Pancreatic carboxypeptidase hydrolysis of bile acid-amino conjugates: selective resistance of glycine and taurine amidates

To find a possible explanation for the selective hepatic conjugation of bile acids with glycine or taurine, the N-acyl amidates of cholic acid and a number of amino acids and amino acid analogues were synthesized, and their susceptibility to hydrolysis by pancreatic juice, gastric juice, serum, or small intestinal mucosal enzymes was measured. Deconjugation by pure carboxypeptidase A and B was also examined, and hydrolysis by these tissue fluids and enzymes was compared with that mediated by a bacterial cholylglycine hydrolase. Human pancreatic juice efficiently hydrolyzed cholyl conjugates of all neutral-L-amino acids (cholyl-L-alanine, cholyl-L-valine, cholyl-L-leucine, and cholyl-L-tyrosine), except cholylglycine. The net hourly rate of hydrolysis (in micromoles per milligram protein per hour) increased when the terminal residue was aromatic or branched aliphatic, and appeared to be specific for L-alpha-amino acids as cholyl-beta-alanine and cholyl-D-valine were not cleaved. From cholyl glycylglycine, only the terminal glycine was efficiently removed. Cholyltaurine and cholyl conjugates with the methyl and propyl analogues of taurine were resistant to hydrolysis. Two basic amino acid conjugates (cholyl-L-lysine and cholyl-L-arginine) were cleaved, whereas conjugates of acidic amino acids (cholyl-aspartate and cholyl-cysteate) were not cleaved. Studies using pure enzymes showed that bovine carboxypeptidase A hydrolyzed the cholyl conjugates of the neutral L-alpha-amino acids with similar specificity as observed for the human pancreatic juice, whereas bovine carboxypeptidase B cleaved the basic amino acid conjugates. Cholyl-L-lysine and cholyl-L-arginine were also cleaved by serum and plasma, which are known to possess carboxypeptidase activity. Cholyl conjugates were not cleaved by gastric juice, by trypsin, or by homogenates of rat small intestinal mucosa. In contrast, all cholyl conjugates were cleaved by a bacterial cholylglycine hydrolase. These experiments indicate that glycine and taurine amidates of cholic acid differ from a number of other conjugates with neutral and basic amino acid in being resistant to hydrolysis by pancreatic and plasma carboxypeptidases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Human intestinal brush border peptidases

Hydrolysis of small peptides, like disaccharide hydrolysis, is an important function of the intestinal brush border, but little is known of the individual human peptidases. The purposes of this study were to detect all human brush border enzymes hydrolyzing dipeptides and tripeptides, identify the most discriminating substrate for each enzyme in order to permit assays in crude mixtures, and begin biochemical characterization of each enzyme. Four brush border peptidases were identified. Enzymes I (aspartate aminopeptidase, E.C. 3.4.11.7) and III (amino-oligopeptidase, E.C. 3.4.11.2) are known brush border enzymes. Enzymes II (membrane Gly-Leu peptidase) and IV (zinc stable Asp-Lys peptidase) have not been identified in human brush border previously. They are distinct from dipeptidyl aminopeptidase IV, carboxypeptidase, and gamma-glutamyl transferase. The substrate most discriminating for each enzyme is alpha-Glu-beta-naphthylamide for I (100% of the brush border activity for this substrate is due to enzyme I), glycylleucine for II (80%), leucyl-beta-naphthylamide for III (91%), and aspartyl-lysine in 5 mM Zn2+ for IV (63%). The enzymes are immunologically distinct and antibodies to each one localize to the brush border on immunohistochemical staining. Purification of 142-, 79-, 158-, and 46-fold was achieved for enzymes I through IV, respectively. Biochemical characteristics include slightly alkaline pH optima, molecular weights of 91,000-190,000, and evidence of metal ion involvement in activity. These studies provide necessary information for determining the role of brush border peptidase deficiencies in human disease.     

