Calf intestinal alkaline phosphatase

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. 

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++. 

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). 

M Husain, C Bieniarz. FC site-specific labeling of immunoglobulins with calf intestinal alkaline phosphatase. Bioconj Chem 5 482-490, 1994. 

B13. Behai, F. J., and Center, M., Heterogeneity of calf intestinal alkaline phosphatase. Arch. Biochem. Biophys. 110, 500-505 (1965). 

E2. Engstrom, L., Studies on calf intestinal alkaline phosphatase. I. Chromatographic purification, microheterogeneity and some other properties of the purified enzyme. Biochim. Biophys. Acta 62, 36-48 (1961). 

FI, Fernley, H. N., and Walker, P. G., Kinetic behavior of calf-intestinal alkaline phosphatase with 4-methylumbelliferyl phosphate. Biochem. J. 97, 95-103 (1965). 

Directed Evolution of Calf Intestinal Alkaline Phosphatase with WalkThrough Recombination... 

Fig. 7.4 Schematic description of the amino acid residue substitutions within the screened calf intestinal alkaline phosphatase (cIAP) variants which are functionally expressed in E. coli. I represents synonymous nucleotide substitution.

Fig. 7.5 Specific activity comparison of the native calf intestinal alkaline phosphatase (clAP), the clAP variant 1A4B, and recombinant human placental alkaline phosphatase (hPAP).

Beumer, C., Wulferink, M., Raaben, W., Fiech-ter, D., Brands, R., Seinen, W. (2003) Calf intestinal alkaline phosphatase, a novel therapeutic dmg for lipopolysaccharide (LPS)-mediated diseases, attenuates LPS toxicity in mice and piglets. /. Pharmacol. Exp. Then 307,... 

Calf intestinal alkaline phosphatase, see clAP Calichemicin 1125 Calmette-guerin vaccine 1430 Calnexin 420, 427 Calogen 477... 

---