Enzymes of the human intestinal brush border membrane. Identification after gel electrophoretic separation

The position of a number of human intestine brush border membrane enzyme activities in polyacrylamide gels after electrophoresis has been determined. These activities are, in order from the origin, maltase/glucoamylase, lactase/phlorizin hydrolase, maltase/sucrase/isomaltase, enteropeptidase, trehalase and γ-glutamyltransferase. Leucylnaphthylamide hydrolyzing activity was inactivated by sodium dodecylsulfate and its position was not determined. The positions of the activities have been correlated with the positions of protein bands previously determined. One such band situated between enteropeptidase and alkaline phosphatase has not been identified.     

Synthesis of maltotriose by maltase purified from rabbit kidney.

To clarify the enzyme participates in maltotriose synthesis, we purified maltase from rabbit kidney using 2-amino-2-hydroxymethylpropane-1,3-diol (Tris) affinity column chromatography. The purified enzyme possessed specific activity of 33.7 mumol/mg/min and estimated molecular weight of 350,000 dalton, as judged by SDS-polyacrylamide gel electrophoresis, comparable with those reported from rat kidney. Moreover this enzyme possessed not only maltase (maltose----glucose) but also amylomaltase (maltose----maltotriose) activity, and both activities were inhibited by Tris in a dose-dependent manner with similar IC50 values. From these results, we concluded that maltotriose was synthesized by maltase in vitro and that kidney maltase may participate in sugar metabolism in vivo.     

Disaccharidase activities after jejunoileal bypass in rat

Digestive enzymatic activities (maltase, lactase and sucrase) have been determined in the intestinal mucosa of rats subjected to a jejunoileal bypass of 45 cm. The weight and protein content of the mucosa (mg/cm) were significantly decreased in the bypassed segment and significantly increased in the unbypassed segment, as compared to control rats. Maltase, lactase and sucrase specific (U/g protein) and total activity (U/cm intestine) were significantly decreased in the bypassed jejunum, compared to sham-operated rats. In the ileum, maltase specific and total activities increased in bypassed animals while the lactase and sucrase activities remained unchanged.     

Effect of Calcium Ions on Enteropeptidase Catalysis

The effects of calcium ions on hydrolysis of low molecular weight substrates catalyzed by different forms of enteropeptidase were studied. A method for determining activity of truncated enteropeptidase preparations lacking a secondary trypsinogen binding site and displaying low activity towards trypsinogen was developed using N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (Z-Lys-S-Bzl). The kinetic constants for hydrolysis of this substrate at pH 8.0 and 25 degrees C were determined for natural enteropeptidase (K(m) 59.6 microM, k(cat) 6660 min(-1), k(cat)/K(m) 111 microM(-1) x min(-1)), as well as for enteropeptidase preparation with deleted 118-783 fragment of the heavy chain (K(m) 176.9 microM, k(cat) 6694 min(-1), k(cat)/K(m) 37.84 microM(-1) x min(-1)) and trypsin (K(m) 56.0 microM, k(cat) 8280 min(-1), k(cat)/K(m) 147.86 microM(-1) x min(-1)). It was shown that the enzymes with trypsin-like primary active site display similar hydrolysis efficiency towards Z-Lys-S-Bzl. Calcium ions cause 3-fold activation of hydrolysis of the substrates of general type GD(4)K-X by the natural full-length enteropeptidase. In contrast, the hydrolysis of substrates with one or two Asp/Glu residues at P2-P3 positions is slightly inhibited by Ca2+. In the case of enteropeptidase light chain as well as the enzyme containing the truncated heavy chain (466-800 fragment), the activating effect of calcium ions was not detected for all the studied substrates. The results of hydrolysis experiments with synthetic enteropeptidase substrates GD(4)K-F(NO(2))G, G(5)DK-F(NO(2))G (where F(NO(2)) is p-nitrophenyl-L-phenylalanine residue), and GD(4)K-Nfa (where Nfa is beta-naphthylamide) demonstrate the possibility of regulation of undesired side hydrolysis using natural full-length enteropeptidase for processing chimeric proteins by means of calcium ions.     

