Abrupt induction of a membrane digestive enzyme by its intraintestinal substrate.

The regulation of amino-oligopeptidase (AOP), an intestinal brush border hydrolase essential for the surface digestion of peptide nutrients, was examined in rats in vivo. Short-term (30-minute) intraintestinal perfusion of a tetrapeptide substrate, Gly-Leu-Gly-Gly, or a synthetic substrate, leucyl-beta-naphthylamide, induced a doubling in the incorporation of [3H]leucine into the AOP in association with intracellular membranes. The subsequent conversion of AOP from nascent to mature enzyme and its membrane-associated transport to the brush border occurred at normal rates.

Folate conjugase: two separate activities in human jejunum.

Previous perfusion studies of the human jejunum suggested that conjugated folate is hydrolyzed on the mucosal surface. The techniques of cell fractionation and DEAE and gel chromatography led to the identification of two separate folate conjugase activities in human jejunal mucosa: one membrane-bound and concentrated in the brush border, the other soluble and intracellular. These enzyme activities exhibit different pH optima, molecular weights, and inhibition characteristics. Folate conjugase in the brush border may accomplish the initial digestion of dietary pteroylpolyglutamates.

Jejunal perfusion of simple and conjugated folates in tropical sprue.

Absorption of labeled simple 3',5',9'-(3)H pteroylmonoglutamate, ([(3)H]PG-1) and conjugated pteroyl-mu[(14)C]glutamyl-gamma-hexaglutamate, ([(14)C]PG-7) folates was assessed in six patients with tropical sprue, before and after 6 mo of treatment, utilizing jejunal perfusion and urinary recovery techniques. Degradation products of [(14)C]PG-7 which were produced during perfusion were identified by DEAE-cellulose column chromatography. Jejunal mucosal activities of folate conjugase, lactase, sucrase, and maltase were measured in every patient. Malabsorption of both [(3)H]PG-1 and [(14)C]PG-7 was found in every untreated patient, with significant improvement after therapy. The urinary excretion of (3)H and (14)C paralleled the luminal disappearance of both isotopes. The chromatographic patterns of intraluminal degradation products of [(14)C]PG-7 obtained during perfusion did not differ from those previously found in normal subjects and were similar in studies performed before and after treatment. The activity of folate conjugase was increased in the mucosa of the untreated patients when compared to the post-treatment levels while the activities of mucosal lactase, sucrase, and maltase were originally low and increased significantly after therapy. These observations suggest that folate conjugase originates at a different mucosal locus than the brush border disaccharidases, and are consistent with previous evidence that folate conjugase is an intracellular enzyme. The present studies have demonstrated unequivocal malabsorption of both simple and conjugated folates in tropical sprue. In tropical sprue, folate malabsorption is the reflection of impaired folate transport and not of impaired hydrolysis.

Jejunal perfusion of simple and conjugated folates in celiac sprue.

The intestinal absorption of [3H]-pteroylmonoglutamate (simple folic acid) and pteroyl-micron[14C]glutamyl-gamma-hexaglutamate ([14C]PG-7, conjugated folic acid) was assessed by the method of jejunal perfusion in five patients with proven celiac sprue who were studied after a gluten-containing or a gluten-free diet, and in nine normal subjects. The luminal disappearance of each folate was markedly impaired after exposure of the patients to dietary gluten and improved by gluten restriction, but not to within the range found in the normal subjects. The luminal disappearance of each folate was markedly impaired after exposure of the patients to dietary gluten and improved by gluten restriction, but not to within the range found in the normal subjects. In each experiment, column chromatography of the luminal aspirates revealed similar spectra of hydrolytic products of [14C]PG-7, whereas the fraction of the distal aspirate chromatogram appearing as pteroyl-micron[14C]glutamyl-gamma-monoglutamate ([14C]-PG-1) was similar in all three groups. By accounting for the variable effects of absorption on the luminal appearance of [14C]PG-1 and by correcting for mucosal hydrolysis which was not followed by release of [14C]PG-1 to the luminal contents, the calculated rate of in vivo hydrolysis of [14C]PG-7 to [14C]PG-1 was found impaired in both celiac sprue groups, with significant improvement on treatment. In mucosal biopsies from the sprue patients, the in vitro activity of folate conjugase in whole homogenates was higher and the activity of disaccharidase lower than in a group of 12 normal mucosal biopsies. These in vitro data suggest that the predominant cellular location of mucosal folate conjugase is different from that of disaccharidase, whereas comparison with the results of in vivo hydrolysis suggests that measurement of the enzyme in whole mucosal homogenates overestimates its significant digestive activity. The present studies indicate that (a) the mucosal lesion of celiac sprue significantly limits the intestinal absorption of both simple and conjugated folate, and (b) malabsorption of conjugated folate results from a combination of impaired hydrolysis and decreased mucosal uptake of hydrolytic product.

