Congenital lactose intolerance is triggered by severe mutations on both alleles of the lactase gene.

BACKGROUND: Congenital lactase deficiency (CLD) is a rare severe autosomal recessive disorder, with symptoms like watery diarrhea, meteorism and malnutrition, which start a few days after birth by the onset of nursing. The most common rationales identified for this disorder are missense mutations or premature stop codons in the coding region of the lactase-phlorizin hydrolase (LPH) gene. Recently, two heterozygous mutations, c.4419C > G (p.Y1473X) in exon 10 and c.5387delA (p.D1796fs) in exon 16, have been identified within the coding region of LPH in a Japanese infant with CLD. METHODS: Here, we investigate the influence of these mutations on the structure, biosynthesis and function of LPH. Therefore the mutant genes were transiently expressed in COS-1 cells. RESULTS: We show that both mutant proteins are mannose-rich glycosylated proteins that are not capable of exiting the endoplasmic reticulum. These mutant proteins are misfolded and turnover studies show that they are ultimately degraded. The enzymatic activities of these mutant forms are not detectable, despite the presence of lactase and phlorizin active sites in the polypeptide backbone of LPH-D1796fs and LPH-Y1473X respectively. Interestingly, wild type LPH retains its complete enzymatic activity and intracellular transport competence in the presence of the pathogenic mutants suggesting that heterozygote carriers presumably do not show symptoms related to CLD. CONCLUSIONS: Our study strongly suggests that the onset of severe forms of CLD is elicited by mutations in the LPH gene that occur in either a compound heterozygous or homozygous pattern of inheritance.

Gata4 and Hnf1alpha are partially required for the expression of specific intestinal genes during development.

The terminal differentiation phases of intestinal development in mice occur during cytodifferentiation and the weaning transition. Lactase-phlorizin hydrolase (LPH), liver fatty acid binding protein (Fabp1), and sucrase-isomaltase (SI) are well-characterized markers of these transitions. With the use of gene inactivation models in mature mouse jejunum, we have previously shown that a member of the zinc finger transcription factor family (Gata4) and hepatocyte nuclear factor-1alpha (Hnf1alpha) are each indispensable for LPH and Fabp1 gene expression but are both dispensable for SI gene expression. In the present study, we used these models to test the hypothesis that Gata4 and Hnf1alpha regulate LPH, Fabp1, and SI gene expression during development, specifically focusing on cytodifferentiation and the weaning transition. Inactivation of Gata4 had no effect on LPH gene expression during either cytodifferentiation or suckling, whereas inactivation of Hnf1alpha resulted in a 50% reduction in LPH gene expression during these same time intervals. Inactivation of Gata4 or Hnf1alpha had a partial effect ( approximately 50% reduction) on Fabp1 gene expression during cytodifferentiation and suckling but no effect on SI gene expression at any time during development. Throughout the suckling period, we found a surprising and dramatic reduction in Gata4 and Hnf1alpha protein in the nuclei of absorptive enterocytes of the jejunum despite high levels of their mRNAs. Finally, we show that neither Gata4 nor Hnf1alpha mediates the glucocorticoid-induced precocious maturation of the intestine but rather are downstream targets of this process. Together, these data demonstrate that specific intestinal genes have differential requirements for Gata4 and Hnf1alpha that are dependent on the developmental time frame in which they are expressed.

Intestinal protein and LPH synthesis in parenterally fed piglets receiving partial enteral nutrition and enteral insulinlike growth factor 1.