Identification of a mucosal form of enteropeptidase in triton X-100 extracts of porcine duodenal mucosa

Porcine enteropeptidase (EC 3.4.21.9) purified from acetone powders of fresh duodenal fluid shows a molecular weight, as determined on Ultragel AcA-34, of 190000. Enteropeptidase has been solubilised from pig intestinal mucosa using 1% (v/v) Triton X-100. When Triton X-100 extracts of freeze-dried mucosa after partial fractionation on DEAE-cellulose were chromatographed on Sephadex G-200, the bulk of the activity eluted in the void volume rather than with an expected Ve/V0 ratio of about 1.24 corresponding to a molecular weight of around 200000. Gel filtration of aqueous mucosal extracts obtained in the absence of Triton X-100 showed two regions of enzymic activity in approximately equal proportions, one in the void volume, and the other with the expected Ve/V0 ratio of 1.24, whereas the Triton X-100 extracts of the residue from the above extract showed the presence of only the macromolecular species of enteropeptidase. This species was excluded from Sepharose 4B. It was confirmed that aminopeptidase was also extracted by Triton X-100 in a molecular form which was excluded from Sepharose 4B. The results suggest that Triton X-100 extracts enteropeptidase with a membrane component attached and in agreement with this it was found that proteolysis rapidly converted the macromolecular form to a stable smaller molecular species corresponding in size to that found in solution in the duodenal fluid. There was full recovery of the enzymic activity following this conversion. Papain and trypsin brought about an almost complete conversion to the smaller form of enteropeptidase whereas chymotrypsin, pancreatin and an intestinal peptidase preparation were only partially effective. It is concluded that membrane bound enzymes such as enteropeptidase and aminopeptidase are bound to the intestinal brush border membrane in a similar manner and are not actively secreted into the lumen but rather are largely released or solubilised by the combined action of the bile and pancreatic secretions.     

The vector containing a signal for specific degradation of chimeric proteins. Synthesis of [Leu5]enkephalin using enteropeptidase

A plasmid vector (pEK1) coding, in framework of beta-galactosidase gene, for the amino acid sequence (Asp)4Lys which is recognized by bovine enteropeptidase has been constructed. Using this vector and chemically synthesized DNA coding for the [Leu5]-enkephalin, a plasmid (pEK-ENK) has been obtained in which the beta-galactosidase gene is fused, through the enteropeptidase linker, with the gene for [Leu5]enkephalin. The chimeric protein produced by expression of this plasmid has been isolated and then cleaved by the enteropeptidase to give [Leu5]enkephalin with the yield 74%.     

Toxicity of gentamicin in organ culture of fetal rat intestine.

The effect of gentamicin in the culture of fetal rat intestine was studied. Fetal rat intestine was cultured with gentamicin or kanamycin at the concentration between 4 to 200 micrograms/ml. Kanamycin did not have influence on lactase, maltase and ALP activities. On the other hand, gentamicin caused decrease of lactase and ALP activities at the concentration of 40 and 200 micrograms/ml compared with the activities at 4 micrograms/ml. Maltase activities did not decrease with gentamicin. Our data suggest that gentamicin could affect lactase and ALP activities and lower concentration should be used in the culture.     

Study of Secondary Specificity of Enteropeptidase in Comparison with Trypsin

A comparative study of secondary specificities of enteropeptidase and trypsin was performed using peptide substrates with general formula A-(Asp/Glu) _n -Lys(Arg)--B, where n = 1-4. This was the first study to demonstrate that, similar to other serine proteases, enteropeptidase has an extended secondary binding site interacting with 6-7 amino acid residues surrounding the peptide bond to be hydrolyzed. However, in the case of typical enteropeptidase substrates containing four negatively charged Asp/Glu residues at positions P2-P5, electrostatic interaction between these residues and the secondary site Lys99 of the enteropeptidase light chain is the main factor that determines hydrolysis efficiency. The secondary specificity of enteropeptidase differs from the secondary specificity of trypsin. The chromophoric synthetic enteropeptidase substrate G₅DK-F(NO₂)G (k _cat/K _m = 2380 mM^–1·min^–1) is more efficient than the fusion protein PrAD₄K-P26 (k _cat/K _m = 1260 mM^–1·min^–1).     

