Alkaline phosphatase, experiment with

Alkaline phosphatase, an enzyme with a molecular weight of approximately 86,000, has been incorporated into a polyanhydride matrix using compression molded PCPP-SA 9 91. Five percent loaded wafers, 50 mg each, were perpared, and measured 1.4 cm in diameter, with a thickness of 0.5 mm. Release experiments were then conducted using techniques similar to those described for carmustine above. As can be seen in Pig. 13, the alkaline phosphatase was released in a well-controlled manner over a prolonged period of time, just over a month, from this polyanhydride. 

As in the alkaline phosphatase example above, p-galactosidase, an enzyme with a molecular weight of approximately 360,000, has also been incorporated into a polyanhydride and released in a well-controlled fashion. As is shown in Fig. 14, the release of 3-galactosidase was quite linear over most of the time examined, and was complete, reaching 100% release in about 800 hr. This experiment utilized 5% loaded, compression-molded wafers of PCPP-SA 9 91, 1.4 cm in diameter and 0.5 mm thick, weighing 50 mg. 

Interest in sol-gel processing was awakened by the work of Avnir et al. in 1990 who performed successful experiments with such enzymes as [1-glucosidasc, alkaline phosphatase, chitinase and aspartase [68]. This gave impetus to their own systematic study of the entrapment of biopolymers in a silica matrix as well as those of other teams [69-79]. The results have been summarized and discussed in numerous review articles (see, e.g., Refs. [41—43,45—49,51,80—85]). 

Considerable ingenuity was required in both the synthesis of these chiral compounds695 697 and the stereochemical analysis of the products formed from them by enzymes.698 700 In one experiment the phospho group was transferred from chiral phenyl phosphate to a diol acceptor using E. coli alkaline phosphatase as a catalyst (Eq. 12-36). In this reaction transfer of the phospho group occurred without inversion. The chirality of the product was determined as follows. It was cyclized by a nonenzymatic in-line displacement to give equimolar ratios of three isomeric cyclic diesters. These were methylated with diazomethane to a mixture of three pairs of diastereoisomers triesters. These dia-stereoisomers were separated and the chirality was determined by a sophisticated mass spectrometric analysis.692 A simpler analysis employs 31P NMR spectroscopy and is illustrated in Fig. 12-22. Since alkaline phosphatase is relatively nonspecific, most phosphate esters produced by the action of phosphotransferases can have their phospho groups transferred without inversion to 1,2-propanediol and the chirality can be determined by this method. 

Neither the occurrence of a constant value of Vmax or a constant product ratio is sufficient proof of the presence of an intermediate. It was seen for alkaline phosphatase that a constant value for Vmax is an artifact, and also that there is no a priori reason why the attack of acceptors on a Michaelis complex should not also give constant product ratios. In order for partitioning experiments to provide a satisfactory proof of the presence of an intermediate, they must be linked with rate measurements. When the rate measurements are restricted to steady state kinetics, the most favorable situation is when the intermediate accumulates. If the kinetics of equations 7.5 to 7.7 hold, it may be concluded beyond a reasonable doubt that an intermediate occurs. The ideal situation is a combination of partitioning experiments with pre-steady state studies, as described for chymotrypsin and amides. 

More recently, isotopic labeling experiments have assumed a major role in establishing the detailed mechanism of enzymic action. It was shown that alkaline phosphatase possesses transferase activity whereby a phos-phoryl residue is transferred directly from a phosphate ester to an acceptor alcohol (18). Later it was found that the enzyme could be specifically labeled at a serine residue with 32P-Pi (19) and that 32P-phosphoserine could also be isolated after incubation with 32P-glucose 6-phosphate (20), providing strong evidence that a phosphoryl enzyme is an intermediate in the hydrolysis of phosphomonoesters. The metal-ion status of alkaline phosphatase is now reasonably well resolved (21-23). Like E. coli phosphatase it is a zinc metalloenzyme with 2-3 g-atom of Zn2+ per mole of enzyme. The metal is essential for catalytic activity and possibly also for maintenance of native enzyme structure. 

In this experiment the Pstl fragment was first digested with DNAase II in sodium acetate buffer, pH 4.7 at room temperature and the reaction halted by chilling and extraction with phenol. After precipitation the DNA was electrophoresed on an 8% polyacrylamide gel and a slice of gel, corresponding to fragments of chain length 150-250 nucleotides cut out and eluted. The 3 -terminal phosphates were removed by treatment with alkaline phosphatase and, after denaturation and removal of the phosphatase with phenol, the DNA was reprecipitated and dissolved in a small volume of water. 

A low detection limit directly influences the sensitivity of the enzyme-based assay. The final enzyme-substrate interaction must yield an ample amount of some end product which can be accurately monitored and, hopefully, quantitated. The authors experiences have been chiefly with enzymatic detection systems which culminate in a visible chromogenic reaction (e.g. alkaline phosphatase, nitroblue tetrazolium, 5-bromo-4-chloro-3-indolyl phosphate). 

To date phosphorothioates have with very few exceptions, been found to be acceptable substrates for stereochemical experiments. They generally react more slowly than naturally occurring substrates, from 1 to 10% of the rates for phosphates in many cases of phosphotransferases and 10 to 100% of the normal rates in the cases of nucleotidyltransferases acting on a-thionucleotides. In a very few cases, notably alkaline phosphatase and phosphoglycerate mutase, phosphorothioates are unacceptable as substrates, necessitating the development of methods for synthesiz-... 

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