Alkaline phosphatase model

PEO-b-PNBOC Polymersomes Alkaline phosphatase Model for theranostics [62]... 

At-MNDP 31-33, 36, 108), whose mechanism of intracellular localization is related to the presence of oncogenically expressed tumor-membrane alkaline phosphatase isoenzymes 42, 108), has been demonstrated strikingly effective in an animal tumor model 33, 34, 38, 39). It has also served as a concomitant analytical probe for identifying the intracellular locus of radiotherapeutic action of this class of drug by a-particle track autoradiography 33,106-109). Phase I and II human therapeutic trials are shortly envisaged 33, 34). 

Liver injury is clinically defined as an increase of serum alanine amino transferase (ALT) levels of more than three times the upper limit of normal and a total bilirubin level of more than twice the upper limit of normal [4]. The clinical patterns of liver injury can be characterized as hepatocellular (with a predominant initial elevation of ALT), cholestatic (with an initial elevation of alkaline phosphatase) or mixed. The mechanisms of drug-induced hepatotoxicity include excessive generation of reactive metabolites, mitochondrial dysfunction, oxidative stress and inhibition of bile salt efflux protein [5]. Better understandings of these mechanisms in the past decades led to the development of assays and models suitable for studying such toxic mechanisms and for selecting better leads in the drug discovery stage. 

In the study of N. A. Gorchakova et al, Enterosgel was used for the treatment of fulminant hepatitis in rats caused by administration of tetrachloromethane [25], It was noted that enterosorption hampered hpid peroxidation in hver tissue of experimental animals, elevated activity of enzymes of the antioxidant pool and decreased activity of serum transaminases, which indicates better preservation of hepatocyte membranes. O.R. Grek et al, used multiple administrations of CCl in combination with drinking of 5% ethanol for modeling of chronic hepatitis in rats. They demonstrated a stable positive effect of Enterosgel administration on activity of serum transaminases and alkaline phosphatase, as well as the rate of hepatic metabolism of xenobiotics [26]. 

A determination of the pH dependence of the lanthanum hydroxide gel-promoted hydrolysis of /3-glyceryl phosphate revealed that the two maxima exist in the pH-rate profile, one at pH 8.6 which presumably involves the species La (OH)+2 and another (smaller) maximum at pH 10.4 which involves the species La (OH) 2+ (4). Presumably the same kind of catalytic mechanism is operative in both cases. These reactions may serve as models for the metal ion-promoted alkaline phosphatases which have been shown to proceed with P—O cleavage (and with no oxygen exchange). 

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. 

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. 

We have already seen a number of models for the zinc(II) containing enzymes such as carbonic anhydrase in Section 11.3.2. Zinc is an essential component in biochemistry, and forms part of the active site of more then 100 enzymes, of which hydrolases (such as alkaline phosphatase and carboxypeptidase A), transferases (e.g. DNA and RNA polymerase), oxidoreductases (e.g. alcohol dehydrogenase and superoxide dismutase) and lysases (carbonic anhydrase) are the most common. In addition, the non-enzyme zinc finger proteins have an important regulatory function. In many of these systems, the non-redox-active Zn2+ ion is present as a Fewis acidic centre at which substrates are coordinated, polarised and hence activated. Other roles of zinc include acting as a template and playing a structural or regulatory role. 

Primary cultured porcine or bovine brain capillary endothelial cells have been used as an in vitro model for the BBB. Recently, an immortalized cell line has been established from mouse, rat, and human brain capillary endothelial cells by infection with Simian virus 40 or transfection of SV40 large T antigen (45 -7). Tatsuta et al. established an immortalized mouse brain capillary endothelial cell line (MBEC4). The activity of y-glutamyl transpeptidase and alkaline phosphatase, specific marker enzymes for brain capillary endothelial cells, was half that in the brain capillary (45). Also, P-gp was expressed on the apical membrane of MBEC4 cells, which corresponds to the abluminal membrane of the brain... 

Another enzyme that was studied extensively in microreactors to determine kinetic parameters is the model enzyme alkaline phosphatase. Many reports have appeared that differ mainly on the types of enzyme immobilization, such as on glass [413], PDMS [393], beads [414] and in hydrogels [415]. Kerby et al. [414], for example, evaluated the difference between mass-transfer effects and reduced effidendes of the immobilized enzyme in a packed bead glass microreactor. In the absence of mass-transfer resistance, the Michaelis-Menten kinetic parameters were shown to be flow-independent and could be appropriately predicted using low substrate conversion data. 

Pavlin D, Dove SB, Zadro R, Gluhak-Heinrich J. Mechanical loading stimulates differentiation of periodontal osteoblasts in a mouse osteoinduction model Effect on type I collagen and alkaline phosphatase genes. Calcif Tissue Int. 2000 67(2) 163-172. 

During the past two decades, essentiality of zinc for man has been established. Deficiency of zinc in man due to nutritional factors and several dls-seased states, has been recognized. A marginal deficiency of zinc appears to be prevalent in many segments of population in developed countries and more severe deficiencies are widespread in many parts of the world. In our experimental human model, a marginal deficiency of zinc was induced by dietary means. Loss of body weight (less than 10% in six months on zinc restricted diet), testicular hypofunction, hyperammonemia and a decrease in plasma, urinary and neutrophil zinc concentration were observed. Changes in zinc dependent enzymes such as deoxythymldine kinase in newly synthesized connective tissue and plasma alkaline phosphatase were also observed as a result of zinc restriction and repletion in our model. 

Kimura and his associates have been preeminent in exploiting the potential of Zn(II) complexes of pendant-arm polyaza macrocycles to act as models for the hydrolytic Zn(II)-containing enzymes. Collectively, their work in this area involves structurally unmodified macrocycles as well as pendant-arm macrocycles, and the reader is referred to a number of reviews 6-15) that summarize their work in its entirety. The particular object of this section is to examine how different types of pendant arm have been introduced onto a macrocyclic framework and how it has been possible to utilize their presence to elicit information of relevance to a particular group of enzymes. The enzyme groups studied using pendant-arm macrocycles have been the alkaline phosphatases and the class II aldolases. 

Ford and Dawson [19] used alkaline phosphatase as a model and found that alkaline phosphatase activity decreased with increasing moisture content. The study involved treated stoppers, flame-sealed glass ampoules, and untreated rubber stoppers. The lowest alkaline phosphatase activity was found in vials with untreated stoppers and relatively high moisture content. The moisture appeared to have originated from the stopper. 

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