Human jejunal brush border folate conjugase. Characteristics and inhibition by salicylazosulfapyridine.

Human jejunal brush border folate conjugase (EC 3.4.22.-) was partially purified and characterized. Three drugs known to be associated with clinical folate deficiency were tested for inhibition of the partially purified enzyme. Using jejunal mucosa from obese patients undergoing intestinal bypass surgery, brush border folate conjugase was purified 50-80-fold by centrifugation, Triton X-100 solubilization and DEAE-Sephadex and Sephacryl S-200 chromatography. Using synthetic pteroyldiglutamyl[14C]glutamate as substrate, the enzyme was found to have a pH optimum of 6.5 and an apparent Km of 1.6 micro M. Incubation of the enzyme with synthetic pteroyl[14C]glutamylhexaglutamate resulted in a spectrum of shorter-chain 14C-labeled pteroylglutamates at 60 min. Pteroyl[14C]glutamate was the major product at 120 min, with quantitative recovery of free glutamate in the incubation medium. Salicylazosulfapyridine was a competitive inhibitor of the enzyme (Ki = 0.13 mM), while ethanol, diphenylhydantoin and salicylazosulfapyridine metabolites had no effect. These data suggest that brush border folate conjugase is an exopeptidase which progressively hydrolyzes glutamyl units from pteroylpolyglutamate, leaving pteroylmonoglutamate as the folate form available for intestinal transport. Inhibition of brush border folate conjugase by salicylazosulfapyridine provides a mechanism for folate malabsorption and deficiency in chronic users of this drug.

Pituitary regulation of postnatal small intestinal ontogeny in the rat: differential regulation of digestive hydrolase maturation by thyroxine and growth hormone.

During the third week of postnatal life, dramatic ontogenic changes occur in the morphology and enzymology of the small intestine of the infant rat, enabling the animal to make the transition from milk to solid food. To investigate the roles of T4 and GH in regulation of these changes, infant rats were hypophysectomized on day 6 of life by the transauricular technique. Hypophysectomy resulted in diminution of somatic and intestinal growth as well as abnormal maturation of the disaccharidases lactase, sucrase, and maltase when measured on day 25. Administration of either T4 or GH to hypophysectomized animals resulted in moderately increased intestinal growth, while complete restoration of small intestinal growth resulted from administration of the combination of both hormones. Although T4, GH, or the combination of hormones reduced lactase activities, T4 alone produced normal maturation of sucrase and maltase. Neither hypophysectomy nor hormone replacement affected aminooligopeptidase. The molecular structure of lactase, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was not altered to a major degree in hypophysectomized animals or animals that received hormone replacement, but minor alterations were evident in sucrase structure in hypophysectomy. These studies indicate that 1) T4 and GH actively participate in postnatal regulation of small intestinal ontogeny; 2) thyroid hormones act directly on developing intestinal tissues to independently produce the normal maturation of the disaccharidases by mechanisms that are not likely to involve alterations in processing of the enzyme-protein; and 3) maturation of aminooligopeptidase is not regulated by pituitary hormones, in distinct contrast to the disaccharidases.

Folate absorption in alcoholic pigs: in vivo intestinal perfusion studies.

To determine if impaired intestinal absorption contributes to the folate deficiency observed in chronic alcoholics, we assessed in vivo folate absorption in Hanford mini-pigs fed ethanol with an adequate diet. Sixteen minipigs were pair-fed diets supplemented with ethanol or sucrose to 60% of total calories for 11 mo. In the ethanol-fed pigs peak blood alcohol concentrations averaged 28 mmol/L, serum alanine transaminase and aspartate transaminase activities were elevated, and liver histology showed a centrilobular distribution of succinate dehydrogenase. Tissue folate concentrations were comparable in both groups. The jejunal uptake of folic acid, measured by intestinal perfusion, was similar in both groups of animals and was not affected by acute exposure to 445 mmol/L ethanol. The in vivo hydrolysis of polyglutamyl folate was reduced by 35% in one ethanol-fed minipig. Decreased hydrolysis of polyglutamyl folate may represent an early step in the development of folate deficiency in chronic alcoholics.

Human intestinal folate conjugase: adaptation after jejunoileal bypass.