BACKGROUND: Providing partial enteral nutrition (PEN) supplemented with insulinlike growth factor-1 (IGF-1) to parenterally fed piglets increases lactase-phlorizin hydrolase (LPH) activity, but not LPH mRNA. The current aim was to investigate potential mechanisms by which IGF-1 up-regulates LPH activity. METHODS: Newborn piglets (n = 15) received 100% parenteral nutrition (TPN), 80% parenteral nutrition + 20% parenteral nutrition (PEN), or PEN + IGF-1 (1.0 mg. kg-1. d-1) for 7 days. On day 7, [2H3]-leucine was intravenously administered to measure mucosal protein and brush border LPH (BB LPH) synthesis. RESULTS: Weight gain, nutrient intake, and jejunal weight and length were similar among the treatment groups. Partial enteral nutrition alone increased mucosal weight, villus width and cross-sectional area, LPH activity, mRNA expression, and high mannose LPH precursor (proLPHh) abundance compared with TPN (P<0.05). Insulinlike growth factor-1 further increased mucosal weight, LPH activity, and LPH activity per unit BB LPH approximately twofold over PEN alone (P < 0.05) but did not affect LPH mRNA or the abundance of proLPHh (one of the LPH isoforms) or mature LPH. Isotopic enrichment of [2H3]-leucine in plasma, mucosal protein, and LPH precursors, and the fractional and absolute synthesis rates of mucosal protein and LPH were similar among the treatment groups. Insulinlike growth factor-1 treatment increased total mucosal protein synthesis (60%, P < 0.05) but not LPH synthesis compared with the other two groups. CONCLUSIONS: Because IGF-1 did not affect the fractional synthesis rate of either mucosal protein or LPH, the authors suggest that enteral IGF-1 increases mucosal protein mass and LPH activity by suppressing mucosal proteolytic degradation.

Molecular and cellular aspects and regulation of intestinal lactase-phlorizin hydrolase.

Carbohydrates are hydrolyzed in the intestinal lumen by specific enzymes to monosaccharides before transport across the brush border membrane of epithelial cells into the cell interior. The enzymes implicated in the digestion of carbohydrates in the intestinal lumen are membrane-bound glycoproteins that are expressed at the apical domain of the enterocytes. Absent or reduced activity of one of these enzymes is the cause of disaccharide intolerance and malabsorption, the symptoms of which are abdominal pain, cramps or distention, flatulence, nausea and osmotic diarrhea. Lactose intolerance is the most common intestinal disorder that is associated with an absence or drastically reduced levels of an intestinal enzyme, in this case lactase-phlorizin hydrolase (LPH). The pattern of reduction of activity has been termed late onset of lactase deficiency or adult type hypolactasia. It was thought that the regulation of LPH was post-translational and was associated with altered structural features of the enzyme. Recent studies, however, suggest that the major mechanism of regulation of LPH is transcriptional. Other forms of lactose intolerance include the rare congenital lactase deficiency and secondary forms, such as those caused by mucosal injury, due to infectious gastroenteritis, celiac disease, parasitic infection, drug-induced enteritis and Crohn's disease. This review will shed light on important strucural and biosynthetic aspects of LPH, the role played by particular regions of the LPH protein in its transport, polarized sorting, and function, as well as on the gene expession and regulation of the activity of the enzyme.

Lactase synthesis is pretranslationally regulated in protein-deficient pigs fed a protein-sufficient diet.

The in vivo effects of protein malnutrition and protein rehabilitation on lactase phlorizin hydrolase (LPH) synthesis were examined. Five-day-old pigs were fed isocaloric diets containing 10% (deficient, n = 12) or 24% (sufficient, n = 12) protein. After 4 wk, one-half of the animals in each dietary group were infused intravenously with [(13)C(1)]leucine for 6 h, and the jejunum was analyzed for enzyme activity, mRNA abundance, and LPH polypeptide isotopic enrichment. The remaining animals were fed the protein-sufficient diet for 1 wk, and the jejunum was analyzed. Jejunal mass and lactase enzyme activity per jejunum were significantly lower in protein-deficient vs. control animals but returned to normal with rehabilitation. Protein malnutrition did not affect LPH mRNA abundance relative to elongation factor-1alpha, but rehabilitation resulted in a significant increase in LPH mRNA relative abundance. Protein malnutrition significantly lowered the LPH fractional synthesis rate (FSR; %/day), whereas the FSR of LPH in rehabilitated and control animals was similar. These results suggest that protein malnutrition decreases LPH synthesis by altering posttranslational events, whereas the jejunum responds to rehabilitation by increasing LPH mRNA relative abundance, suggesting pretranslational regulation.