Stimulation of thyroidal thiol endopeptidases by thyrotropin

Rabbit thyroids contain cathepsin D (CD) and several thiol endopeptidases including cathepsin B and three newly described enzymes (cathepsins 180K, 110K, and 45K). The present paper assesses the relative physiological importance of these enzymes in thyroglobulin degradation in rabbits. Thyroidal thiol endopeptidase [thiol thyroglobulin hydrolase (thiol TgH)] activity increased in the absence of changes in CD activity in animals treated with 10 U bovine TSH. Peak enzyme activity occurred 24 h after injection of hormone. After 20 U bovine TSH, thiol endopeptidase activity increased by approximately 100%, whereas CD increased by 50%. The increase in thiol enzyme activity was attributed both to cathepsin B and to the other thiol endopeptidases. The lysosomal acid hydrolases acid phosphatase and dipeptidyl peptidase II were unaffected by TSH at either dose level. Thiol TgH activity, but not CD activity, was decreased in thyroids of rabbits treated with T4 [5 micrograms/(100 g BW X day)] for 1 week. All thyroidal acid hydrolases examined were suppressed in animals receiving T4 for 3 weeks. Thiol TgH activity was localized primarily to a lysosome-enriched fraction of thyroid homogenates. Our results suggest that the thiol proteases probably are the most important endopeptidases in thyroglobulin hydrolysis in vivo and that their activities are influenced by TSH.     

Effect of bile acids and pH on the release of enteropeptidase in man

Bile acids increase the release of human enteropeptidase as well as other brush-border enzymes (alkaline phosphatase, leucine aminopeptidase) from duodenal mucosa, as had been shown earlier in experimental animals. The action of bile acids is independent of their known enhancing effect on enteropeptidase activity. The pH of duodenal juice is an important, hitherto unrecognized, factor in the release mechanism of brush-border enzymes. All of the above enzymes tested were released to a markedly greater extent at pH 8.2 than 6.3, regardless of the presence or absence of bile acid. Contrary to some results obtained with animal tissue, by other investigators, our experiments with human duodenal mucosa indicate that enteropeptidase, under all conditions tested, is released at a rate considerably greater than that for alkaline phosphatase or leucine aminopeptidase. The looser association of enteropeptidase with cellular components relative to that of other brush-border enzymes, as indicated by our observations, may be related to the unique function of enteropeptidase as the trigger enzyme of protein digestion.This work was supported by the Medical Research Service of the Veterans Administration.     

Mechanism of action and control in the digestion of proteins and peptides in humans

This review aims to report the major control mechanisms of protein and peptides digestion of special interest in human patients. Regarding protein assimilation its digestive process begins at the stomach with some not so indispensable actions comparatively to those of duodenal/jejunal lumen. However even the intestine processes are partially under gastric secretion control. Proteolytic enzyme activities are related to protein structure and amino acid constituents, tertiary and quartenary structures need HCl denaturation prior to enzymatic hydrolysis. Thereafter the exopeptidases are guided by either NH2 (aminopeptidases) or COOH (carboxypeptidases) terminals of the molecule while endopeptidases are oriented by the specific amino acids constituents of the peptide. Both dietary and luminal secreted proteins and polypeptides undergo to either limited or complete proteolysis resulting basic or neutral free-amino acids (40%) or dioctapeptides. The brush border peptidases continue to degrade oligopeptide to di-tripeptides and neutral free-amino acids. Some peptides are uptaked by the enterocytes whose cytosolic peptidases complete the hydrolysis. Hence the digestive products flowing in the portal vein are mainly free-amino acids from either luminal or cytosolic hydrolysis and some di-tripeptides intactly absorbed. Both mechanical and chemical processes of digestion are under neural (vagal), neuroendocrinal (acetilcholine), endocrinal (gastrin, secretin and cholecystokinin) or paracrinal (histamine) controls. The gastric phase (hydrochloric acid and pepsinogen secretions) is activated by gastrin, histamine and acetilcholine which respond to both dietary-amino acids (tryptophan and phenylalanine) and mechanic distention of stomach. The pancreatic secretion is stimulated by either cephalic or gastric phases and has influence on the intestinal phase of digestion. The intestinal types of cells S and I release secretin and cholecystokinin respectively in response of acid quimo (cells S) or amino acids and peptides (cells I) in the lumen. Secretin stimulates the releasing of water, bicarbonate and enteropeptidases whereas cholecystokinin acts on pancreatic enzymes.     