During intestinal absorption, dietary polyglutamyl folates are hydrolyzed to monoglutamyl folates by pteroylpolyglutamate hydrolase (folate conjugase). This enzyme is present in the brush border and intracellular fractions of human jejunal mucosa. We compared the activities of brush border and intracellular folate conjugase (BBFC and ICFC), and other mucosal enzymes in the jejunum in continuity and bypassed loop in nine patients who underwent intestinal reconstitution operations 3 to 9 yr after jejunoileal bypass. Control jejunum was obtained from seven obese patients undergoing gastric bypass surgery. Jejunal morphometry showed mucosal hyperplasia with taller villi, larger crypts, and no change in cell size in the jejunum in continuity as compared to control or bypass jejunum. In brush border fractions, specific activities of sucrase and BBFC were significantly greater in the jejunum in continuity than in the control or bypass jejunum. In contrast, the specific activities of the other brush border and intracellular enzymes, including ICFC, were similar in all three segments. This is the first evidence that BBFC, like sucrase, adapts to luminal nutrition. The difference in response of BBFC and ICFC to diet and/or pancreato-biliary secretions provides evidence that these are distinct enzymes which are regulated by different mechanisms.

Affinity labelling of the folate-binding protein in pig intestine.

A specific transport system for folate and a high-affinity folate-binding protein have been identified in pig intestinal brush-border membranes. To determine if the binding protein plays a role in folic acid (PteGlu) uptake in to the cell, the inactivation of folate binding and transport by N-hydroxysuccinimide esters of folic acid (NHS-PteGlu) was compared. In addition, the number of brush-border proteins modified by the affinity reagent was assessed. Brush-border vesicles were incubated with various concentrations of NHS-PteGlu or NHS-methotrexate. Transport and binding of [3H]PteGlu by the vesicles were measured at 37 and 4 degrees C respectively by using the vacuum-filtration technique. NHS-methotrexate and NHS-PteGlu specifically inhibited PteGlu transport. Incubating the vesicles with 1 microM-NHS-PteGlu inactivated [3H]PteGlu transport by 60% and binding by 80%. Half-maximal inhibition of both transport and binding was observed at similar concentrations of the affinity reagent (0.05 and 0.07 microM-NHS-PteGlu respectively). Treating the vesicles with radiolabelled NHS-PteGlu followed by gel electrophoresis and autoradiography revealed a specifically labelled protein with an Mr of 56,000. These results indicate that the intestinal folate-binding and transport proteins are identical and that the function of the folate-binding protein is to transport folate into the cell.

Regulation of the ontogenic and regional expression of intestinal aminooligopeptidase.

Aminooligopeptidase (AOP) is the predominant peptidase in the small intestinal epithelium. During postnatal development in the rat, characteristic ontogenic and regional patterns of AOP activities become established. To investigate the molecular mechanisms regulating the maturational changes in AOP activity, we compared AOP synthesis and assembly in the jejunum of suckling (14-day-old) and weaned (28-day-old) rats. AOP synthesis was assessed in vivo by intraperitoneal labeling with [35S]methionine. Both AOP activity and AOP synthesis doubled at weaning, while its posttranslational processing remained unaltered. To examine the mechanisms responsible for generating the regional differences in AOP activity in weaned rats, we contrasted the de novo synthesis, kinetic properties, total immunoreactive protein, and degradation of the jejunal and ileal peptidases. Although AOP catalytic activity was significantly greater in the jejunum than in the ileum, its synthesis rate and substrate affinity (Km) were identical at both sites. However, the ileal peptidase was degraded more rapidly than the jejunal enzyme (t1/2 = 4 and 10 h, respectively). In summary, our data show that increased synthesis accounts for the ontogenic rise in intestinal AOP activity but that altered enzyme turnover produces the jejunal-ileal gradient in AOP activity in weaned rats. Thus the ontogenic and regional expressions of intestinal AOP are defined by transcriptional/translational and posttranscriptional regulatory mechanisms, respectively.

Folate binding and hydrolysis by pig intestinal brush-border membranes.