Influence of the diet made by its family ancestors in the hereditary component of an individual: study in six generations of rats.

BACKGROUND: The prevalence of the deficiency of enzyme Lactase/phlorizin hydrolase (LPH) varies widely between different countries, with a low of 1-3% in Scandinavia and close to 100% in most of South-East Asia. Various carbohydrates are capable of enhancing LPH mRNA levels in the small intestine, and that transcriptional control plays a major role in the carbohydrate-induced alterations of LPH mRNA expression. MATERIAL AND METHODS: A generation of rats was randomized and assigned to two groups; those that were fed a high-carbohydrate diet and, those that were fed standard rodent meal. The sixth generation of these animals was fed standard rodent meal. Transfection experiments using Caco-2 cells were performed using sperm samples of the sixth generation of rats. RESULTS: This study suggests that the feeding with a high-carbohydrate diet during five generations of rats increases the capacity of production of enzyme LPH, LPH mRNA levels and the transcription rate of the LPH gene in the sixth generation of these animals, and this fact happens independently of the diet that this generation of rats had. Transfection experiments show that this influence has had to take place necessarily within the hereditary component which the last generation of rats received from its family ancestors. CONCLUSION: The feeding received by the ancestors of a generation of rats could influence within hereditary component received by them.

Interaction between the homeodomain proteins Cdx2 and HNF1alpha mediates expression of the lactase-phlorizin hydrolase gene.

Lactase-phlorizin hydrolase is a brush-border enzyme which is specifically expressed in the small intestine where it hydrolyses lactose, the main carbohydrate found in milk. We have previously demonstrated in transgenic mice that the tissue-specific and developmental expression of lactase is controlled by a 1 kb upstream region of the pig lactase gene. Two homeodomain transcription factors, caudal-related homeodomain protein (Cdx2) and hepatic nuclear factor 1alpha (HNF1alpha), are known to bind to regulatory cis elements in the promoters for several intestine-specific genes, including lactase, and are present in mammalian intestinal epithelia from an early stage in development. In the present study, we examined whether Cdx2 and HNF1alpha physically interact and co-operatively activate transcription from the lactase-phlorizin hydrolase promoter. We show that the presence of both factors leads to a much higher level of transcription than the sum of the activation by either factor alone. The N-terminal activation domain of Cdx2 is required for maximal synergy with HNF1alpha. With the use of pull-down assays, we demonstrate a direct protein-protein interaction between Cdx2 and HNF1alpha. The interaction domain includes the homeodomain region of both proteins. This is the first demonstration of a functional interaction between two transcription factors involved in the activation of a number of intestine-specific genes. Synergistic interaction between tissue-restricted factors is likely to be an important mechanism for reinforcing developmental and tissue-specific gene expression within the intestine.

Dietary carbohydrates enhance lactase/phlorizin hydrolase gene expression at a transcription level in rat jejunum.

