Hydrolase activities, fractionation

Drugs may also undergo hydrolysis by intestinal esterases (hydrolases), more specifically carboxylesterases (EC 3.1.1.1) in the intestinal lumen and at the brush border membrane [58, 59]. It has been shown that intestinal hydrolase activity in humans was closer to that of the rat than the dog or Caco-2 cells [60]. In these studies, six propranolol ester prodrugs and p-nitrophenylacetate were used as substrates, and the hydrolase activity found was ranked in the order human > rat Caco-2 cells > dog for intestinal microsomes. The rank order in hydrolase activity for the intestinal cytosolic fraction was rat > Caco-2 cells = human > dog. The hydrolase activity towards p-nitrophenylacetate and tenofovir disoproxil has also been reported in various intestinal segments from rats, pigs and humans. The enzyme activity in intestinal homogenates was found to be both site-specific (duodenum > jejunum > ileum > colon) and species-dependent (rat > man > Pig)-... 

FAAH has been found mainly in microsomal and mitochondrial fractions of rat brain and liver (Deutsch and Chin, 1993 Desarnaud et al., 1995), and of porcine brain (Ueda et al., 1995). Recent studies performed with confocal microscopy, showed that FAAH is localized intracellularly as a vesicular-like staining, that has no association with the plasma membranes and is partially co-localized with the endoplasmic reticulum (Fig. 4.5). These morphological data were corroborated by biochemical assays of FAAH activity in subcellular fractions, showing that AEA hydrolysis was primarily confined to the endomembrane compartment (Oddi et al., 2005). Moreover, by means of reconstituted vesicles derived from purified membrane fractions, it was demonstrated that transport activity is retained by plasma membrane vesicles devoid of FAAH, thereby indicating that AEA hydrolase activity is not necessary for AEA membrane transport. Overall, by means of confocal microscopy, subcellular fractionation, and... 

Table VII. Fractionation of Phosphorylase and Hydrolase Activities in Cell-free Extracts of Cellvibrio gilvus...

Gil, R, M. C. Gonzalvo, A. F. Hernandez, E. Vilanueva, and A. Pla. 1994. Differences in the kinetic properties, effect of calcium and sensitivity in inhibitors of paraoxon hydrolase activity in rat plasma and microsomal fraction of the liver. Biochemical Pharmacology 48 1559-1568. 

Collectively, these experiments show that APS is the substrate reduced and that PAPS is reduced only if hydrolyzed to APS. Three hydrolase activities are associated with the fraction from Chlorella containing the 3 -nucleotidase activity (Tsang and Schiff, 1976b). These activities, which copurify and yield only one protein band on gel electrophoresis, catalyze the following reactions ... 

Since the PHA depolymerases have ndo-hydrolase activity, as mentioned above, the random copolymers with 3HB unit as a major constituent are degradable by PHA depolymerase even though enzymatically inactive monomeric units are introduced. The effects of chemical structure of second monomer units and copolymer compositions on the rate of enzymatic erosion have been examined through the enzymatic degradation of solution-cast films of random copolymers of 3HB with various HA units in the presence of PHA depolymerase. " The enzymatic degradation of solution-cast films of these PHA copolymers was performed in an aqueous solution of purified PHA depolymerase from R. pickettii T1 at 37 °C. The rate of enzymatic erosion of solution-cast PHA films increased markedly with an increase in the fraction of second monomer units up to 10-20 mol.% to reach a maximum value followed by a decrease in the erosion rate (Figure 18). The highest rates of enzymatic erosion were 5-10 times that of the P(3HB) homopolymer film. 

An enzyme from the flowers of Sinningia cardinalis Reichsteinia cardinalis, Gesneriaceae) has hydrolase activity associated with microsomal fractions and requires NADPH as an essential cofactor (hydrolyase activity II). This enzyme converts naringenin (10) and apigenin (5) to eriodictyol (17) and luteolin (4), respectively (Dewick, 1989). The flavone synthase activity of this enzyme was abolished completely by treatment with the cytochrome P-450 inhibitor ancymidol, but the flavonoid 3 -monooxygen-ase activity was not altered. 

The action of hydrolases released on physical damage has been discussed earlier. The results of such action may be rapid and extensive, as shown with bean leaves and potato tubers (see above). To illustrate this, it can be calculated that, if all the endogenous substrates (membrane phospholipids and ga-lactolipids) in potato tuber were available to the acyl hydrolase enzyme, then at pH 6 all the lipid could be deacylated within 1 s at 25°C. Hence the need for care in the isolation of lipids or of subcellular fractions from such tissues. Free fatty acids may be respiratory substrates in storage tissue slices the liberation of free fatty acids by acyl hydrolase activity on cutting the tissue can provide the substrate, as shown by Hasson and Laties (1976a) in potato tuber slices. 