The intestinal absorption of dietary polyglutamyl folate involves hydrolysis, binding, and transport at the brush-border surface of the enterocyte. We studied the binding of [3H]folic acid ([3H]PteGlu) and the hydrolysis of [14C]pteroyltriglutamate ([14C]PteGlu3) by use of brush-border vesicles prepared from pig jejunal mucosa. [3H] PteGlu associated with the vesicles was not affected by increasing the osmolarity of the incubation solution, verifying that the experiments describe binding and not transport of PteGlu. The binding of [3H]PteGlu was saturable (Kd = 0.08 microM) and pH dependent with maximal binding at pH 5.2. Binding was competitively inhibited by PteGlu3 (Ki = 0.25 microM) and 5-methyltetrahydrofolate (Ki = 0.8 microM) but was not affected by components of the PteGlu molecule. ZnCl2, MgCl2, and MnCl2 enhanced the binding capacity but not the affinity of the binding component for PteGlu. We distinguished the processes of folate binding and hydrolysis by demonstrating differences in metal ion requirements and susceptibilities to various inhibitors. In addition, the binding component and the hydrolytic enzyme had distinct affinities for PteGlu (Ki = 45 microM for PteGlu3 hydrolysis). These data demonstrate pH-dependent, specific, and saturable binding of PteGlu to the intestinal brush-border membrane and suggest that the binding component is separate from the hydrolytic enzyme.

Ontogeny of intestinal lactase: posttranslational regulation by thyroxine.

To assess the molecular mechanisms underlying the regulation of lactase ontogeny by thyroxine (T4), we performed an in vivo study of lactase catalytic activity, synthesis, subunit structure, degradation, and enterocyte migration rates in propylthiouracil-induced hypothyroid rat pups, hypothyroid pups injected with T4, and normally weaned rats. Although lactase catalytic activity remained elevated in the hypothyroid rats and declined normally in the other two groups, lactase synthesis was constant among the groups. Lactase subunit structure was identical in normally weaned and T4-injected animals, but the 100-kDa moiety, characteristic of weaned rats, was absent in the hypothyroid pups. The turnover of lactase enzyme was more rapid in euthyroid and T4-injected rats than in hypothyroid animals (t1/2 = 17, 20, and 30 h, respectively). In addition, enterocyte migration was accelerated in the T4-injected rats and reduced in the hypothyroid group compared with controls. However, transit rate was not directly related to lactase activity. Our results suggest that T4 regulates lactase ontogeny by posttranslational mechanisms that include altered processing and increased degradation of the lactase enzyme.

Ontogeny of membrane and soluble amino-oligopeptidases in rat intestine.

Intestinal amino-oligopeptidase (AOP) plays an essential role in protein digestion. To characterize its postnatal development, we measured AOP activity in intestinal membrane and cytosolic fractions in suckling and weaned rats, compared the subunit structures of the membrane and soluble enzymes, and assessed the biochemical relationship of these peptidases. At weaning, jejunal membrane AOP activity doubled while soluble AOP activity in the ileum fell abruptly. The maturational increase in the molecular mass of ileal membrane AOP was due to alterations in the N-linked glycosylation of this protein. Ileal membrane and soluble AOP exhibited similar substrate affinities, pH optima, inhibition characteristics, and antigenic epitopes. However, soluble AOP was 25-35 kDa smaller than the membrane enzyme. Peak incorporation of [35S]methionine into ileal brush-border AOP preceded maximal radioactivity in soluble AOP, suggesting that the membrane peptidase is a precursor of the soluble enzyme. We conclude that membrane and soluble AOP are closely related proteins with distinct developmental profiles and that the soluble peptidase may be derived from endocytosis of the membrane enzyme.

Comparison of folate conjugase activities in human, pig, rat and monkey intestine.

Folate conjugase, an intestinal enzyme that hydrolyzes dietary polyglutamyl folate to the absorbable monoglutamyl derivative, is present in two locations in human jejunal mucosa: one on the brush border membrane, the other soluble and intracellar. Although the brush border enzyme has not been found in the rodent, a recent study demonstrated both brush border and intracellular folate conjugases in the pig with properties similar to the human enzymes. To confirm and expand these data, we compared folate conjugase activities in intestinal brush border and 30,000 X g supernatant fractions in human, pig, rat and monkey mucosa. In both the human and pig, brush border folate conjugase was active from pH 4.5 to 8.5, and activity was significantly increased by zinc acetate. In contrast, folate conjugase activity was negligible in rat and monkey mucosal brush borders. Intracellular folate conjugase was maximally active at pH 4.5 in the human, pig and monkey, whereas this enzyme was equally active from pH 4.0 to 7.5 in the rat. In each species, supernatant activity at pH 4.5 was completely inhibited by p-hydroxymercuribenzoate. Although folate conjugase was quantitatively more active in the human than in the pig intestine, the brush border and intracellular enzymes exhibited similar properties. Sucrase, lactase and aminopeptidase activities were also similar in the two species. Our data show marked species differences in mucosal folate conjugase activities and indicate that the pig may be a suitable experimental animal for further studies of folate hydrolysis and absorption in humans.

