A-D-Mannosidase

A case similar to the slow, practically irreversible inhibition of jack bean a-D-mannosidase by swainsonine is represented by the interaction of castanospermine with isomaltase and rat-intestinal sucrase. Whereas the association constants for the formation of the enzyme-inhibitor complex were similar to those of other slow-binding glycosidase inhibitors (6.5 10 and 0.3 10 M s for sucrase and isomaltase, respectively), the dissociation constant of the enzyme-inhibitor complex was extremely low (3.6 10 s for sucrase) or could not be measured at all (isomaltase), resulting in a virtually irreversible inhibition. Danzin and Ehrhard discussed the strong binding of castanospermine in terms of the similarity of the protonated inhibitor to a D-glucosyl oxocarbenium ion transition-state, but were unable to give an explanation for the extremely slow dissociation of the enzyme-inhibitor complex. 

Group (c), a-D-mannosidase from jack beans and from almonds, and a-D-galactosidase from coffee beans, showed no inactivation. The results with these enzymes can possibly be explained by the formation of a (weak) non-covalent complex in which glycosylation is too slow to cause inactivation within the time period of measurements, or, less likely, rapid hydrolysis of the glycosyl-enzyme intermediate. 

A. E. Hakansson, J. van Ameijde, L. Guglielmini, G. Home, R. J. Nash, E. L. Evinson, A. Kato, and G. W. J. Fleet, Looking glass inhibitors Synthesis of a potent naringinase inhibitor l-DIM (l,4-dideoxy-l,4-imino-L-mannitol), the enantiomer of DIM (l,4-dideoxy-l,4-imino-D-mannitol) a potent a-D-mannosidase inhibitor, Tetrahedron Asymmetry, 18 (2007) 282-289. 

One particular example of the early extraction of a chemical from a plant, a small tree found in the rainforests of eastern Queensland, would be that of acronycine 1 [1,2], an alkaloid currently undergoing clinical trials as an anticancer agent. More recently, a group in Perth at Murdoch University succeeded in extracting the alkaloid, swainsonine 2 from a desert shrub which causes poisoning in cattle [3]. Swainsonine has been found to exhibit an interesting spectrum of biological activity [4], especially as a potent, reversible inhibitor of various a-D-mannosidases [5]. 

There are two attributes of a-D-mannosidase (EC 3.2.1.24) that receive particular attention in this article, namely, its behavior as a zinc-containing metalloenzyme, and its action on naturally occurring, D-mannose-containing molecules. However, such more-systematic considerations as the kinetics of action and the distribution of the enzyme in Nature are not overlooked. 

Early work on a-D-mannosidase of plant origin, notably in almond emulsin, has been described.1 There has been a revival of interest in the enzyme from all sources, and an extensive survey of plant... 

Since Lewy and coworkers5 first showed that a-D-mannosidase occurs in mammals, its presence has been detected in virtually every... 

Ovariectomy caused a pronounced fall in the a-D-mannosidase activity of mouse uterus, and the normal level was restored by administration of estrone.28 Although the ovary is not particularly rich in a-D-man-nosidase, the greater proportion of the activity in cattle, pig, and human ovaries was found to be concentrated in the corpus luteum.14,28... 

Results are expressed as fig of p-nitrophenol liberated per g of seed from 6 mM p-nitrophenyl a-D-mannoside in 1 hr at 37° and pH 5. Coarsely ground beans. Defatted sweet-almond meal. A commercial /3-D-glucosidase preparation purified from emulsin. a-D-Mannosidase activity is given per g of purified material. 

Distribution of a-D-Mannosidase Activities" in Sucrose Homogenates of Mammalian Tissues15... 

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. 

The theory has been advanced that there exists in the cell a particulate structure somewhat smaller than the mitochondrion, the lyso-some, that contains certain autolytic enzymes in a latent situation. The lysosome theory55,56 is very largely based upon measurements made in sucrose homogenates of rodent liver. Although the results for a-D-mannosidase in this tissue (see Table IV) are not incompatible with the theory, the results for other tissues do not always conform to it. In particular, the contrast between mouse and rat spleen argues against a universal single particle to which a-D-mannosidase is confined. Apart from the results quoted in Table IV, not much work has been done on the intracellular location of a-D-mannosidase. 

For the assay of a-D-mannosidase, the incubation mixture employed in our laboratory contained 0.5 ml of M acetate buffer at a pH appropriate for the particular enzyme preparation, 1.5 ml of 16 mM p-nitrophenyl a-D-mannopyranoside, 1.5 ml of water (which could be replaced by other additives as required), and 0.5 ml of suitably diluted, enzyme preparation. After 1 hour at 37°, the reaction was terminated, and the color was developed by adding 4 ml of 0.4 M glycine-sodium hydroxide buffer, pH 10.5. The mixture was centrifuged for 15 minutes at 1500 g, and the color intensity of the liberated p-nitrophenol (25-150 fig) in the supernatant liquor was measured on a Spekker photoelectric absorptiometer, with use of Ilford No. 601 violet filters having maximal transmission at 430 nm, and a 1-cm light path. Separate control-experiments for enzyme and sub-... 

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