Dietary modulation of intestinal enzymes of the house sparrow (Passer domesticus): testing an adaptive hypothesis

Insectivorous/frugivorous passerine species studied so far lack the ability to modulate intestinal maltase activity, in contrast to galliformes. We tested for dietary modulation of small intestine (SI) enzymes including maltase in house sparrows to understand whether the difference between the galliformes on the one hand, and the passerines on the other, reflects a phylogenetic pattern (maltase modulated in galliformes but not passerines), a dietary pattern (maltase modulated in granivores but not insectivore/frugivores), some other pattern, or chance. We also tested the prediction that intestinal peptidase activity would be increased on a high protein (HP) diet. Birds were fed three diets high in starch, protein, or lipid for 10 days. For birds on the HP diet (60.3% protein) we observed the predicted upward modulation of aminopeptidase-N activity, as compared with the lower-protein, high starch (HS) (12.8% protein) diet. In contrast, birds eating the HS diet had similar maltase and sucrase activities, and only slightly higher isomaltase activity, compared with birds eating the high protein (HP), starch-free diet. Birds eating high lipid (HL) diet had low activities of both carbohydrases and peptidase. Considering that the statistical power of our tests was adequate, we conclude that house sparrows show little or no increase in carbohydrases in response to elevated dietary carbohydrate. We cannot reject the hypothesis that maltase lability among avian species has a phylogenetic component, or that high dietary fat has a depressing effect on both carbohydrase and peptidase activities.     

Production and Purification of Recombinant Enteropeptidase Expressed in an Insect–Baculovirus Cell System

Enteropeptidase (EC 3.4.21.9) is the glycoprotein enzyme in the small intestine that triggers the activation of the zymogens in pancreatic juice by converting trypsinogen into trypsin. Because of its physiological significance, there have been many studies on the expression, purification, and characterization of enteropeptidase from different species. The baculovirus expression system has been commonly used in research communities and scientific industries for the production of high levels of recombinant proteins, which require posttranslational modifications for functional activity. In the present study, we isolated bovine enteropeptidase catalytic subunit gene from Bos taurus indicus (GenBank accession no. KC756844), and cloned it in pFast Bac HT “A” baculovirus expression donor vector, under the polyhedrin promoter. Recombinant bovine enteropeptidase was expressed in SF-9 insect cells with high expression levels. Recombinant enteropeptidase was purified using Ni-NTA affinity chromatography. A 6-mg quantity of pure active protein was obtained from 100 mL culture using this approach. Its activity and kinetic parameters were determined by cleavage of its fluorogenic substrate Gly-(Asp) 4-Lys-β-naphthylamide. The recombinant bovine enteropeptidase showed a K _m value of 0.75 ± 0.02 mM with K _cat 25 ± 1 s.     

The level and distribution of disaccharidases in the camel (Camelus dromedarius) intestine

1. The disaccharidases, cellobiase, isomaltase, lactase, maltase, sucrase and trehalase were investigated for presence in the camel (Camelus dromedarius) intestine and pancreas. All, except sucrase, were present. 2. Their levels of activities were measured at different positions of the small and large intestines and the location of maximum level of activity for each enzymes along the intestinal tract was established. 3. High levels of activities were determined in the contents of the intestinal lumen and, therefore, it is absorbed into the cells of the epithelial villi and hydrolyzed there. 4. The possibility of carbohydrate digestion in camel intestine is discussed.     

小鼠小肠四种消化酶活力的胎后发育模式

为明确晚成型小鼠胎后发育肠道消化酶活力的建立过程和发育模式,探讨其与适应性调节假说的关系,测定了从出生后至27日龄小鼠小肠前、中、后段的乳糖酶、蔗糖酶、麦芽糖酶和氨基肽酶的酶活力。结果发现单位组织酶活力方面,乳糖酶活力先增后降,小肠前段在9日龄而中后段在12日龄达到最高,至27日龄时仅中段有微弱的酶活力;蔗糖酶活力12日龄始出现,前段和后段自15日龄迅速升高,至18日龄达最高,但随后显著降低,而中段在15日龄后持续升高至21日龄达到最高,此后维持在较高水平;麦芽糖酶出生时已具有活力,但在15日龄前维持较低水平,此后迅速升高,前后段在18日龄,中段在21日龄达到峰值,此后下降;小肠前段的氨基肽酶活力出生后至27日龄持续下降,而后段和中段从出生到断乳前则持续升高,断乳后略有下降。除乳糖酶总酶活力先增后降,在15日龄达峰值外,其余3种酶的总酶活力均持续增加。在小肠不同位置4种酶活力的分布具有显著差异,且日龄对不同位置酶活力的影响趋势不同。总之,小鼠小肠4种消化酶的酶活力随时间的变化能够与其食物转变的消化需求相匹配,部分地支持适应性调节假说。     