We have previously shown that dietary sucrose stimulates the lactase/phlorizin hydrolase (LPH) mRNA accumulation along with a rise in lactase activity in rat jejunum [Goda, Yasutake, Suzuki, Takase and Koldovský (1995) Am. J. Physiol. 268, G1066-G1073]. To elucidate the mechanisms whereby dietary carbohydrates enhance the LPH mRNA expression, 7-week-old rats that had been fed a low-carbohydrate diet (5.5% of energy as starch) were given diets containing various monosaccharides or sucrose for 12h. Among carbohydrates examined, fructose, sucrose, galactose and glycerol elicited an increase in LPH mRNA accumulation along with a rise in lactase activity in the jejunum. By contrast, glucose and alpha-methylglucoside were unable to elicit a significant increase in LPH mRNA levels. To explore a transcriptional mechanism for the carbohydrate-induced increases in LPH mRNA levels, we employed two techniques currently available to estimate transcriptional rate, i.e. RNA protection assays of pre-mRNA using an intron probe, and nuclear run-on assays. Both assays revealed that fructose elicited an increase in transcription of the LPH gene, and that the transcription of LPH was influenced only slightly, if at all, by glucose intake. These results suggest that certain monosaccharides such as fructose or their metabolite(s) are capable of enhancing LPH mRNA levels in the small intestine, and that transcriptional control might play a major role in the carbohydrate-induced increase of LPH mRNA expression.

Protein kinetics determined in vivo with a multiple-tracer, single-sample protocol: application to lactase synthesis.

Precise analysis of the kinetics of protein/enzyme turnover in vivo has been hampered by the need to obtain multiple tissue samples at different times during the course of a continuous tracer infusion. We hypothesized that the problem could be overcome by using an overlapping (i.e., staggered) infusion of multiple stable amino acid isotopomers, which would take the place of multiple tissue samples. We have measured, in pigs, the in vivo synthesis rates of precursor (rapidly turning over) and mature (slowly turning over) polypeptides of lactase phlorizin hydrolase (LPH), a model for glycoprotein synthesis, by using an overlapping infusion of [2H3]leucine, [13C1]leucine, [13C1]phenylalanine, [2H5]phenylalanine, [13C6]phenylalanine, and [2H8]phenylalanine. Blood samples were collected at timed intervals, and the small intestine was collected at the end of the infusion. The tracer-to-tracee ratios of each isotopomer were measured in the plasma and jejunal free amino acid pools as well as in purified LPH polypeptides. These values were used to estimate kinetic parameters in vivo using a linear steady-state compartmental model. The fractional synthesis rates of the high-mannose, complex glycosylated and mature brush-border LPH polypeptides, so determined, were 3.3 +/- 1.1%/min, 17.4 +/- 11%/min, and 0.089 +/- 0.02%/min, respectively. We conclude that this multiple-tracer, single-sample protocol is a practicable approach to the in vivo measurement of protein fractional synthesis rates when only a single tissue sample can be obtained. This method has broad application and should be particularly useful for studies in humans.

GATA-6 stimulates a cell line-specific activation element in the human lactase promoter.

Lactase-phlorizin hydrolase (LPH) synthesis is restricted to differentiated small intestinal enterocytes and is highly regulated during development. Analysis of expression of LPH promoter segments fused with luciferase transfected in Caco-2 cells, a line that uniquely expresses LPH mRNA, mapped an 18-base pair (bp) segment 100 bp upstream of the transcription start site that is required for transactivation. Remarkably, the LPH upstream element (LUE) has no stimulatory activity in both human intestinal and nonintestinal lines in which LPH mRNA is absent. Electrophoretic analysis of sequence-specific DNA-nuclear protein complexes demonstrated the presence of a Caco-2 cell-specific protein(s) (CCP), which is uniformly absent in LPH nonproducer cell lines. Mutational analysis of the LUE demonstrated that bases contained within a GATA consensus motif are critical for both CCP binding and transcription from the LPH promoter. Caco-2 cells express high levels of GATA-6 mRNA in a cell line-specific manner, suggesting that GATA-6 is a CCP that complexes with the LUE. When expressed by a plasmid, GATA-6 transactivated the LPH promoter. The stimulation was abrogated with mutations in the GATA consensus motif as well as mutations in a flanking downstream element. These studies are consistent with an important role of an intestinal GATA binding protein in cell type-specific transactivation of the LPH promoter.

Parenteral nutrition selectively decreases protein synthesis in the small intestine.

