Diesterase activity

Although in no case has the enzyme been purified to homogeneity, much evidence exists that the ribonudeoside 2, 3 -cyclic phosphate diesterase activity and the 3 -nucleotidase activity reside in the same protein. Thus, in all cases the ratio of the two activities remained constant throughout purification which has varied from 130-fold for the P. mirabilis enzyme (4) to 2000-fold for the enzyme from V. alginolyticus (6). Anraku (S) found that both activities from E. coli B had the same optimal pH, both showed the same behavior to activators such as Co2+, and to inhibitors [Zn2+, Cu2+, ethylenediaminetetraacetate (EDTA)], both were activated simultaneously by heating at 55° for 5 min and... 

Prior to 1961, ribonucleoside 2, 3 -cyclic phosphate diesterase activity had been reported from two vertebrate tissues, calf spleen (24) and beef... 

Tsukada (30) have examined developmental changes of the enzyme in chick brain and spinal cord. Enzymic activity appears at about the eighteenth day of incubation and increases rapidly until 3 days after hatching in the brain and between 18 and 21 days of incubation in the spinal cord. These are precisely the periods of active myelination in the brain and spinal cord of the chick, respectively. Similarly, brain tissue of the newborn rat is devoid of cyclic phosphate diesterase activity it appears at about 8 days after birth and increases dramatically between the tenth and thirty-fifth day of life (29). This coincides precisely with the development of myelin in this species. The diesterase is essentially absent in the brain of the jimpy mouse (31), a lethal mutant devoid of myelin in the central nervous system. It is also absent from the spinal cord of this mutant. The enzyme is about 50% deficient in brain tissue of the quaking mouse (29), a mutant with partial deficiency of myelin. There is no activity in nerve fibers and ganglia from a variety of invertebrates such as squid, octopus, crab, shrimp, and starfish. Nerve tissue in these organisms is nonmyelinated. All these observations point to an intimate association of the enzyme with myelin in vivo. 

When prostatic acid phosphatase is purified so that it is completely free of diesterase activity, it can be used in a variety of structural... 

The reaction mixture contained the diesterase activity from brain homogenate (about 10 fig of protein), Tris-HCl buffer (pH 7.4), and MgS04. The reaction was started by addition of substrate. The reactions were terminated at suitable intervals with ethanol and extracted with chloroform. After centrifugation for 10 minutes, 1 fiL of the supernatant solution was analyzed. Figure 9.962 shows the results of the assay, illustrating an increase in the height of the peak of the reaction product, 2 -AMP, with incubation. 

Cerebral tissues were homogenized with a glass homogenizer and used as the source of the diesterase activity. 

Calmodulin has been hybridized with phospho diesterase with retaining its activity to respond to calcium ion in changing its conformation so as to activate phospho diestelase. Intelligent molecular materials have been designed using calmodulin. 

II. Ribonudeoside 2, 3 -Cydic Phosphate Diesterase with 3 -Nudeotidase Activity from Microorganisms... 

UMP becomes bound to site B which catalyzes the hydrolysis of the phosphomonoester bond. Adenosine and 3 -AMP by binding at site B could interfere with the breakdown of cyclic 2, 3 -UMP. Similarly, binding of bis (p-nitrophenyl) phosphate at site A could interfere with the breakdown of 3 -AMP. Cyclic 2, 3 -UMP and bis(p-nitrophenyl) phosphate compete for site A while adenosine competes with 3 -AMP for site B. Unemoto et al. (7) have examined the mutual inhibition of substrates and substrate analogs for the enzyme from halophilic V. alginolyticus. They also concluded that 3 -ribonucleotides and ribonucleo-side 2, 3 -cyclic phosphates are hydrolyzed at different sites. However, because of the nature of the mutual inhibition between 3 -AMP and bis(p-nitrophenyl) phosphate, they suggested that part of the site for the latter substrate overlaps with the 3 -nucleotidase site. At this time the precise mechanism of action of the enzyme is not settled, but clearly there are two active sites, one a 3 -nucleotidase site and a cyclic phosphate diesterase site. Anraku (18) has described this protein as a double-headed enzyme. 

Early studies revealed that the 3, 5 -cyclic phosphate diesterase is present in all mammalian tissues (38, 33, 36), being most active in cerebral cortex (36, 37). It has also been identified in extracts of liver fluke (Fasciola hepatica), the common earthworm (Lumbricus terrestris), and fly larvae (36) and it has been studied in marine organisms (38), the cellular slime mold Dictyostelium discovdeum (39, AO), and in E. coli (Al). The enzyme has been partially purified from beef heart (30), dog heart, (A8) and bovine brain (37, A3). No highly purified preparations have yet been obtained and most studies have been performed with relatively crude preparations. 

AMP is related to certain mental diseases and may be involved in the action of tranquilizers and antidepressant drugs (60). Whether the ability of diuretic agents such as ethacrynic acid and chlorthalidone to inhibit the enzyme in kidney (68) is related to their diuretic action is also not known. It has been suggested that inhibition of diesterase by diazoxide (59) may explain the hyperglycemic activity of this agent. Several materials are known to activate the enzyme. Imidazole produces strong activation of the enzyme from mammalian tissues (36, 38, 42) but not from E. coli (41). It has been reported (61) that insulin activates the beef heart enzyme in vitro, but it is not known if this has relevance... 

In addition to the proposed regulatory role of ATP and pyrophosphate, some possibility exists that 3, 5 -cyclic phosphate diesterase is under physiological control. Such ideas arose through observations of Cheung (43, 62) that the partially purified enzyme from beef brain was markedly activated by snake venom. The stimulatory factor was labile at extreme pH it was not dialyzable and appeared to be a protein. A similar activating factor is also present in brain tissue (63) and is removed during purification of the diesterase. It seems to interact stoichiometrically with the enzyme. The activator is destroyed by trypsin and is not proteolytic itself. The precise role of this protein in regulating the phosphodiesterase in vivo is not yet established, however. 

It must be emphasized that much of the information on the enzyme to date comes from partially purified preparations. Even though it seems uniquely specific for nucleoside 3, 5 -cyclic phosphates, the possibility cannot be excluded that in some tissues more than one diesterase may be present. In fact, Hardman and Sutherland (64) have shown that a second 3, 5 -cyclic phosphate diesterase exists in heart muscle which is primarily specific for uridine 3, 5 -cyclic phosphate. This enzyme was found to be more sensitive to inhibition by theophylline and to activation by imidazole. Further studies are needed to clarify its precise status other such enzymes may be discovered in the future. 

---