Genetic control of maltase formation in yeast. III. Isolation and characterization of temperature-sensitive mutants affecting maltase induction and maltose utilization

Temperature-sensitive mutants affecting maltose utilization in the yeast Saccharomyces cerevisiae have been isolated. Two such mutants although failing to ferment maltose at the restrictive temperature, have normal induced level of maltase. The third mutant (UNT-37) not only failed to ferment maltose but has 5-6 fold less induced level of maltase at the restrictive temperature than the parental strain. The genetic control mechanisms of maltase induction and maltose utilization have been discussed.     

Precocious alteration of digestive enzyme activities in small intestine and pancreas by chronic oral administration of protease inhibitor in suckling rats.

1. The role of endogenous CCK in the development of digestive enzyme activities in small intestine and pancreas was investigated in suckling rats. Synthetic protease inhibitor (camostat 100 micrograms/g bwt) was orally administered twice daily for 5 days from 11 days of age. 2. Pancreatic hypertrophy and hyperplasia, and alteration of pancreatic enzyme composition, especially decreases in amylase activity and increases in trypsin and chymotrypsin activities were produced by camostat treatment. These changes were completely suppressed by simultaneous administration of the potent CCK receptor antagonist L-364,718 (1 microgram/g bwt). 3. With camostat treatment, intestinal lactase activity decreased to 41%, while maltase and sucrase activities increased 3 and 2.5 times respectively. These changes in enzyme activities were not affected by the application of L-364,718. 4. The mucosal disaccharidase and pancreatic enzyme activities could not be modified by chronic subcutaneous injection of camostat. The precocious induction of maltase and sucrase activities by camostat treatment was also observed in the adrenalectomized pups. 5. These results indicate that pancreatic growth accompanied by alteration of digestive enzyme composition in the suckling rats is regulated by endogenous CCK, but the precocious induction of disaccharidase activities is not mediated by endogenous CCK released by camostat treatment.     

p26--a calcium binding protein from photoreceptor cells in the bovine retina: primary structure and expression in E. coli]

The primary structure of the bovine retinal calcium binding protein P26 has been determined by the parallel analysis of the protein and the corresponding cDNA. This protein is identical to recovering and shares 59% homology with visinin, a cone specific calcium binding protein from chicken retina. P26 was expressed in E. coli as a fusion protein and, after purification by affinity chromatography on IgG-Sepharose 6, cleaved off with enteropeptidase.     

Test of the adaptive modulation hypothesis in rodents: dietary flexibility and enzyme plasticity.

The phenotypic response of digestive enzymes was assessed in two species of rodents with different foods habits. Species were Phyllotis darwini (omnivorous) and Octodon degus (herbivorous). The activity of sucrase, maltase and aminopeptidase-N were determined in vitro in animals feeding two contrasting diets. No effect of dietary chemistry on sucrase and maltase activities was observed. Nevertheless, aminopeptidase-N showed a reversible response to diet in P. darwini but not in O. degus. Through Principal Component Analysis we separated the specific and non-specific modulation of the enzymes. The analysis showed that aminopeptidase-N activity is up-regulated by dietary protein in P. darwini. Differences in the phenotypic response of this species apparently reflect the historic levels of specific substrates of the natural diets for this enzyme, linking dietary flexibility and digestive plasticity in an evolutionary context.     

Dissecting structural basis of the unique substrate selectivity of human enteropeptidase catalytic subunit

Enteropeptidase is a key enzyme in the digestion system of higher animals. It initiates enzymatic cascade cleaving trypsinogen activation peptide after a unique sequence DDDDK. Recently, we have found specific activity of human enteropeptidase catalytic subunit (L-HEP) being significantly higher than that of its bovine ortholog (L-BEP). Moreover, we have discovered that L-HEP hydrolyzed several nonspecific peptidic substrates. In this work, we aimed to further characterize species-specific enteropeptidase activities and to reveal their structural basis. First, we compared hydrolysis of peptides and proteins lacking DDDDK sequence by L-HEP and L-BEP. In each case human enzyme was more efficient, with the highest hydrolysis rate observed for substrates with a large hydrophobic residue in P2-position. Computer modeling suggested enzyme exosite residues 96 (Arg in L-HEP, Lys in L-BEP) and 219 (Lys in L-HEP, Gln in L-BEP) to be responsible for these differences in enteropeptidase catalytic activity. Indeed, human-to-bovine mutations Arg96Lys, Lys219Gln shifted catalytic properties of L-HEP toward those of L-BEP. This effect was amplified in case of the double mutation Arg96Lys/Lys219Gln, but still did not cover the full difference in catalytic activities of human and bovine enzymes. To find a missing link, we studied monopeptide benzyl-arginine-β-naphthylamide hydrolysis. L-HEP catalyzed it with an order lower K _m than L-BEP, suggesting the monopeptide-binding S1 site input into catalytic distinction between two enteropeptidase species. Together, our findings suggest structural basis of the unique catalytic properties of human enteropeptidase and instigate further studies of its tentative physiological and pathological roles.     