We investigated the effects of an elemental diet fed parenterally or enterally on total mucosal protein and lactase phlorizin hydrolase (LPH) synthesis. Catheters were placed in the stomach, jugular vein, and carotid artery of 12 3-day-old pigs. Half of the animals were given an elemental regimen enterally and the other half parenterally. Six days later, animals were infused intravenously with [2H3]leucine for 6 h and killed, and the midjejunum of each animal was collected for analysis. The weight of the midjejunum was 8 +/- 1.5 and 17 +/- 1.6 g in parenterally fed and enterally fed piglets, respectively. LPH activities (mumol.min-1.g protein-1) were significantly higher in parenterally vs. enterally fed piglets. Total small intestinal LPH activities were lower in parenterally vs. enterally fed animals. The abundance of LPH mRNA relative to elongation factor-1 alpha mRNA was not different between groups. The fractional synthesis rate of total mucosal protein and LPH was significantly lower in parenterally fed animals (67 +/- 7 and 66 +/- 7%/day, respectively) than in enterally fed animals (96 +/- 7 and 90 +/- 6%/day, respectively). The absolute synthesis rate (the amount of protein synthesized per gram of mucosa) of total mucosal protein was significantly lower in parenterally fed than in enterally fed piglets. However, the absolute synthesis rate of LPH was unaffected by the route of nutrient administration. These results suggest that the small intestine partially compensates for the effects of parenteral feeding by maintaining the absolute synthesis rate of LPH at the same levels as in enterally fed animals.

Lactase phlorizin hydrolase synthesis is decreased in protein-malnourished pigs.

We have examined the effect of protein malnutrition on brush border (BB) lactase phlorizin hydrolase (LPH) synthesis in young pigs. Two groups of four 3-wk-old pigs were fed diets containing either 19 g soy protein, 63 g carbohydrate and 5 g fat per 100 g diet (a protein-sufficient diet) or 3 g soy protein, 85 g carbohydrate and 5 g fat per 100 g diet (a protein-deficient diet). After 8 wk of consuming the diets, pigs were infused intravenously with 2H3-leucine for 8 h, then killed. The jejunum was collected for measurement of lactase activity, LPH mRNA abundance and the rate of LPH post-translational synthesis. Lactase activities did not differ between groups (mean 8.1 +/- 1.2 micromol x min(-1) x g mucosa(-1)). LPH mRNA abundance relative to elongation factor-1alpha mRNA (the constitutive/reference mRNA) was significantly (P < 0.05) higher in well-nourished pigs (0.36 +/- 0.03%) than in protein-malnourished pigs (0.21 +/- 0.02%). The rate constants of BB LPH post-translational synthesis were also significantly higher in the well-nourished (103 +/- 9% x d(-1)) than in the protein-malnourished pigs (66 +/- 8% x d(-1)). Further, the absolute synthesis rate of BB LPH, a measure of the amount of enzyme synthesized per gram of tissue, was significantly higher in well-nourished than in protein-malnourished pigs (in arbitrary units, 892 +/- 90 vs. 450 +/- 34, respectively). Thus, protein malnutrition affects both LPH mRNA abundance and post-translational processing in young pigs.

Regulation of lactase-phlorizin hydrolase gene expression by the caudal-related homoeodomain protein Cdx-2.

Lactase-phlorizin hydrolase is exclusively expressed in the small intestine and is often used as a marker for the differentiation of enterocytes. The cis-element CE-LPH1 found in the lactase-phlorizin hydrolase promoter has previously been shown to bind an intestinal-specific nuclear factor. By electrophoretic mobility-shift assay it was shown that the factor Cdx-2 (a homoeodomain-protein related to caudal) binds to a TTTAC sequence in the CE-LPH1. Furthermore it was demonstrated that Cdx-2 is able to activate reporter gene transcription by binding to CE-LPH1. A mutation in CE-LPH1, which does not affect Cdx-2 binding, results in a higher transcriptional activity, indicating that the CE-LPH1 site contains other binding site(s) in addition to the Cdx-2-binding site.

