Release of intestinal surface-membrane glycoproteins associated with enzyme activity by brief digestion with papain.
Rat intestinal surface-membrane glycoproteins were labelled by intraperitoneal injection of [1-(14)C]glucosamine 4h before the animals were killed. At this time, density-gradient centrifugation of disrupted brush borders indicated that glycoprotein radioactivity was distributed identically with sucrase, a plasma-membrane marker. Labelled brush borders were digested by papain for brief time-intervals known to release surface-enzyme particles without disruption of the unit membrane. Digestion for 5min released 90% of the surface sucrase, and almost one-half of the brush-border glycoprotein and label. On Sepharose 4B column chromatography most of the glycoprotein and label emerged as a single peak. This peak contained the most actively labelled glycoprotein in the brush border and was closely associated with maltase, sucrase, beta-naphthylamidase and alkaline phosphatase. The peak was partially resolved on polyacrylamide-gel electrophoresis into three bands. Each band contained a distinctive enzyme or enzyme pair, and was labelled by [1-(14)C]glucosamine. No periodic acid-Schiff-negative protein was observed in the peak material. Glycoproteins susceptible to brief digestion with papain are therefore closely linked to released surface-enzyme particles. Intestinal surface glycoproteins are heterogeneous with respect to molecular weight, electrophoretic mobility and function.
D000469:Alkaline Phosphatase / Q000032:analysis; D000581:Amidohydrolases / Q000032:analysis; D000818:Animals; D002247:Carbon Isotopes; D002462:Cell Membrane / Q000201:enzymology*; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D004589:Electrophoresis, Disc; D005944:Glucosamine / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis*; D007413:Intestinal Mucosa / Q000201:enzymology*; D008297:Male; D008320:Maltose / Q000032:analysis; D010206:Papain; D051381:Rats; D013393:Sucrase / Q000032:analysis
D002247:Carbon Isotopes; D006023:Glycoproteins; D008320:Maltose; D000469:Alkaline Phosphatase; D013393:Sucrase; D010206:Papain; D000581:Amidohydrolases; D005944:Glucosamine