On the other hand, there are other processes that may participate as well, to some extent, in the initial metabolism and hydrolysis of chylomicron cholesteryl esters in the liver. Liver homogenates and homogenate fractions display cholesteryl ester hydrolase activity at neutral pH, and the enzyme(s) responsible for such activity have been partially purified and characterized (Deykin and Goodman, 1962 Stein et al., 1969 Tuhackova et al., 1980). It is possible that some uptake of cholesteryl esters can occur without uptake of the entire remnant particle [see, e.g., Chajek-Shaul et al. (I981a,b) for such evidence in other tissues]. It is also possible that dissociation of the constituents of the remnant can occur to some extent, permitting cholesteryl ester hydrolysis to take place before remnants are delivered to lysosomes. The extent to which these alternative processes might occur in normal physiology is not known. 

Protein synthesis and membrane formation in embryonic and postnatal rat liver occurs faster than in the liver of adult individuals. It was demonstrated that an increase in transferase activity coincided with an increase in hydrolase activity. However, the ratio between these two activities was not constant. This ratio increased from 0.8 (day of delivery) to 2.6 (4-7 days after delivery) and subsequently decreased to 1-2 (adult rat). These fluctuations were caused by the binding of enzyme molecules with microsomal membranes, and differences in the expression of catalytic properties were caused by the relative proportions of membrane-bound and free fractions. It was demonstrated with the aid of enzyme extractions with the nonionic detergent Triton X-100 and sodium deoxycholate (Duck-Chong and Poliak, 1973 Fig. 38). There are bacterial mutants with reduced esterase activity. In some of them esterase activity is reduced because of low levels of enzyme synthesis. Other mutants have low esterase activity due to the weakening of the association of the membrane-enzyme compound (Frehel et al., 1974). 

The leaves and tubers of potatoes contain high levels of lipolytic acyl hydrolase activities (1,2,3). These enzymes are capable of hydrolyzing all endogenous phospholipids and galactoliplds. It was recently reported that all of the lipolytic acyl hydrolase activity in potato tubers is associated with a glycoprotein fraction called "patatin" which comprises about 30% of the soluble protein in tubers (4). This study was undertaken in order to verify whether this finding was valid for other varieties of potatoes (in ref 4 Kennebec was the only variety studied). 

M NaCl DEAE eluate and the Con A Sepharose effluent, indicating that it was not associated with patatin. Much lower levels of C,-NDB-PC hydrolase activity were detected. Although most of this activity also was found in the Con A Sepharose effluent, significant levels were also found in the 0.5 M NaCl DEAE eluate, and the patatin fractions. This experiment indicates that unlike potato tubers, very little of the lipolytic activity in potato leaves is associated with patatin or other glycoproteins which bind to Con A. 

In contradistinction to the decrease in lysosomal enzyme activity observed in alveolar macrophages and bronchial lavage fluid, Dillard et al. reported that continuous ozone exposure (0.70-0.79 ppm for 5-7 days) resulted in an increase in the activity of some lysosomal hydrolases in rat whole-lung homogenates and lung fractions, including the soluble supernatant. The tocopherol concentrations of the diet had no effect on the findings. 

In mouse liver and kidney and in rat liver, a-D-mannosidase activity appeared to be equally distributed between the two cytoplasmic-granule fractions. With mouse spleen and cancer tissue, a considerable proportion of the enzyme was found free in the cytoplasm. Rat spleen, on the other hand, lacked this cytoplasmic fraction. Inasmuch as the enzyme within the cytoplasmic granules was not fully active in a sucrose homogenate until the membranes had been disintegrated, a-D-mannosidase conforms to the definition of a lysosomal hydrolase. 

Bacillus subtilis /zNB esterase is a member of the a./(3 hydrolase fold family (Moore and Arnold, 1996 Ollis et al., 1992). The canonical a/j3 hydrolase fold consists of a mostly parallel eight-stranded [3 sheet surrounded on both sides by a helices (Nardini and Dijkstra, 1999). p B esterase contains 489 amino acids arranged in a central thirteen-stranded f3 sheet that is surrounded by fifteen a helices (Fig. 12, see color insert). Similar to the structure of acetylcholine esterase (Sussman et al., 1991), a large fraction of the pSB esterase structure has no defined secondary structure (52% random coil, 33% a helix, and 14% /3 sheet). This high degree of random coil structure is allowed in the a/(3 hydrolase fold, where large insertions in loops were found to be tolerated while still maintaining catalytic activity (Nardini and Dijkstra, 1999). 

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