Publisher Summary This chapter discusses the procedures employed for the analysis of the sugar components of glycoproteins. Most of the methods used for the estimation of the monosaccharides present in glycoproteins are colorimetric in nature. Because of the relatively low specificity of some of these color reactions, it is often important to apply them only after appropriate separation of the constituents. In many cases complete identification, preferably by chromatographic means, of all the sugar and amino acid constituents of a glycoprotein should precede any colorimetric analysis so that potentially interfering substances may be taken into account. The hydrolytic release of the monosaccharides is also considered in the chapter. The optimal conditions of acid hydrolysis for the monosaccharides vary substantially because of differences both in the stability of their glycosidic bonds and in their susceptibility to destruction during the hydrolysis. One of the prerequisites for an accurate analysis of the monosaccharide components of a polymer is finding hydrolysis conditions under which all of a given sugar is released and yet is not significantly destroyed.

[1] Analysis of sugars found in glycoproteins

Physiological functions of mineral micronutrients (Cu, Zn, Mn, Fe, Ni, Mo, B, Cl).

Objective: To determine whether extended transthoracic esophagectomy for adenocarcinoma of the mid/distal esophagus improves long-term survival. Background: A randomized trial was performed to compare surgical techniques. Complete 5-year survival data are now available. Methods: A total of 220 patients with adenocarcinoma of the distal esophagus (type I) or gastric cardia involving the distal esophagus (type II) were randomly assigned to limited transhiatal esophagectomy or to extended transthoracic esophagectomy with en bloc lymphadenectomy. Patients with peroperatively irresectable/incurable cancer were excluded from this analysis (n = 15). A total of 95 patients underwent transhiatal esophagectomy and 110 patients underwent transthoracic esophagectomy. Results: After transhiatal and transthoracic resection, 5-year survival was 34% and 36%, respectively (P = 0.71, per protocol analysis). In a subgroup analysis, based on the location of the primary tumor according to the resection specimen, no overall survival benefit for either surgical approach was seen in 115 patients with a type II tumor (P = 0.81). In 90 patients with a type I tumor, a survival benefit of 14% was seen with the transthoracic approach (51% vs. 37%, P = 0.33). There was evidence that the treatment effect differed depending on the number of positive lymph nodes in the resection specimen (test for interaction P = 0.06). In patients (n = 55) without positive nodes locoregional disease-free survival after transhiatal esophagectomy was comparable to that after transthoracic esophagectomy (86% and 89%, respectively). The same was true for patients (n = 46) with more than 8 positive nodes (0% in both groups). Patients (n = 104) with 1 to 8 positive lymph nodes in the resection specimen showed a 5-year locoregional disease-free survival advantage if operated via the transthoracic route (23% vs. 64%, P = 0.02). Conclusion: There is no significant overall survival benefit for either approach. However, compared with limited transhiatal resection extended transthoracic esophagectomy for type I esophageal adenocarcinoma shows an ongoing trend towards better 5-year survival. Moreover, patients with a limited number of positive lymph nodes in the resection specimen seem to benefit from an extended transthoracic esophagectomy.

Extended transthoracic resection compared with limited transhiatal resection for adenocarcinoma of the mid/distal esophagus: five-year survival of a randomized clinical trial.

Analog approaches to the structure of the transition state in enzyme reactions

Publisher Summary This chapter discusses the results of the studies that permit some generalizations on the catalytic mechanism of glycoside hydrolases from widely differing sources and with different sugar and aglycon specificities and that have become available over the past 15 years. The strong inhibition of glycosidases by aldonolactones was first mentioned in 1940 by Japanese workers who studied β- D -glucosidases from Aspergillus (Taka-diastase) and almonds. Even though both of these groups of compounds are derived from normal substrates by only a minor modification of the glycon moiety, they are discussed together with pseudosubstrates because their reactions with glycosidases show, in many cases, unusual kinetic features. The information relevant to the mechanism of an enzyme-catalyzed reaction can, in general, only be obtained from irreversible inhibitors that react specifically at the active site, and thereby inactivate the enzyme. In many cases, inhibition studies were not carried out to obtain information on the reaction mechanism, but for other purposes. Thus, only inhibitors were tested that were considered suitable for the particular project, for example, studies on the biological function of the enzyme where glycosylamines and aldonolactones are unsuitable.

Glycoside Hydrolases: Mechanistic Information from Studies with Reversible and Irreversible Inhibitors

Publisher Summary This chapter discusses the preparation and properties of β–glucuronidase. All mammalian tissues contain a group-specific enzyme that catalyzes the hydrolysis of the biosynthetic β-D-glucopyranosiduronic acids of all types to the aglycons and D-glucuronic acid. This enzyme is commonly known as “β-glucuronidase,” a term that will be employed in the following pages. In the same way, for the sake of brevity, β-D-glucopyranosiduronic acids will often be referred to as β-glucuronides. Enzymes that decompose β-glucuronides have been obtained from diverse sources other than the mammalian cell. β-Glucuronidase finds its major application at the present time as a reagent for the hydrolysis of urinary steroid D-glucuronides, for which purpose it has certain advantages over mineral acids, and the rapid changes in tissue β-glucuronidase activity induced in vivo by certain steroid and pituitary hormones offer possibilities for the rapid measurement of the hormones. The enzyme might perhaps be employed with advantage as a hydrolytic agent in carbohydrate chemistry. Most of the assay methods depend upon colorimetric determination of the amount of aglycon liberated from a specified concentration of substrate at a fixed pH, the results being read from a standard curve. Unit β-glucuronidase activity is then almost universally expressed as that which liberates 1 μg. of aglycon in 1 hr. at 38°; a similar type of unit is employed when the liberation of D-glucuronic acid is followed.