Intestinal disaccharidase activity following pancreatic duct occlusion in the rat

The influence of pancreatic secretions on growth and brush-border enzyme activity, throughout the entire small intestine, was examined in the rat. Pancreatic secretions were excluded from the gut lumen by stapling the pancreatic ducts, without interruption of bile flow. The entire small intestine was studied as four segments; the duodenum and three distal segments of equal length. Weight of intestine and mucosa, and mucosal sucrase, isomaltase, lactase, and alkaline phosphatase activity were measured 10-15 days following pancreatic duct occlusion, or sham-operation. The duodenum of pancreatic duct-occluded animals exhibited significant hypertrophy. In general, specific and total disaccharidase activities were greater in duct-occluded animals than in controls throughout the intestine. The increase was more pronounced in distal than in proximal segments. The sucrase/isomaltase ratio was significantly greater in pancreatic duct-occluded animals than in controls in the two distal segments. Alkaline phosphatase activity was not affected by pancreatic duct occlusion. The greater relative increase of disaccharidase activities and sucrase/isomaltase activity ratios in the distal segments of duct-occluded animals, indicates a more important regulatory role of pancreatic enzymes in the distal small intestine. It is concluded that regulation of intestinal brush-border enzyme activity by pancreatic secretion is selective for enzyme and site as follows: disaccharidases, but not alkaline phosphatase, are regulated; the sucrase subunit of the sucrase/isomaltase complex is most sensitive to regulation, while lactase is least sensitive; and the regulatory effect on disaccharidases is greater in distal than in proximal intestine.     

Intestinal mucosa in diabetic rats: Studies of microvillus membrane composition and microviscosity

In experimental diabetes, a number of intestinal brush-border hydrolases and transport systems are stimulated. In this study, we assessed possible effects of diabetes on the composition and membrane fluidity of rat intestinal brush-border membranes that might correlate with these functional changes. We found similar proportions of lipid and protein in the diabetic and control preparations, although there was a considerable increase in total membrane from the diabetic rats, presumably reflecting mucosal hyperplasia. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane protein revealed an increase in the bands corresponding to sucrase-isomaltase, consistent with an increased enzyme activity of sucrase. Membrane lipid analysis revealed only a decrease in fatty acids of the neurtral lipid fraction of diabetics—a change that may well have occurred during membrane preparation. 1-6-Diphenyl-1,3,5-hexatriene fluorescence polarization data, obtained as a function of temperature, was similar for the diabetic and control rats, with a three-phase linear model superior to one- and two-phase linear or quadratic models. The overall composition of the intestinal brush-border membrane, unlike other plasma membranes, appears little affected by experimental diabetes.     

Duodenal brush-border mucosal glucose transport and enzyme activities in aging man and effect of bacterial contamination of the small intestine

Duodenal biopsies were collected from 38 subjects (24 female and 14 male) ranging in age from 55 to 91 years. Evidence of bacterial contamination of the small bowel (BCSB) was sought at the same time by bacterial culture of duodenal aspirates and by hydrogen and [¹⁴C]glycocholic acid breath tests; subjects were considered to be positive for BCSB if any one of the three tests was abnormal. Biopsies were analyzed for six brush-border membrane enzyme activities: maltase, sucrase, lactase, alkaline phosphatase, leucine aminopeptidase, and -glucosidase. Analysis of covariance with age as the covariate indicated no significant effect of age on the specific activities of these enzymes. Mucosal Na⁺-dependent glucose transport was quantified in brush-border membrane vesicles prepared from the biopsies. In all groups, glucose transport at 20–30 sec was greater (ranging from mean values of 2.45 to 3.66 times) than at 45 min, consistent with Na⁺-coupled glucose transport, and no significant effect of age was observed. BCSB had no significant effect on specific activities of any of the duodenal mucosal hydrolases but was associated with reduced (P=0.05) brush-border glucose transport. None of the variables studied was significantly affected by the gender of subjects. In conclusion, these biochemical data do not support the contention that reduced capacity for carbohydrate absorption in the elderly is explained by reductions in duodenal brush-border mucosal disaccharidase activities or glucose transport.This work was supported by a grant from Research into Ageing. J.L.W. was supported by a Postgraduate Studentship from Research into Ageing.     

The enzymology and intracellular organization of peptide precursor processing: the secretory vesicle hypothesis

The 'secretory vesicle hypothesis of precursor processing' states that the initial endopeptidase cleavages which excise the nascent, biologically active peptides from their protein precursors occur primarily in secretory vesicles (or granules). Hence, all the processing steps subsequent to these cleavages must also occur within these organelles. Two types of evidence are presented in support of this view: (1) cell biological studies which implicate the secretory vesicle as the site of precursor conversion to peptides, and (2) enzymological studies which locate and characterize putative processing enzymes in secretory vesicles. The processing enzymes reviewed include the 'prohormone-converting enzymes' which cleave at pairs of basic amino acids, other endopeptidases, carboxypeptidase-B-like enzymes and aminopeptidase, and N-acetylation and alpha-amidation enzymes. The properties of these enzymes in relation to the nature of the processing micro-environment in the secretory vesicles is discussed.     

