Nasi Mian

Factors influencing the viscous properties of chicken tracheal mucins

1. Reduced viscosities, in water, of different types of mucin, such as fibrillar, gelatinous and soluble phase, separated from chicken tracheal secretions were measured. 2. H-bond breaking agents caused a significant decrease in the reduced viscosity of these mucins, but thiol-reagents alone did not have any effect. 3. Papain and Pronase did not cause any decrease in the reduced viscosity of these mucins. Neuraminidase decreased the reduced viscosity of soluble phase mucin by 50% by removing about 30% of its N-acetylneuraminic acid but had no effect on fibrillar and gelatinous mucins. Sulphatase neither removed any sulphate ester groups nor decreased the reduced viscosity. Due to some nonspecific intermolecular interaction, mixtures of mucins and enzymes or ovalbumin exhibited elevated reduced viscosities. 4. Ionic strength of the solutions appeared to decrease the reduced viscosity of these mucins. Increasing concentrations of Ca2+ in solutions of ionic strength of approx. 0.1 caused significant decrease in the reduced viscosity, but had no such effect in solutions of ionic strength of more than 0.1. 5. N-Acetylneuraminic acid and sulphate ester residues were 46.6 +/- 0.2, 43.4 +/- 0.6, 27.9 +/- 3.3 mg/g and 66.0 +/- 2.0, 34.2 +/- 3.3, 2.5 +/- 0.8 mg/g for fibrillar, gelatinous and soluble phase mucins, respectively. There appeared to be a good correlation between viscosity and N-acetylneuraminic acid contents among mucins of low reduced viscosities and between viscosity and sulphate ester residues among mucins of high reduced viscosities.

The multiple forms and kinetic differences of rat colonic β-N-acetylhexosaminidases

Rat colonic beta-N-acetylhexosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30) has been separated into three forms by DEAE-cellulose chromatography with an increasing salt gradient. It was not possible to separate the glucosaminidase activity from the galactosaminidase activity by a variety of chromatographic procedues, but the ratio of the two specific activities varied during purification. The pH optima were however identical, for both activities and all three forms. Kinetic measurements including inhibition by substrate analogues showed differences between the two activities as well as among the three forms. A common active site model was inconsistent with the results. Data from mixed substrate experiments were consistent with a model wherein the two activities reside in seperate active sites, each able to be inhibited by the substrate for the other site. The effect of acetate and SH reagents confirmed the two-site model. Treatment with neuraminidase, thimerosal, p-hydroxymercuribenzoate, HgCl2 and AgNO3 or heating at 50 degrees C did not produce any effect on the A form that could be identified as a conversion to the B form. Measurement of the effects on both activities supported the two-site model. It is concluded that the relationship between the A and B forms in the rat colonic mucosa hexosaminidases must be different from that reported for such enzymes from other sources.

Studies on the kinetics of glycosidases from chemically-induced rat colonic tumours and normal rat colon

K-m values of beta-N-acetylglucosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase EC 3.2.1.30), beta-N-acetylgalactosaminidase (EC 3.2.1.53), beta-galactosidase (beta-D-galactoside galactohydrolase EC 3.2.1.23) and alpha-L-fucosidase (alpha-L-fucoside fucohydrolase EC 3.2.1.51) of distal colonic tumours, induced in rats by 1,2-dimethylhydrazine, were found to be significantly different compared with the values for the enzymes of the colonic mucosa of the control and tumour-bearing animals and of the proximal colonic tumours. The inhibition kinetics data also showed a significant difference between the enzymes of the distal colon tumours and of other experimental tissues. The data on the effect of pH on enzyme kinetics (pK values) showed no significant difference in the catalytic groups of the active centres of enzymes from tumours and from the control colonic mucosa. Tumour beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase compared with the enzymes from other experimental tissues were found to be different in their thermal inactivation kinetics. K-m values of 14 days old foetal intestinal beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase were significantly different from the values obtained for the adult mucosal enzymes but were similar to those of the distal colonic tumour enzymes.

Kinetic evidence for two types of active site in the N-acetyl-β-D-hexosaminidase of bovine kidney

N-Acetylhexosaminidases have been reported to hydrolyse the terminal N-acetylglucosamine and N-acetylgalactosamine from fl-glycosidic linkages at comparable rates. In general, it has proved impossible to separate the N-acetyl-/3-D-glucosaminidase activity (2-acetamido-2-deoxy&D-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30) from the N-acetyl-D-Dgalactosaminidase activity (2-acetamido-2-deoxy-flD-galactoside acetamidodeoxygalactohydrolase, EC 3.2.1 S3) by conventional protein fractionation procedures, and this fact, together with the close similarity between their pH and thermal inactivation profiles, has established the view that the two activities are carried by one enzyme that has been designated N-acetylhexosaminidase . Previous kinetic investigations, some rather limited, have concluded that the same active sites are responsible for the two activities [l-7]. However, a detailed kinetic study from this laboratory on the enzymes of rat colonic tissues strongly suggested the presence of two separate active sites, each able to be inhibited by the substrate for the other site [8]. In view of this apparent difference between the rat colonic enzymes and those reported from other sources, it seemed to us desirable to carry out a similar detailed kinetic investigation on a readily available preparation of N-acetylhexosaminidase.

Role of directional Ca2+ effect on reduced viscosities of mucus secretions from chicken trachea in vitro

Reduced viscosities of fibrillar and gelatinous type mucins produced in response to high submucosal Ca2+ and low luminal Ca2+ effects were significantly higher than those of corresponding types of normal mucins. The increased reduced viscosity of experimental mucin samples was due to their aggregation with unique low molecular weight (mr 325,000 and 46,200) sulphate-rich components. The Ca2+ appeared to exert two opposing effects on viscosity properties of mucins; whereas Ca2+-dependent complexes between different types of mucins appeared to be a selective phenomenon between sulphate-rich mucins and components. Ester sulphate residue content rather than N-acetylneuraminic acid residue content of these mucins and low molecular weight components showed a very good correlation with their reduced viscosity and Ca2+-binding values.

Directional Ca2+ effect on stimulation of secretion of common mucins and unique sulphate-rich components from chicken trachea in vitro

High submucosal Ca2+ (3.6-18 mM) significantly increased the secretion of a common high molecular weight fibrillar mucin (approx. Mr is greater than 2.10(6)) and also elicited the secretion of an additional low molecular weight component (approx. Mr 325,000). Low luminal Ca2+ (0.018 mM) also significantly increased the secretion of a common high molecular weight gelatinous mucin (approx. Mr is greater than 2.10(6)) and elicited the secretion of an additional low molecular weight component (approx. Mr 46,200). The additional low molecular weight components were more heavily sulphated (6.7 and 4.2%) than common high molecular weight mucins (2.1 and 1%). The low molecular weight components and high molecular weight mucins were secreted as aggregates which could be dissociated by EGTA. The low molecular weight components and high molecular weight mucins were different in the number of their glycoprotein constituents and in the ion-exchange chromatographic profiles and the carbohydrate and ester sulphate residue content of their acidic glycoproteins.