Preparation and Properties of β-Glucuronidase

1. α-Mannosidase from jack-bean meal was purified 150-fold. β-N-Acetyl-glucosaminidase and β-galactosidase were removed from the preparation by treatment with pyridine. Zn2+ was added during the purification to stabilize the α-mannosidase. 2. At pH values below neutrality, α-mannosidase undergoes reversible spontaneous inactivation at a rate dependent on the temperature, the degree of dilution and the extent of purification. The enzyme is also subject to irreversible inactivation, which is prevented by the addition of albumin. 3. Reversible inactivation of α-mannosidase is accelerated by EDTA and reversed or prevented by Zn2+. Other cations, such as Co2+, Cd2+ and Cu2+, accelerate inactivation; an excess of Zn2+ again exerts a protective action, and so does EDTA in suitable concentration. 4. Neither Zn2+ nor EDTA has any marked effect in the assay of untreated enzyme. In an EDTA-treated preparation, however, Zn2+ reactivates the enzyme during assay. 5. It is postulated that α-mannosidase is a dissociable Zn2+–protein complex in which Zn2+ is essential for enzyme activity.

Purification and properties of α-d-mannosidase from jack-bean meal

1. A method was devised for the preparation of (1-->5)-lactones from pyranose sugars and uronic acids by platinum-catalysed oxidation with gaseous oxygen in aqueous solution at acid pH. It was applied to mannose, N-acetylglucosamine, N-acetylgalactosamine, glucuronic acid, galacturonic acid, galactose, l-arabinose and d-fucose. 2. Only the first three yielded products that could be obtained in the solid state without decomposition. In every case, however, the oxidation product in aqueous solution behaved as the aldono-(1-->5)-lactone, and was more inhibitory towards the appropriate glycosidases than any aldonolactone preparation hitherto examined. 3. The stabilities of the oxidation products were studied, and their interconversion with the (1-->4)-lactones was demonstrated. Ring-opening does not appear to be mandatory for this isomeric change, which in some instances is very rapid. 4. To explain all the inhibitory effects observed with aldonolactones on glycosidases of corresponding configuration, it is tentatively postulated that inhibition may be due entirely to the (1-->5)-lactone, and that any inhibitory effect seen with the (1-->4)-lactone is a measure of the extent and speed of its conversion into the (1-->5)-lactone in aqueous solution.

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Inhibition of glycosidases by aldonolactones of corresponding configuration: Preparation of (1-->5)-lactones by catalytic oxidation of pyranoses and study of their inhibitory properties.

1. α-d-Mannosidase from rat epididymis was purified 300-fold. β-N-Acetyl-glucosaminidase and β-galactosidase were removed from the preparation by treatment with pyridine. Zn2+ was added during the purification to stabilize the α-mannosidase. 2. Mammalian α-mannosidase is most stable at pH6. At lower pH values it undergoes reversible spontaneous inactivation. The enzyme is also subject to irreversible inactivation, which is delayed by the addition of albumin. 3. Reversible inactivation of α-mannosidase is accelerated by EDTA and reversed or prevented by Zn2+. Other cations, such as Co2+, Cd2+ and Cu2+, accelerate inactivation and the action of a toxic cation can be prevented by Zn2+ or by EDTA in suitable concentration. 4. The enzyme is stabilized by substrate and neither Zn2+, EDTA nor a toxic cation has more than a small effect in the assay of an untreated preparation. The addition of Zn2+ is necessary, however, for a constant rate of hydrolysis during prolonged incubation of the enzyme with substrate. In an EDTA-treated preparation, Zn2+ reactivates the enzyme during the assay. 5. Evidence is presented that α-mannosidase is a dissociable Zn2+–protein complex, in which Zn2+ is essential for enzyme activity.

Purification and properties of α-d-mannosidase from rat epididymis

Publisher Summary This chapter discusses the methyl and phenyl glycosides of the common sugars. Several new general methods or modified procedures for glycoside synthesis have been introduced. The O -acylglycosyl halides, where the acyl group at the potential reducing group in a fully acylated reducing sugar has been replaced by halogen, are the most generally useful intermediates in the synthesis of alkyl and aryl glycosides, and the O -acetylglycosyl bromides (“acetobromosugars”) are by far the most commonly employed. They are more reactive than the chlorides and more stable than the iodides. In some instances, however, the bromide is so unstable that the chloride is preferred. Yields of O -acetylglycosyl bromides obtained from other sugars by this method were: lactose 85%, arabinose 50%, maltose 60%, cellobiose 72%, and galactose 75%. With cellobiose, some glacial acetic acid has to be added at the start to maintain solution. In general, the products are pure enough to be used without recrystallization in the next stage of glycoside synthesis. Apart from their use as starting materials for the preparation of O -acetyl-glycosyl halides, polyacetylated sugars are employed extensively in glycoside synthesis by the Helferich reaction, in which they are condensed directly with low-melting phenols.

G. A. Levvy

Papers

Preparation and Properties of β-Glucuronidase

Purification and properties of α-d-mannosidase from jack-bean meal

Inhibition of glycosidases by aldonolactones of corresponding configuration: Preparation of (1-->5)-lactones by catalytic oxidation of pyranoses and study of their inhibitory properties.

Purification and properties of α-d-mannosidase from rat epididymis

Methyl and phenyl glycosides of the common sugars.