Human intestinal lactase-phlorizin hydrolase: isolation and preparation of a specific antiserum.

Human intestinal lactase-phlorizin hydrolase (lactase) was selectively isolated with monospecific polyclonal antibodies to rat lactase. In addition to their immunologic similarities indicated by this isolation, human and rat lactase have similar kinetic characteristics but different subunit structure when analyzed by gel electrophoresis under reducing conditions. Rabbits immunized by injecting human lactase complexed with anti-rat lactase produced specific antibodies to human lactase that exhibited little cross-reactivity to the rat enzyme. The simple single-step procedure allows isolation of human lactase in high purity from small biologic samples and preparation of specific antisera to the human enzyme.

Availability of monoglutamyl and polyglutamyl folates in normal subjects and in patients with coeliac sprue.

Intestinal folate absorption was assessed in six normal subjects and in four patients with coeliac sprue who were studied before and after treatment by dietary gluten exclusion. Comparisons were made of the luminal disappearance from the perfused jejunum of 3H-pteroylmonoglutamate and pteroyl 14C-glutamylhexaglutamate, and of the 48-hour urinary recovery of each isotope after perfusion and a tissue saturating dose of folic acid. The labelled urinary folates consisted of folic acid, 10-formyltetrahydrofolate, and 5-methyltetrahydrofolate. In each group urinary recovery of 3H was significantly greater than that of 14C, confirming the evidence from jejunal perfusion that the availability of monoglutamyl folate is greater than that of polyglutamyl folate. According to the urinary recovery data, both folates were poorly absorbed in untreated coeliac sprue, but were normally absorbed after treatment. Assuming uniform displacement of the absorbed labelled folates by the parenteral flushing dose, the finding of greater urinary isotope recovery than of luminal folate disappearance from the perfused proximal jejunal segment suggests an adaptation of the distal small bowel for folate absorption in coeliac sprue.

The effect of chronic alcohol ingestion on free radical defense in the miniature pig.

Cellular protection from free radical reactions was measured in hepatic tissue from controls and miniature pigs fed ethanol. The activities of copper-zinc superoxide dismutase (CuZnSOD) and glutathione peroxidase were lower and the activity of manganese superoxide dismutase (MnSOD) was higher in the pigs fed ethanol than in controls. Glutathione concentration was lower in the pigs fed ethanol than in controls. Ethanol consumption did not result in increased lipid peroxidation as assessed by thiobarbituric acid-reactive substances. The ethanol-induced effects on the activities of the superoxide dismutases may be a reflection of available metals and/or a response to increased hepatic concentration of oxygen radicals. Because the protection afforded by the glutathione system was altered, pigs fed ethanol may be at risk of peroxidative damage with continued ethanol exposure.

Intestinal lactase in the neonatal rat. Maturational changes in intracellular processing and brush-border degradation.

The mechanism of decline in the catalytic activity of intestinal lactase during neonatal maturation has not been defined, but a shift in the lactase subunit synthesis from an active 130-kDa subunit to an inactive 100-kDa species has now been noted in the adult rat (Quan, R., Santiago, N. A., Tsuboi, K. K., and Gray, G. M. (1990) J. Biol. Chem. 265, 15882-15888). The subunit structure, synthesis, intracellular assembly, and subsequent degradation of lactase from the brush-border surface membrane was examined in 15-day-old pre-weaned and 30-day-old post-weaned intact rats. Lactase was labeled intraintestinally with [35S]methionine, isolated from Triton-solubilized membranes with monospecific polyclonal anti-lactase, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The protein-stained gel revealed subunits of 225 and 130 kDa, the latter species predominating in both the pre- and post-weaned state. The distinct adult-type 100-kDa moiety was present in post-weaned animals while only a trace of a slightly larger (approximately 110 kDa) species was observed in pre-weaned animals. Quantitation of radioactivity in newly synthesized lactase revealed an increasing prominence of the 100-kDa species in post-weaned rats (130/100 incorporation ratio: pre-weaned 6.2; post-weaned 3.3). Accumulation of newly labeled lactase in brush-border membranes after intraperitoneal [35S]methionine labeling was similar in both groups at 3 h. Despite these comparable rates of lactase synthesis, assembly and insertion in the pre- and post-weaned state, subsequent removal of the 130-kDa unit was more rapid in post-weaned animals (t1/2 = 11 h; pre-weaned t1/2 = 37 h). In intact rats, the neonatal maturational decline in lactase catalytic activities involves both a shift to production of the inactive 100-kDa subunit and increased membrane surface degradation of the active 130-kDa subunit.