Two recently identified immunodominant epitopes from alpha-gliadin account for most of the stimulatory activity of dietary gluten on intestinal and peripheral T lymphocytes in patients with celiac sprue. The proteolytic kinetics of peptides containing these epitopes were analyzed in vitro using soluble proteases from bovine and porcine pancreas and brush-border membrane vesicles from adult rat intestine. We showed that these proline-glutamine-rich epitopes are exceptionally resistant to enzymatic processing. Moreover, as estimated from the residual peptide structure and confirmed by exogenous peptidase supplementation, dipeptidyl peptidase IV and dipeptidyl carboxypeptidase I were identified as the rate-limiting enzymes in the digestive breakdown of these peptides. A similar conclusion also emerged from analogous studies with brush-border membrane from a human intestinal biopsy. Supplementation of rat brush-border membrane with trace quantities of a bacterial prolyl endopeptidase led to the rapid destruction of the immunodominant epitopes in these peptides. These results suggest a possible enzyme therapy strategy for celiac sprue, for which the only current therapeutic option is strict exclusion of gluten-containing food.

The GATA family of transcription factors regulate tissue-specific patterns of gene expression during development. We have characterized the interaction between GATA proteins and the lactase gene promoter. Nuclear protein bound to the lactase gene GATA region cis element (-97 to -73) was analyzed by electrophoretic mobility shift assays (EMSA) and supershift assays with GATA antibodies. Lactase promoter activities were assayed in Caco-2 cells transfected with wild-type and mutated luciferase promoter-reporter constructs and GATA-4/5/6 expression constructs. EMSA with the GATA region probe yields a specific DNA-protein complex that requires the GATA factor binding site WGATAR. The complex is recognized by GATA-4- and GATA-6-specific antibodies. GATA-4/5/6 expression constructs are able to activate transcription driven by the wild-type promoter, but not by a promoter in which the GATA binding site is mutated, in Caco-2 and nonintestinal QT6 cells. GATA factor binding to the lactase cis element correlates with functional promoter activation. We conclude that each of the GATA family zinc finger proteins expressed in the intestine, GATA-4, -5, and -6, can interact with the lactase promoter GATA element and can function to activate the promoter in Caco-2 cells.

The mechanism of decline of intestinal lactase during mammalian development remains uncertain. Despite a major loss of catalytic activity, lactase mRNA appears to persist at detectable concentrations in adult rats. We quantified lactase activity, total lactase protein, and lactase mRNA in rats aged 7, 11, 15, 18, 22, 30, and 60 days using the 7S ribosomal RNA as the developmental control. The active lactase fraction was 0.81 of total lactase for all age groups except 60-day-old animals, in which it declined to 0.60 (P = 0.004), indicating that conversion of active lactase to inactive species contributed to the lower activity in the adult. Northern blots revealed a single discrete 6.8-kb message at all ages. Although lactase activity and immunoprotein decreased coordinately to a minimum by day 30 (20% of the 7-day value), lactase mRNA doubled to a maximum at day 22 and was maintained at 7-day concentrations even in 60-day adults. The lactase mRNA-to-protein ratio was low at 7 days (0.19) but more than doubled (0.50) by 22 days, achieved a fivefold increase (1.0) by 30 days, and persisted at 0.77 in adults. The relative excess of lactase message during maturation suggests that translational or post-translational events may be paramount in the developmental regulation of lactase gene expression.

Intestinal amino-oligopeptidase (AOP) is an essential brush-border hydrolytic enzyme required for the surface digestion of nutrient oligopeptides produced from luminal pancreatic protease action on dietary protein. There is an abrupt rise in AOP catalytic activity during postnatal rat development, but the mechanism has not been defined. AOP expression was examined in rats 11 to 60 days of age by measurement of AOP mRNA, catalytic activity, and total AOP protein (by quantitative rocket immunoassay). Specific catalytic activity began increasing at 18 days, achieved a maximum by 22 days (+125% over 11 days), and remained stable thereafter. A 1.1-kb AOP cDNA, generated by the polymerase chain reaction and used to quantify specific mRNA, identified a single 3.8-kb species at all ages on Northern blots. The abundance of beta-actin mRNA, which increased slightly (+40%), and 7S RNA, which did not change, was also measured as developmental controls. The AOP mRNA-to-7S RNA ratio increased dramatically (+410%) between 11 and 60 days of age. A comparable initial rise in AOP activity (+130%) and in its mRNA (+170%) was observed between 11 and 22 days, followed by a divergence of the two curves, with a marked relative excess of mRNA compared with catalytic activity in the 60-day-old adult. The ratio of catalytically active to total immunoreactive AOP protein was higher in 60-day-old adults compared with both 11- to 15-day-old preweaned (65% of 60-day value) and 22- to 30-day-old postweaned (61% of 60-day value) animals.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

With the goal of isolating and identifying plasma membrane vesicle populations from epithelial cells of the rat exorbital lacrimal gland, we have designed an analytical fractionation of homogenates of the gland parenchyma. This fractionation utilizes separation procedures based on three independent physical properties of subcellular particles: sedimentation coefficient, density, and density after interaction of membrane cholesterol with digitonin. A commonly accepted marker for basal-lateral membranes, Na-K-ATPase, is associated with at least two physically distinct membrane populations. One population can be identified as basal-lateral membrane fragments on the basis of its fractional and specific contents of Na-K-ATPase; it accounts for 50% of the total Na-K-ATPase activity, enriched 29-fold with respect to the initial homogenate. With these values we calculate that the sample of basal membranes has been purified 60-fold with respect to the initial homogenate. The remaining Na-K-ATPase activity appears to be associated, at three- to fivefold lower specific activities, with intracellular membrane populations. We speculate that these populations have been derived from the Golgi complex.

The intracellular synthesis and processing of intes- tinal aminooligopeptidase, a brush-border surface glycoprotein that is essential for the surface hydrolysis of peptide nutrients, was examined in rats labeled intraintestinally with amino acid or carbohydrate precursors followed by isolation with a specific anti-aminooligo-peptidase globulin. After a 5-min pulse with [36S]methi- onine, incorporation into a nascent form of the enzyme was maximal in the endoplasmic reticulum-Golgi frac- tion after 15 min of nonradioactive chase; nascent aminooligopeptidase declined appreciably by 45 min coincident with its apparent stoichiometric conversion to a larger mature species. Nascent aminooligopeptidase could be cleaved to a smaller form by endo-/I-glycosi-dase H indicating it contains high mannose carbohy- drate chains. Mature aminooligopeptidase was also transiently endo-/I-glycosidase H-sensitive but rapidly became resistant, either prior to or shortly after its transfer to Golgi. Separation of endoplasmic reticulum from Golgi membranes, achieved by subjecting the en- doplasmic reticulum-Golgi fraction to two-phase counter-current distribution, revealed that both nas- cent and mature aminooligopeptidase were present in endoplasmic Intestinal Organelle Fractionation-Two methods 4) 5.5 buffer, sorbitol, on 25-6570 linear sorbitol gradient, centrifuged to equilibrium (SW-27 rotor, 85,000 X g for 18 Brush-borders from pellet by resuspension in 2.5 ml of isolation buffer, 40% sorbitol and centrifuged (85,000 X g a 40-80% linear sorbitol gradient. collected, assayed for marker enzymes, and appropriate fractions brush-border