Lactase phlorhizin hydrolase turnover in vivo in water-fed and colostrum-fed newborn pigs.

We have estimated the synthesis rates in vivo of precursor and brush-border (BB) polypeptides of lactase phlorhizin hydrolase (LPH) in newborn pigs fed with water or colostrum for 24h post partum. At the end of the feeding period, piglets were anaesthetized and infused intravenously for 3h with L-[4-3H]- phenylalanine. Blood and jejunal samples were collected at timed intervals. The precursor and BB forms of LPH were isolated from jejunal mucosa by immunoprecipitation followed by SDS/PAGE, and their specific radioactivity in Phe determined. The kinetics of precursor and BB LPH labelling were analysed by using a linear compartmental model. Immunoisolated LPH protein consisted of five polypeptides [high-mannose LPH precursor (proLPHh), complex glycosylated LPH precursor (proLPHe), intermediate complex glycosylated LPH precursor (proLPH1i) and two forms of BB LPH]. The fractional synthesis rate (Ks) of proLPHh and proLPHc (approx. 5%/min) were the same in the two groups but the absolute synthesis rate (in arbitrary units, min-1) of proLPHh in the colostrum-fed animals was twice that of the water-fed animals. The Ks values of proLPHi polypeptides were significantly different (water-fed, 3.89%/min; colostrum-fed, 1.6%/min), but the absolute synthesis rates did not differ. The Ks of BB LPH was not different between experimental treatment groups (on average 0.037%/min). However, the proportion of newly synthesized proLPHh processed to BB LPH was 48% lower in colostrum-fed than in water-fed animals. We conclude that in neonatal pigs, the ingestion of colostrum stimulates the synthesis of proLPHh but, at least temporarily, disrupts the processing of proLPH polypeptides to the BB enzyme.

Prenatal ethanol exposure alters the expression of intestinal hydrolase mRNAs in newborn rats.

To gain insight into the postnatal growth delay induced by ethanol in utero, we characterized functional impairments of the small intestine of neonatal rats prenatally exposed to ethanol using a well-described model of gestational alcoholism (25% ethanol w/v in the drinking water). Expression of the intestinal enzymes-lactase-phlorizin hydrolase (LPH) and intestinal alkaline phosphatase (IAP)-that are critical for enteral nutrition of neonates was studied. Characteristic patterns of LPH and IAP expression along the proximal-distal (horizontal) and crypt-villus (vertical) axes of the small intestine, as well as the intracellular localization of LPH and IAP mRNAs and immunoreactive proteins within absorptive enterocytes, were not altered by prenatal exposure to ethanol. However, a 10- to 15-fold increase in the number of LPH and IAP mRNA molecules per absorptive enterocyte was found throughout the intestine of ethanol-exposed neonates, compared with controls, whereas lactase and alkaline phosphatase activities per enterocyte remained unchanged. These findings suggest that ethanol in utero alters the mRNA abundance of epithelial enzymes in newborn rat small intestine. Changes in mRNA abundance could be an important aspect of enterocyte adaptation to high ethanol concentrations in gastrointestinal amniotic fluid of ethanol-exposed fetuses.

Maturation of human intestinal lactase-phlorizin hydrolase: generation of the brush border form of the enzyme involves at least two proteolytic cleavage steps.

