Enzymic activities of the brush border membrane of rat intestine hydrolyzing β-naphthylamides of amino acids, leucinamide and dipeptides

doi:10.1016/0005-2736(72)90188-5

Biochimica et Biophysica Acta (BBA) - Biomembranes

Volume 274, Issue 2, 9 August 1972, Pages 420-425

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Abstract

Brush border membranes isolated from rat intestine hydrolyzed β-naphthylamides of many amino acids, l-leucinamide and l-phenylalanyl-l-alanine with high specific activities.

These enzymic activities, appearing to be intrinsic to that subcellular fraction, were characterized as regards pH optimum, ion dependence and puromycin inhibition.

Only the enzyme hydrolyzing γ-l-glutamyl-β-naphthylamide was not activated by metal ions, or inhibited by puromycin; after papain solubilization, it was separated by gel filtration on Sephadex G-200 from the enzyme(s) responsible for the hydrolysis of the other studied substrates.

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Cited by (27)

Aminopeptidase activity in homogenates of various absorptive mucosae m the albino rabbit: implications in peptide delivery
1986, International Journal of Pharmaceutics
A methodology was developed to determine the type and activity of aminopeptidases in the conjunetival, nasal, buccal, rectal and vaginal homogenates, relative to duodenal and ileal homogenates, so as to define the aminopeptidase barrier to peptide absorption from non-oral routes. 4-Methoxy-2-naphthylamides of leucine, alanine, arginine and glutamic acid were used as substrates. Based on the pattern of substrate hydrolysis and the effect of activators and inhibitors on the rate of substrate hydrolysis, four to five aminopeptidases were estimated to be present in the mucosal homogenates studied. Aminopeptidase N was present in all these mucosae to the extent of 50–100% of ileal activity, whereas aminopeptidase A was present to the extent of 4–20% of ileal activity. Overall, the differences in aminopeptidase activity among the various non-oral routes were not large. This suggests that, before guidelines can be established to select a given route for optimal peptide delivery, further work is necessary to determine if the non-oral routes differ in non-aminopeptidase activity and in membrane permeability.
Peptide hydrolases of the human small intestinal mucosa: Identification of six distinct enzymes in the brush border membrane
1980, Clinica Chimica Acta
For this investigation highly purified brush border membranes from human small intestine, prepared according to the method described in the preceding paper [I], have been used. Solubilisation of brush border membrane proteins by sodium dodecyl sulphate, Triton X-100 and papain followed by electrophoresis in polyacrylamide gels revealed six distinct peptide hydrolases. These included the enzymes aminopeptidase A (EC 3.4.11.7), dipeptidylpeptidase IV (EC 3.4.14.-), γ-glutamultranspeptidase (EC 2.3.2.2) and aminopeptidase M (EC 3.4.11.2), which were clearly separable on polyacrylamide gels after solubilisation with Triton X-100 or papain. Activity recovered in the aminopeptidase M peak in the above gel system could be resolved into two distinct peptidases in addition to aminopeptidase M, by SDS-gel-electrophoresis. One of these peptidases was most active towards aliphatic tripeptide (aminopeptidase 1) while the other appeared to be specific for dipeptides (aminopeptidase 2).
Peptide hydrolases of the human small intestinal mucosa: Distribution of activities between brush border membranes and cytosol
1980, Clinica Chimica Acta
Brush border membranes from frozen human small intestine have been purified using a method which did not involve the use of EDTA-containing buffers or the disruption of brush border fragments with high concentrations of Tris. On average a 24-fold increase in specific activity of α-glucosidase (brush border marker) was obtained in the final preparation which contained insignificant traces of enzyme marker activities from cytosol and lysosomes.
The homogenates of human small intestinal mucosa were shown to contain enzymes capable of hydrolysing di-, tri-, and tetrapeptides as well as amino acid- and peptide-2-nephthylamides. Assuming a 100% location of α-glucosidase in the brush border membrane, distribution studies indicated that activities against tetrapeptides and leucyl-2-naphthylamide were located exclusively in the brush border membrane. A large proportion of activity against α-glutamyl-2-naphthylamide, γ-glutamyl-2-naphthylamide and glycyl-prolyl-2-naphthylamide were also recovered in the brush border membrane fraction. Depending on the substrate utilized, 33–87% of tripeptidase activity was located in the brush border membrane. However, 58–87% of dipeptidase activity was recovered in the soluble fraction.
