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

The postition 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 gamma-glutamyl-transferase. 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.     

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.     

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。     

Crystal structure of enteropeptidase light chain complexed with an analog of the trypsinogen activation peptide.

Enteropeptidase is a membrane-bound serine protease that initiates the activation of pancreatic hydrolases by cleaving and activating trypsinogen. The enzyme is remarkably specific and cleaves after lysine residues of peptidyl substrates that resemble trypsinogen activation peptides such as Val-(Asp)4-Lys. To characterize the determinants of substrate specificity, we solved the crystal structure of the bovine enteropeptidase catalytic domain to 2.3 A resolution in complex with the inhibitor Val-(Asp)4-Lys-chloromethane. The catalytic mechanism and contacts with lysine at substrate position P1 are conserved with other trypsin-like serine proteases. However, the aspartyl residues at positions P2-P4 of the inhibitor interact with the enzyme surface mainly through salt bridges with the Nzeta atom of Lys99. Mutation of Lys99 to Ala, or acetylation with acetic anhydride, specifically prevented the cleavage of trypsinogen or Gly-(Asp)4-Lys-beta-naphthylamide and reduced the rate of inhibition by Val-(Asp)4-Lys-chloromethane 22 to 90-fold. For these reactions, Lys99 was calculated to account for 1.8 to 2.5 kcal mol(-1) of the free energy of transition state binding. Thus, a unique basic exosite on the enteropeptidase surface has evolved to facilitate the cleavage of its physiological substrate, trypsinogen.     

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.     

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.     

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.     

Estrogen-induced changes in the maltase activity of rat uterus

The effect of estrogen administration on uterine maltase activities at pH 4.3, 5.6 and 6.2 have been studied in the ovarleotomized immature rat. Estradiol (in total doses of 0.5, 1.0 and 2.0 /ug) for 3 consecutive days resulted in increased uterine maltase levels at pH 4.3 and pH 5.6, though the activity at pH 6.3 remained unchanged. A single injection of 2/ug estradiol resulted in an increase in the enzyme activities at pH 4.3 and pH 5.6; this was detectable in 36 hours and reached peak levels 48 hours after the hormone injection. Actinomycin and cycloheximide blocked the estradiol-induced increase in the enzyme activities. Pretreatment with clomiphene also neutralized estradiol-induced increases in the enzyme activities.     

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%.     

Neutral maltase of human granulocytes: localization on the extracytoplasmic side of the plasma membrane and some properties

Neutral maltase is an alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) which is present in human granulocytes and B-lymphocytes but not in T-lymphocytes. These cells have been reported to contain a renal-type neutral maltase which cross-reacts with an antiserum raised against kidney brush-border enzyme. No study has been performed to assess the subcellular localization of the enzyme. Molecular properties of leukocyte neutral maltase from any species are unknown. We report in this paper that neutral maltase is present on the extracytoplasmic side of human granulocyte plasma membrane. These results are supported by subcellular fractionation on Percoll gradient and by papain digestion of intact granulocytes. The enzyme is probably an integral membrane protein. The anchorage to the lipid bilayer may be similar to that of the stalked brush-border hydrolases. Some properties of granulocyte neutral maltase were also determined on a plasma membrane-enriched fraction. The enzyme cleaves maltose and nigerose but not other glucosides disaccharides and oligosaccharides. The Km for maltose is (+/- SD) 0.78 (+/- 0.06) mM, that for nigerose 21.05 (+/- 1.43) mM. The Vmax for nigerose is 0.83-fold that for maltose. Tris, maltotriose, maltotetraose, and maltopentaose were inhibitors of granulocyte neutral maltase.     

Carbohydrase activities in the bovine digestive tract

1. The carbohydrase activities of homogenates of mucosa from the abomasum, small intestine, caecum and colon, and of the pancreas of cattle were studied. 2. The disaccharidase activities were located mainly in the small intestine and showed a non-uniform pattern of distribution along the small intestine; trehalase activity was highest in the proximal part, lactase and cellobiase activities were highest in the proximal and middle parts and maltase activity was highest in the distal part. 3. The intestinal lactase and cellobiase activities were highest in the young calf and decreased with age, whereas the intestinal maltase and trehalase activities, which were very low compared with the lactase activity, did not change with age. 4. No intestinal sucrase or palatinase activity was detected in the calf or in the adult cow. 5. Homogenates of intestinal mucosa also exhibited amylase and dextranase activity. 6. Homogenates of the pancreas possessed a strong amylase activity and a weak maltase activity. The maltase activity did not change with age, whereas the amylase activity increased with age. 7. No marked differences were observed between the carbohydrase activities of calves fed solely on milk and those of calves given a concentrate-hay diet from 6 weeks of age.     

Lack of circadian rhythmicity of rat fetal intestinal enzymes as compared to the dams

The 24-hr activity patterns of intestinal maltase, lactase, leucylnaphthylamine hydrolyzing activity, γ-glutamyltransferase, and alkaline phosphatase were determined in pregnant rats maintained on a 12-12 light-dark cycle, with feeding during the dark period (1800-0600 hr, EST). The activities of these enzymes plus those of lysosomal maltase and lactase were followed during the same time period in 19- to 20-day-old fetuses. The activity patterns in the dams followed circadian rhythms, with peak activities occurring during the feeding-dark period. These rhythms are similar to the feeding schedule-cued rhythms observed in male rats and, therefore, are assumed to be feeding schedule cued also. In the fetuses, which obtained nutrients through the placenta, the activities increased in a somewhat nonlinear manner throughout the entire 24-hr period, but did not display a defined rhythm. It is concluded that endogenous intestinal enzyme rhythms do not exist in utero, and that oral and/or intermittent feeding is necessary for these rhythms to occur.     

Effects of hydrocortisone on carbohydrase concentrations, de novo synthesis and turnover patterns in immature rat intestine

Hydrocortisone administration to infant rats enhanced cellobiase and maltase activities and induced precocious expression of sucrase and trehalase activities along the length of the small intestine. These activity changes reflected proportional concentration increases in the enzymes lactase (EC 3.2.1.23), maltase/glucoamylase (EC 3.2.1.20) and sucrase-isomaltase (EC 3.2.1.48/10). Administration of an equivalent tracer dose of [3H]leucine (by body weight) to control and hydrocortisone-treated infant rats resulted in greater accumulation of label in the carbohydrase pools of the treated rats, suggesting their increased de novo synthesis. The increased concentrations of lactase and maltase/glucoamylase induced by exogenous hydrocortisone were matched by the presence of corresponding greater amounts of label in their brush border pools. Accumulation of label in each of the lactase, maltase/glucoamylase and sucrase-isomaltase pools was generally similar in the hydrocortisone-treated rats, suggesting equivalent stimulation of their synthesis as a group by the humoral agent. The turnover rates of the carbohydrases as a group were found to be similar and did not appear to differ in control and hydrocortisone-treated rats. Total protein synthesis rates were slightly greater in the intestine of the hydrocortisone-treated group of rats.