Human lactase-phlorizin hydrolase (LPH), a brush border membrane hydrolase of the small intestine, is synthesized as a precursor molecule that undergoes proteolytic cleavage to yield mature LPH (LPHbeta) by a trypsin-like protease (Naim et al., 1987, 1991). Arg868-Ala869 has been previously proposed to be the putative cleavage site for this processing step. Site-directed mutagenesis of this monobasic site does not lead to the generation of an uncleaved proLPH species, which strongly suggests the existence of an additional cleavage site. Further analyses of LPH synthesized in different cell lines lend support to this hypothesis. Biosynthetic labeling of human intestinal biopsy samples in the presence of trypsin reveals an LPHbeta species that is slightly smaller than the intracellularly cleaved molecule. When the proLPH molecule is screened for potential cleavage sites, two dibasic pairs are revealed upstream of the N-terminal end of brush border LPH at Lys851-Arg852 and Arg830-Lys831. Treatment of proLPH with trypsin for different periods of time supports the idea of at least two cleavage steps, whereby Arg868-Ala869 represents the final cleavage site that generates LPHbeta. We propose that the initial cleavage of proLPH takes place intracellularly at a site further away from Arg868-Ala869, to generate LPHbeta initial; LPHbeta is subsequently cleaved extracellularly in the gut lumen, presumably by trypsin, at Arg868-Ala869 to mature brush border LPH (LPHbeta initial).

Folding of human intestinal lactase-phlorizin hydrolase.

The folding of human intestinal prolactase-phlorizin hydrolase (pro-LPH) has been analyzed in a cell-free transcription/translation system. In the presence of the thiol oxidant GSSG, disulfide bond formation in pro-LPH can be promoted concomitant with the binding of the molecule to a conformation-specific monoclonal anti-LPH antibody. Under these conditions, pro-LPH does not bind to the molecular chaperone BiP. In the absence of GSSG, on the other hand, pro-LPH does not bind to the monoclonal anti-LPH antibody, but can be immunoprecipitated with a polyclonal antibody that is directed against a denatured form of the enzyme. In this case, interaction of pro-LPH with immunoglobulin heavy chain binding protein can be discerned. The results demonstrate the existence of intramolecular disulfide bonds that are essential for the promotion of pro-LPH to a native conformation. Furthermore, BiP is involved in the folding events of pro-LPH.

Further characterization of the 5'-flanking region of the rat lactase-phlorizin hydrolase gene.

The lactase-phlorizin hydrolase gene is widely used as a marker of intestinal differentiation. Recent evidence demonstrating that transcription plays a major role in the regulation of this gene suggests that study of the 5'-flanking region will allow an understanding of how the expression of this gene is controlled. However, sequence, restriction, and primer extension analysis of a rat genomic clone has revealed that previously published data are incomplete. In the present study, we used a directed sequencing strategy to carefully analyze this region. Our expanded analysis of the 5'-flanking region of the lactase-phlorizin hydrolase gene should facilitate future studies of its structure and function.

Brush-border disaccharidase synthesis in infant pigs measured in vivo with [2H3]leucine.

Conscious unrestrained piglets were fasted overnight and infused intravenously with [2H3]leucine for 6 h. Sucrase isomaltase and lactase phlorizin hydrolase were immunoprecipitated from jejunal mucosal membranes, and the immunoprecipitates were electrophoresed on polyacrylamide gels. Bands corresponding to the pro and mature isoforms of both enzymes were acid hydrolyzed. [2H3]leucine isotopic enrichment was measured by gas chromatography-mass spectrometry using negative chemical ionization. Plasma leucine reached isotopic steady state within 90 min. The isotopic enrichment of mucosal leucine was 73% of that of plasma leucine. The high mannose and complex glycosylated forms of prolactase were in isotopic equilibrium, and their isotopic enrichment was 94% of mucosal leucine. The fractional synthesis rates of total and membrane protein were 0.45 and 0.65 days-1, whereas the processing rates of mature lactase, sucrase, and isomaltase were 0.90, 0.23, and 0.21 days-1, respectively. Approximately 65% of the label in the sucrase isomaltase immunoprecipitate was in the complex glycosylated precursor, whereas 73% of the label in lactase phlorizin hydrolase was in the mature (160 kDa) form. We conclude that the low rate of brush-border sucrase synthesis reflects a slow rate at which the complex glycosylated precursor is processed to the brush-border form.