Alkaline phosphatase calf intestine

Alkaline phosphatase (calf intestinal) Horseradish peroxidase... 

Phosphatase alkaline Origin calf intestinal mucosa... 

Phosphatase, alkaline Origin calf intestine or kidney... 

Alkaline phosphatases [AP, orthophosphoric-monoester phosphorylase (alkaline optimum) EC 3.1.3.1] represent a large family of almost ubiquitous isoenzymes found in organisms from bacteria to animals. In mammals, there are two forms of AP, one form present in a variety of tissues and another form found only in the intestines. They share common attributes in that the phosphatase activity is optimal at pH 8-10, is activated by the presence of divalent cations, and is inhibited by cysteine, cyanides, arsenate, various metal chelators, and phosphate ions. Most conjugates created with AP utilize the form isolated from calf intestine. 

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. 

Enzymatic markers used in immunohistochemistry Horseradish peroxidase (HRP) and calf intestinal or E.coli alkaline phosphatase (AP). Glucose oxidase from Aspergillus niger and E.coli /3-galactosidase are only rarely applied. 

Morton, R. K. 1955. Some properties of alkaline phosphatase of cows milk and calf intestinal mucosa. Biochem. J. 60, 573-582. 

Different tissues contain different isoenzymes of alkaline phosphatase, and the intestinal isoenzyme is not inhibited by levamisole The enzyme used in immu-nohistochemistry is extracted from calf intestine, so that levamisole can be used as an inhibitor without affecting the desired reaction. For labile antigens in the intestine, it is better to switch to the peroxidase method. 

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. 

Perhaps the most characteristic feature of alkaline phosphatase is the way in which the pH optimum changes with increasing substrate concentration. A typical set of curves for calf intestinal phosphatase and phenyl phosphate is given in Fig. 1. Other examples of this type of behavior are found with -glycerophosphate and chicken intestinal... 

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).

As with Km, the effect of pH on Fmax cannot be described by a simple ionization curve. With calf intestinal phosphatase, the log ym8X curve for a monoester substrate is sigmoid (143, 162) or, in the case of synovial phosphatase, extremely shallow (76). Both curves approach a maximum value at alkaline pH. Barman and Gutfreund, however, found that milk phosphatase had an optimum at pH 10 with only 60% activity at pH 11 (83). This is by no means typical since placental phosphatase has been shown to be fully active with the same substrate, p-nitrophenyl phosphate at pH 11.5 (85). With PP as substrate there is evidence that an optimum in Vmax is reached at considerably lower pH values (8.5-9.2) (116, 117, 164). A pH-activity curve for calf intestinal phosphatase is given in Fig. 3. Features to note are the plateau in activity around pH 7, corresponding to a minimum in the phosphorylation rate constant, and a change in rate determining step at about pH 6 (165). 

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. 

Alkaline phosphatase (EC 3-1.3-1) Sigma type VII from calf intestine at pH 10.5 was equilibrated with a mix in Eq. 2 at 25°C for 30 min. The 31p NMR spectrum was recorded at pH 8.10. There was no R-l-P left and there were three Pj s found containing and The integration and lack of detectable... 

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++. 

---