A novel mucin sulphatase from human faeces: its identification, purification and characterization.
1. Colonic mucus is heavily sulphated and it is likely that this contributes considerably to its resistance to degradation by bacterial enzymes. The presence of a mucin-desulphating enzyme in faeces could therefore be very important in determining the rate of degradation of secreted mucus and hence the level of protection of the mucosa. 2. A novel assay for mucin sulphatase has been developed using biologically labelled human colonic [35S]sulphomucin as a substrate and a mucin sulphatase has been purified from faeces by sequential high-performance gel filtration and ion-exchange chromatography. 3. The mucin sulphatase has been shown to have a pH optimum of 4.5 and activity over the pH range 3-7. It has a pI of 4.0 and is inhibited by inorganic sulphate and phosphate. The purified enzyme preparation gave a single band on electrophoresis with a molecular mass of 15,000 Da. It has a Km of 41.9 mmol/l and a Vmax. of 1.17 katal/kg for glucose 6-sulphate. The enzyme was also shown to enhance fivefold the deglycosylation of [3H]glucosamine-labelled mucin by a faecal mucin glycosidase preparation. 4. Two bacteroides spp. isolated from normal human faeces, Bacteroides fragilis and B. thetaiotaomicron, were found to be producers of mucin-desulphating enzymes. 5. Mucin sulphatase is likely to be critical in determining the rate of enzymic degradation of secreted colonic mucin.
Tsai
H H
HH
Department of Medicine, University of Liverpool, U.K.
Sunderland
D
D
Gibson
G R
GR
Hart
C A
CA
Rhodes
J M
JM
D001441:Bacteroides fragilis / Q000302:isolation & purification; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D005243:Feces / Q000201:enzymology* / Q000382:microbiology; D006026:Glycoside Hydrolases / Q000032:analysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D009077:Mucins / Q000378:metabolism*; D013429:Sulfatases / Q000302:isolation & purification*
D009077:Mucins; C012564:sulfomucin; D013429:Sulfatases; C074449:mucin sulfatase; D006026:Glycoside Hydrolases