Dipeptidylaminopeptidase and carboxypeptidase activities of the brush border of rabbit small intestine
1978, Gastroenterology
Two peptidases have been found in the brush border of rabbit small intestine: dipeptidylaminopeptidase IV, which is able to hydrolyze glycyl-l-proline from glycyl-l-prolyl-β-naphthylamide, and a carboxypeptidase, which is able to hydrolyze N-carbobenzoxy-l-prolyl-l-alanine and is activated by Co²⁺. These enzymes have the highest levels of specific activity in the distal ileum. The enzymatic activities hydrolyzing these substrates were found to be almost totally localized in the brush border: 90 and 99% of carboxypeptidase and dipeptidylaminopeptidase activities, respectively, were recovered in the brush border, assuming 100% recovery of the sucrase activity in this subcellular fraction. Both enzymes were present in the brush border of rabbit fetuses at the end of gestation as well as in the brush border of rabbits 7 days after ligation of the pancreatic duct. These results demonstrate that they are not bacterial or pancreatic enzymes absorbed on the luminal surface of the brush border. The proteins of brush border of rabbit intestine were solubilized by papain digestion and filtered on Sephadex G-200: the electrophoresis on gradient of polyacrylamide gel of the filtered proteins identified six peptidases: the dipeptidylaminopeptidase IV; the carboxypeptidase; the oligoaminopeptidase (substrate l-leucyl-β-naphthylamide); the aminopeptidase A (substrate α-l-glutamyl-β-naphthylamide); and two other peptidases hydrolyzing glycyl-l-leucine and l-methionyl-l-leucine. The demonstration in the brush border of these different peptidases suggests that this subcellular organelle plays a major role in terminal protein digestion, complementary to intraluminal digestion and intracellular digestion.
Gamma-glutamyl transferase of rat and human intestine: Greater enhancement of activity by dipeptides than by amino acids or longer peptides
1978, Biochemical medicine
A number of amino acids and peptides are substrates for gamma-glutamyl transferase (γ-GT) and their presence increases the activity of this membrane-bound enzyme. The enzyme has been postulated to play a role in intestinal transport of amino acids and peptides. In this study the degree of activation of both human and rat intestinal brush border γ-GT activity by various amino acids and peptides was assessed. Of the 22 l-amino acids tested, rat γ-GT was activated by 13, inhibited by isoleucine, and not significantly affected by 8. Human γ-GT was activated by 7 amino acids, inhibited by isoleucine, and unaffected by 14. Meaningful correlation of γ-GT activation with known characteristics of human intestinal amino acid tansport was not possible. Results obtained when γ-GT was tested for activation by peptides revealed that significant errors may occur due to hydrolysis of test peptides by contaminating peptide hydrolases. Thus, peptide hydrolases present in intact brush borders result in significant underestimation of γ-GT activation by dipeptides and overestimation of activation by tripeptides. The magnitude of the error varied greatly with the individual peptide used.
Maximal activation of both rat and human peptide hydrolase-free γ-GT by dipeptides was more than 2-fold greater than that observed for amino acids white the four tripeptides tested were generally less activating than amino acids. The pattern of activation of both enzymes by peptides is compatible with the currently known specificity of the peptide transport system.
Postnatale Entwicklung des Dünndarmepithels beim Meerschweinchen
1978, Progress in Histochemistry and Cytochemistry

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Enzymic activities of the brush border membrane of rat intestine hydrolyzing β-naphthylamides of amino acids, leucinamide and dipeptides

Abstract

Life Sci.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Anal. Biochem.

J. Biol. Chem.

Biochim. Biophys. Acta

Science

Acta Biol. Med. Germ.

Biochem. J.

Clin. Chim. Acta