The enzymatic activity from the sediment of the Gilau dam reservoir - Cluj county

The enzymological studies on the sediment of the accumulation lake that has the main purpose of supplying drinking water to the city of Cluj-Napoca and the nearby villages, were aimed at the comprehensive understanding of the complex processes that happen in these habitats of special significance. In the sediment samples the following enzymatic activities have been quantitatively determined: phosphatase, actual and potential dehydrogenase, catalase, urease and protease. Non-enzymatic catalytic activity was also measured. Based on the relative values for the enzymatic activities, the enzymatic indicator of the sediment quality (EISQ) was calculated (ranging from 0.1 to 0.7). The enzymatic activities have been qualitatively determined for maltase, saccharase, lactase, cellobiase, amylase, dextranase, levanase, cellulase and inulinase. The correlation between the enzymatic and bacteriologic potential was statistically calculated.     

Specificity studies on enteropeptidase substrates related to the N-terminus of trypsinogen.

The specificity of the synthetic substrate Gly-[L-Asp]4-L-Lys 2-naphthylamide originally developed for the assay of enteropeptidase (EC 3.4.21.9), was investigated with partially purified aminopeptidase. Our results indicate that, not only enteropeptidase, but also the concerted action of the aminopeptidases of the rat small intestine, can rapidly release 2-naphthylamine from the substrate. A previously undescribed, highly active, dipeptidylaminopeptidase, which hydrolyses a Gly-Asp dipeptide from the N-terminus of the substrate, was detected in rat small intestine. The resulting [L-Asp]3-L-Lys 2-naphthylamide fragment is then degraded by a combination of aminopeptidase A and N to yield free 2-naphthylamine. Thus the present substrate cannot be regarded as being specific for enteropeptidase, and its use leads to an over-estimation of enteropeptidase activity in homogenates and extracts of intestinal tissue. In order to prevent this non-specific hydrolysis by aminopeptidases, stereoisomeric substrates with the sequence L-Ala-D-Asp-[L-Asp]3-L-Lys methyl ester, D-Ala-[L-Asp]4-L-Lys methyl ester and L-Ala-[Asp]4-L-Lys methyl ester were synthesized and tested as alternative substrates by their ability to inhibit the enteropeptidase-catalysed activation of trypsinogen.     

In vitro study of the intestinal brush border enzyme activities in developing anuran amphibian: effects of thyroxine, cortisol, and insulin

The effects of several hormones on intestinal brush border membrane enzymatic activities have been investigated in intestinal explants taken from the amphibian midwife toad at different developmental stages. Explants were treated for at least 2 days with thyroxine (0.1 microgram/ml of culture medium) or for 2 days with cortisol (25 micrograms/ml) or insulin (6 mU/ml). The hydrolases examined were maltase, trehalase, glucoamylase, and alkaline phosphatase. In the explants from tadpoles in prometamorphosis, thyroxine had no effect on hydrolase activities; cortisol increased the activity of only glucoamylase, and insulin increased activity of maltase, glucoamylase, and alkaline phosphatase. When the explants were taken from tadpoles at the beginning of climax, cortisol and insulin generally stimulated the enzyme activities studied. When taken from tadpoles at the end of climax, at the moment when the embryonic cells under the degenerating epithelium divide, cortisol and insulin had little effect on these activities. When the animals terminate their metamorphosis, the intestinal epithelium of the explants is totally newly formed (secondary epithelium). At this time, cortisol stimulated the activities of maltase, glucoamylase, and alkaline phosphatase, while insulin decreased the activities of maltase and glucoamylase.