Phosphatase calf intestinal

Alkaline phosphatase (calf intestinal) Horseradish peroxidase... 

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. 

Scheme 3 Dispensable phosphorylation of HDAC1. HDAC1 is posttranslationally phosphorylated. Upon phosphorylation, HDAC1 forms complexes with RbAp48 and mSinSA, which generate active HDAC1. When the protein complex is treated with calf intestinal phosphatase (CIP), the phosphates are removed, but activity and complex formation remain.

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. 

Center and Behai (49) have resolved 5 -nucleotidase from calf intestinal mucosa into three fractions using DEAE-cellulose chromatography. One of these was obtained free of nonspecific phosphatase. It had a pH optimum of 6-6.5, Mn2+, Mg2+, and Co2+ (1-10 mill) all enhanced activity and complete inactivation was produced with 1 mM EDTA. This enzyme hydrolyzes all 5 -ribonucleotides at similar rates and hydrolyzes 5 -deoxribonucleotides more slowly. These properties indicate that it is strikingly similar to the one obtained from acetone powder preparations of chicken and rat liver (32, 33) and from soluble supernatants of rat liver (36). The other two activities (which were not fully characterized) (49) could possibly have originated from particulate material or membranes because the authors employed deoxycholate in the early phase of purification. 

Amino acid compositions of placental phosphatase have been published (28, 73, 86). Two are in good agreement and are given in Table II. The overall composition is remarkably similar to that of E. coli phosphatase (37)—the relative percentages of amino acid residues on a molar basis are (E. coli values in parentheses) acidic, 21 (21) basic, 14 (12) hydrophilic, 15 (17) and nonpolar, 51 (51). The amino acid sequence around the reactive serine group of calf intestinal phosphatase is Asp-... 

Ultraviolet absorption spectra have been published for enzymes from the following sources calf intestine (90), horse kidney (69a), and human placenta (28). For crystalline placental phosphatase E]%"s nm = 7.8 (in 0.05 M phosphate buffer pH 7.0). Titration curves for calf intestinal phosphatase (range pH 4-10) indicate an isoelectric point of 5.7 which is invariant with respect to temperature (15°-25°) and ionic strength (0.02-0.5) (91). 

Cloetens (98) dialyzed pig kidney phosphatase against 0.01 M KCN for 6 days and found a considerable loss in activity. However, several minutes preincubation with Mg2+ before assay gave up to 40% recovery of activity. Of a series of metal iqns tested, Zn2+ was the most effective giving 70% recovery. Hofstee investigated the effects of glycine, EDTA, and metal ions on calf intestinal phosphatase (99) and concluded that dialysis against EDTA produced an inactive enzyme. Addition of Zn2+... 

The whole question of the specificity was reopened with the discovery that E. coli phosphatase, contrary to an earlier statement (114), hydrolyzed a variety of polyphosphates including metaphosphate of average chain length 8 (97). It was subsequently reported that partially purified phosphatases from several mammalian tissues had appreciable PPi-ase activity at pH 8.5 (115). This was confirmed (116) and extended to include ATPase and fluorophosphatase activities (117). Proof that the same enzyme is responsible for the monoesterase and PPi-ase activities was afforded by heat inactivation studies, cross inhibition experiments, and inhibition of PPi-ase activity by L-phenylalanine, a specific inhibitor of intestinal phosphatase. It was also found that calf intestinal phosphatase couid be phosphorylated by 32P-PP and the number of sites so labeled agreed with the number of active sites determined with a monoester substrate using a stopped-flow technique (118). It would seem that the main reason for the confusion with regard to the PPi-ase activity results from the inclusion of Mg2+ in the assay. This stimulates the monoesterase activity but almost completely inhibits PPi-ase activity (117). 

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.

For the hydrolysis of p-nitrophenyl phosphate by placental phosphatase at pH 10.5 the corresponding figure is 10,380 cal/mole (101a). Taking into account changes in ionization of the enzyme, a value of 9800 cal/mole for 4-methylumbelliferyl phosphate and calf intestinal phosphatase was derived (143). The comparable values for nonenzymic hydrolysis of monoanions of aryl phosphates are 27,000-31,000 cal/mole... 

A detailed study of the effects of dioxane and ethanol on calf intestinal phosphatase showed that Fmax for p-nitrophenyl phosphate decreased... 

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).

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