Alkaline phosphatase properties

The major enzymes used in ELISA technology include horseradish peroxidase (HRP), alkaline phosphatase (AP), (3-galactosidase (P-gal), and glucose oxidase (GO). See Chapter 26 for a detailed description of enzyme properties and activities. HRP is by far the most popular enzyme used in antibody-enzyme conjugates. One survey of enzyme use stated that HRP is incorporated in about 80 percent of all antibody conjugates, most of them utilized in diagnostic assay systems. 

The RPIA technology has been enhanced in the Stratus CS system by utilization of a dendrimer-antibody complex in which the analyte-specific capture antibody is covalenty coupled onto a dendrimer. The test packs in the Stratus CS system include dendrimer-capture antibody complex reagent, the alkaline phosphatase labeled antibody conjugate reagent, the substrate-wash reagent and a piece of glass fiber filter paper as the solid phase. Preparation and unique properties associated with these dendrimer-coupled antibody complexes are described below. 

Table 2 Effects of alkaline phosphatase concentration on the gelation time and mechanical properties of the peptide hydrogels of Emoc-Y-OH (entries 1-4) and...

The frequent occurrence of sialylated enzymes, or even of multiple forms, which are sometimes tissue-dependent, with a varying number of sialyl residues as, for example, in y-glutamyltranspeptida.se (EC 2.3.2.2),456,457 is not yet fully understood. Although the activity of most of these enzymes is not influenced by removal of sialic acid,454 the activity of monoamine oxidase A (EC 1.4.3.4) of outer mitochondrial membranes of rat liver has been shown to be destroyed by treatment with sialidase438 the substrate specificity of acetylcholinesterase (EC 3.1.1.7) is altered,459 the kinetic properties of human acid and alkaline phosphatases (EC 3.1.3.1 and 3.1.3.2) are changed, and the stability of a-D-galactosidase (EC 3.2.1.22) is drastically lowered.415 In these cases, an influence of sialyl residues on the conformation of the enzyme is assumed, but awaits firm evidence. 

The non-specific alkaline phosphatases present in bone and calcifying cartilage have several properties in common. The ATPases concerned in the formation of different hard tissues seem to be isozymes. It could be shown that two enzymes capable of degrading ATP exist. One of them can be inhibited by levamisole and R 8231 and is probably identical with non-specific alkaline phosphates. The activity of the other enzyme, tentatively named Ca-ATPase , is dependent on the presence of Ca2+ or Mg2+ and is activated by these ions. The Ca-ATPase is unaffected by ouabain and ruthenium red. It may be speculated that the Ca-ATPase is concerned with the transmembranous transport of Ca2+-ions to the mineralization front229. ... 

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

We shall now briefly outline some of the features of the zinc metalloenzymes which have attracted most research effort several reviews are available, these are indicated under the particular enzyme, and for more detailed information the reader is referred to these. Attention is focussed here, albeit briefly, on carbonic anhydrases,1241,1262,1268 carboxypeptidases, leucine amino peptidase,1241,1262 alkaline phosphatases and the RNA and DNA polymerases.1241,1262,1462 Finally, we examine alcohol dehydrogenases in rather more detail to illustrate the use of the many elegant techniques now available. These enzymes have also attracted much effort from modellers of the enzymic reaction and such studies, which reveal much interesting coordination chemistry and often new catalytic properties in their own right—and often little about the enzyme system itself (except to indicate possibilities), will be mentioned in the next section of this chapter. 

Ironically, AP is the enzyme of choice for some applications due to its stability. Since it can withstand the moderately high temperatures associated with hybridization assays, AP is the enzyme of choice for labeling oligonucleotide probes. Alkaline phosphatase also is capable of maintaining enzymatic activity for extended periods of substrate development. Increased sensitivity can be realized in ELISA procedures by extending the substrate incubation time to hours and sometimes even days. These properties make AP the second most popular choice for antibody—enzyme conjugates (behind HRP), being used in almost 20% of all commercial enzyme-linked assays. 

Recently, two papers have appeared reporting the spectral properties of cobalt alkaline phosphatase. According to Simpson and Vallee (115) addition of two equivalents of Co2+ to the apoenzyme yields only a small absorption increase around 500 nm, characteristic of octahedral complexes. Only slight activation is observed in this process. Further addition of two more equivalents of Co2+ results in full activation and in the formation of an intense visible absorption. Addition in excess of four equivalents has no further effect on activity or spectrum. 

If E. coli is grown in the presence of relatively high concentrations (3.5 fjt,M) of 60Co2+, an active 60Co(II) enzyme is synthetized (122). Its properties resemble those of the Co(II) enzyme reconstituted from the apoenzyme. Certain mutants of E. coli produce defective alkaline phosphatases. The Co(II) enzyme from the mutant U47 lacks the characteristic bands of the Co (II)-complex of the normal enzyme, and the spectrum is broad and poorly resolved (123). 

The most important aspect of the study of Co(II) metalloenzymes is the possibility of using the metal ion as a functional, built-in reporter of the dynamics of the active site. The spectral and magnetic properties of Co (II) carbonic anhydrase have given valuable clues to the catalytic function of this enzyme. The recent studies of Co(II) alkaline phosphatase and Co (II) carboxypeptidase A indicate the general applicability of this approach to enzymes where the probe properties of the constitutive metal ion are poor. The comparison of the absorption spectra of these enzymes and low-molecular weight models have shown that the proteins provide irregular, and in some cases nearly tetrahedral environments. It is obvious, however, that a knowledge of the crystal structures of the enzymes is necessary before the full potential of this method can be exploited. 

Taxol (paclitaxel) is a cytotoxic drug that has been shown to have potent antileukemic and tumor inhibitory properties. Taxol suffers from very poor water solubility (0.25 pg/mL), and hence, a few phosphate ester prodrugs of taxol have been synthesized. While great improvement of water solubility was achieved (>10 mg/mL), these prodrugs were resistant to degradation by alkaline phosphatase, probably due to hindered enzyme access, and no free taxol was formed [50],... 

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

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