segment of the proximal tubule. The intestinal alkaline phosphatase, which is released in urine, has its origin in the kidney. As a result, intestinal alkaline... 

KidoT, Hayano M, Kobayashi E, Nogawa K, Nishijo M,Tabata M, Nakagawa H, Tsuritani I. Significance of elevated urinary human intestinal alkaline phosphatase in Japanese people exposed to environmental cadmium.Toxicol Lett 1995 80 49-54. 

FEurea fractional excretion of urea lAP intestinal alkaline phosphatase... 

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