Acid phosphatase problems

There are stability problems in urines stored for analysis. Fifty percent of delta-aminolevulinic acid was lost in specimens stored without preservative and exposed to light for 24 hours (V3). The loss increased to 80% in 48 hours, 85% in 72 hours, and 95% in 2 weeks. However, the same specimens acidified with tartaric acid and stored in the dark lost 2% of the aminolevulinic acid in 72 hours and 6% in 2 weeks (V3). The destruction of catecholamines collected in nonacidified urine specimens is well documented (Cll). Urinary acid phosphatase was destroyed on freezing (S15). The effect was related to increasing salt concentration during freezing and was prevented by the addition of albumin (S15). 

Stor e characteristics of the native enzyme are generally good, with activities maintained over years. Alkaline phosphatase conjugates are usually prepared via amino or carboxylic acid side chains and purified by gel filtration chromatography. Conjugates are very stable, but the enzyme is costly due to the limited supply of calf intestine. Alkaline (and also neutral and acid) phosphatase enzymes in biological samples are a potential problem with the use of this enzyme label. Careful washing of solid phases may be required to ensure no interference in assays. 

The acid phosphatase activity of leukocytes was studied by Valentine and Beck (B8, VI) in 1951. There appear, however, to have been no significant attempts to purify the enzyme from this source, or to describe its characteristics. Recently, Szajd and Pajdak (S32) indicated the isoenzyme characteristics of leukocyte acid phosphatase, and Li and his associates (L7, L8) studied this problem in greater detail. They suspended a leukocyte preparation, carefully separated from blood, in 5% Triton X-100 to yield a final concentration of 10 X 10 cells per milliliter and subjected the suspension to six cycles of alternate freeze-thaw treatment. The suspension was then centrifuged at lOOOp for 15 minutes at 4°C, and the supernatant was used for electrophoretic studies. Specimens centrifuged at 100,000p for 15 minutes gave the same results. Electrophoresis was carried out at 4°C for 60 minutes on a 7.5% acrylamide gel matrix containing 0.5% Triton X-100 at pH 4.0 with a current of 4 mA per tube. The substrate was -naphthyl phosphate. 

The isomerism existing between the pairs of nucleotides was attributed to the different locations of the phosphoryl residues in the carbohydrate part of the parent nucleoside,49 63 since, for instance, the isomeric adenylic acids are both hydrolyzed by acids to adenine, and by alkalis or kidney phosphatase to adenosine. Neither is identical with adenosine 5-phosphate since they are not deaminated by adenylic-acid deaminase,68 60 and are both more labile to acids than is muscle adenylic acid. An alternative explanation of the isomerism was put forward by Doherty.61 He was able, by a process of transglycosidation, to convert adenylic acids a" and 6 to benzyl D-riboside phosphates which were then hydrogenated to optically inactive ribitol phosphates. He concluded from this that both isomers are 3-phosphates and that the isomerism is due to different configurations at the anomeric position. This evidence is, however, open to the same criticism detailed above in connection with the work of Levene and coworkers. Further work has amply justified the original conclusion regarding the nature of the isomerism, since it has been found that, in all four cases, a and 6 isomers give rise to the same nucleoside on enzymic hydrolysis.62 62 63 It was therefore evident that the isomeric nucleotides are 2- and 3-phosphates, since they are demonstrably different from the known 5-phosphates. The decision as to which of the pair is the 2- and which the 3-phosphate proved to be a difficult one. The problem is complicated by the fact that the a and b" nucleotides are readily interconvertible.64,64... 

Heterogeneous phosphorylation is often a problem when kinases are expressed in insect cells. Multiple approaches have been used to solve this problem. Proteins have been completely dephosphorylated by incubation with A protein phosphatase or alkaline phosphatase [38, 39, 56]. Ion exchange and isoelectric focusing chromatography have been used to separate proteins with multiple phosphorylation states. An y-aminophenyl ATP-sepharose column was used to separate different phosphory-lated states of human c-Src [34]. Alternatively, serine/threonine or tyrosine phosphorylation sites can be mutated to alanine or phenylalanine, respectively [42]. For tyrosine kinases with multiple autophosphorylation sites, the active site aspartic acid can be mutated to an asparagine, creating a kinase dead mutant [57]. 

Another common enzyme used m these procedures is alkaline phosphatase. Alkaline phosphatase will cleave phosphates off of a donor molecule, which then in turn acts as a mediator of a color change involving a third molecule. This system is often used because alkaline phosphatase can create more of the color-producing molecules per enzyme molecule than can peroxidase, resulting in better sensitivity. Alkaline phosphatase systems are especially sensitive for examining protein or nucleic acid blots with enzyme labels. The problem when examining tissue is the presence of the endogenous enzyme m the tissues examined. 

Enzyme DNA hybridization assays with electrochemical detection can offer enhanced sensitivity and reduced instrumentation costs in comparison with their optical counterparts. Efforts to prevent non-specific binding of the codissolved enzyme and to avoid fouling problems by selecting conditions suitable to amplify the electrode response have been reported by Heller and co-workers [107]. A disposable electrochemical sensor based on an ion-exchange film-coated screen-printed electrode was described by Limoges and co-workers for an enzyme nucleic acid hybridization assay using alkaline phosphatase [108] or horseradish peroxidase [109]. In another methodology to improve sensitivity, a carbon paste electrode with an immobilized nucleotide on the electrode surface and methylene blue as hybridization indicator was coupled, by Mascini and co-workers [110], with PGR amplification of DNA extracted from human blood for the electrochemical detection of virus. 

Microcystins have caused the poisoning of wild and domestic animals worldwide, and in 1996, they caused the death of 76 people in Caruaru, Brazil, which was attributed to the use of microcystin-contaminated hemodialysis water. Microcystins, like the well-documented tumor promoter, okadaic acid, strongly and specifically inhibit the protein phosphatases 1 and 2A and have a tumor-promoting activity in the rat liver. In addition to acute hepatotoxicity, microcystins pose problems to human health—which could result from low-level, chronic exposure to microcystins in drinking water, as suggested by the high incidence of primary liver cancer in the... 

Diagnosis of renal problems, xanthinuria, and toxemia of pregnancy via determination of the ratio of hypoxanthine to xanthine in plasma is facilitated by the use of biosensors. Xanthine oxidase immobilized on aminopropyl-CPG (controlled pore glass) activated with glutaraldehyde oxidizes hypoxanthine first to xanthine and then to uric acid. Use of an IMER with biosensors for hypoxanthine, xanthine, and uric acid provides the necessary data. Pre- or postcolumn enzymatic reactions catalyzed by creatinine deiminase, urease, alkaline phosphatase, ATPase, inorganic pyrophosphatase, or arylsufatase facilitate analysis of uremic toxins (simultaneous detection of electrolytes, serum urea, uric acid, creatinine, and methylguanidine). 

As noted in the introduction, the effects of multiple modes of catalysis are often multiplicative rather than simply additive. Consequently, it is not surprising that a number of hydrolytic metalloenzymes have evolved that utilize a constellation of three metal ions in catalysis. Perhaps not coincidentally, all well-characterized examples of this class catalyze the hydrolytic cleavage of phosphate ester or phosphoric acid anhydride bonds, which represent a difficult and long-standing chemical problem. In every case but one, the metal ions in the trimetal centers are all zinc. As we shall see, alkaline phosphatase utilizes a Zn2Mg trinuclear center. It should be pointed out that in the older literature many of the enzymes discussed in this section have been described as containing dinuclear metal centers. Only in the last few years has it become clear that three metal ions are present and participate in catalysis by these systems. 

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