Intestinal lactase. Shift in intracellular processing to altered, inactive species in the adult rat.
The regulatory mechanism of decline in catalytic activity for intestinal lactase (lactase-phlorizin hydrolase, beta-galactosidase) as mammals mature has not been defined. Solubilized intestinal brush-border membranes from adult male rats (greater than 4 months of age, 200-400 g) were examined by high performance liquid Zorbax GF-450 chromatography, subjected to denaturing acrylamide electrophoresis, blotted to nitrocellulose, and identified by specific polyvalent anti-lactase. Three major species were present within the 235-kDa active lactase peak (225, 130, and 100 kDa). The 100-kDa moiety was also prominent in the approximately 300-kDa region of the GF-450 effluent, suggesting it is a catalytically inactive oligomer. In vivo synthesis and assembly of lactase by intraintestinal pulse [( 35S]methionine, 5 min) and chase (15-120 min) revealed rapid (15 min of chase; maximum, 60 min) intracellular synthesis in the endoplasmic reticulum-Golgi fraction of multiple species (64, 100, 130, 175, and 225 kDa). The 64-kDa species disappeared from the intracellular membrane compartment and was not transferred to the brush-border surface. The 175-kDa moiety appeared to be processed to the 225-kDa unit prior to relocation to the surface membrane. By 120 min, the 100-kDa species became the predominant (approximately 60%) radiolabeled unit in both endoplasmic reticulum-Golgi and brush border. In the adult rat, lactase is assembled in multiple molecular forms that are differentially processed: (a) intracellular degradation (64-kDa unit) or (b) transfer to the brush-border surface as catalytically active (225 and 130 kDa) or inactive (100 kDa) species. Although substantial synthesis of lactase proteins prevails, major changes in processing appear to serve as an important regulatory mechanism producing the maturational decline of catalytic activity. The accompanying article (Castillo, R. O., Reisenauer, A. M., Kwong, L. K., Tsuboi, K. K., Quan, R., and Gray, G. M. (1990) J. Biol. Chem. 265, 15889-15893) extends our studies to synthesis and assembly during the neonatal period of maturation.
Quan
R
R
Department of Medicine (Gastroenterology), Stanford University School of Medicine, California 94305.
Santiago
N A
NA
Tsuboi
K K
KK
Gray
G M
GM
D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003902:Detergents; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005696:Galactosidases / Q000096:biosynthesis*; D006056:Golgi Apparatus / Q000201:enzymology; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D007583:Jejunum / Q000201:enzymology*; D007700:Kinetics; D043322:Lactase; D008297:Male; D008715:Methionine / Q000378:metabolism; D008871:Microvilli / Q000201:enzymology*; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000201:enzymology; D010206:Papain; D011499:Protein Processing, Post-Translational; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility; D001616:beta-Galactosidase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D003902:Detergents; D007527:Isoenzymes; D008715:Methionine; D005696:Galactosidases; D043322:Lactase; D001616:beta-Galactosidase; D010206:Papain