Glucose absorption from starch hydrolysates in the human jejunum.

The intestinal absorption and mucosal hydrolysis of a partial and a complete alpha-amylase hydrolysate of corn starch, simulating the normal intermediary and end products of luminal starch digestion, was studied using an in vivo steady state jejunal perfusion technique in normal human subjects. Alpha-amylase was excluded from the test segment by proximal balloon occlusion. Products of hydrolysis during intestinal perfusion were identified using gel permeation chromatography. Three isocaloric, isotonic sugar saline solutions containing 140 mM glucose, 70 mM maltose and the partial amylase hydrolysate of starch (51.5 +/- 1.4% of glucose content comprising glucose polymers of more than 10 glucose units) were perfused in the first study. Net glucose absorption during perfusion of the partial hydrolysate and free glucose was similar, but significantly faster from maltose (p less than 0.05). Hydrolysis of the polymer fraction containing more than 10 glucose units was significantly slower (29.5 +/- 2.0% of infused load) than the lower molecular weight fraction (56.4 +/- 3.8%, p less than 0.001). As net glucose absorption from the partial hydrolysate was similar to that from glucose, despite the slow hydrolysis of the higher molecular weight fraction, it seemed likely that oligosaccharides in the more rapidly hydrolysed lower molecular weight fractions were exerting a kinetic advantage on glucose absorption. This was confirmed in a second study, where glucose absorption from a complete amylase hydrolysate consisting predominantly of maltose, maltotriose and alpha-limit dextrins, occurred significantly faster (81.8 +/- 4.8 mmol/h/25 cm) than from isocaloric free glucose (55.8 +/- 4.9 mmol/h/25 cm, p less than 0.001). Chromatograms of intestinal aspirates suggested that (1->4), but not 1->6) linked oligosaccharides liberated during luminal and brush-border hydrolysis of dietary starch conferred a kinetic advantage on glucose absorption.     

Identification of proline-specific carboxypeptidase localized to brush border membrane of rat small intestine and its possible role in protein digestion

A proline-specific carboxypeptidase (carboxypeptidase P, EC 3.4.12.-) was identified and partially characterized in the brush border membrane fraction of rat intestinal enterocytes and shown to be distinct from pancreatic proteases. The carboxypeptidase activity of isolated brush border membranes, with Z-Gly-Pro-Leu as substrate, was 43 nmol/min/mg protein representing a 16-fold purification when compared with mucosal cell homogenates. Activity was maximal in the middle region of the small intestine, and villus cells had twice the activity of crypt cells. Carboxypeptidase activity was maximal at pH 7.0, was stimulated by divalent cations, and was inhibited by metal chelating agents, suggesting that it is a metalloenzyme. The enzyme had the highest activity with synthetic peptides containing proline penultimate to the carboxy terminus. In vivopatterns of hydrolysis and absorption of amino acids from Z-Pro-Trp were examined using an intestinal perfusion technique. These studies indicate that brush border membrane carboxypeptidase may play an important role in the digestion of prolinecontaining peptides and proteins.This work was supported by grant AM17938 from the National Institutes of Health and by the Veterans Administration Medical Research Service.     

Receptor-like function of heparin in the binding and uptake of neutral lipids

Molecular mechanisms regulating the binding, amphipathic stabilization, and metabolism of the major neutral lipids (e.g., cholesteryl esters, triglycerides, and fatty acids) are well studied, but the details of their movement from a binding compartment to a metabolic compartment deserve further attention. Since all neutral lipids must cross hydrophilic segments of plasma membranes during such movement, we postulate that a critical receptor-like site exists on the plasma membrane to mediate a step between binding and metabolism and that membrane-associated heparin is a key part of this mediator. For example, intestinal brush border membranes containing heparin bind homogeneous human pancreatic 125I-labeled cholesterol esterase (100 kDa) and 125I-labeled triglyceride lipase (52 kDa). This interaction is enzyme concentration-dependent, specific, and saturable and is reversed upon addition of soluble heparin. Scatchard analysis demonstrates a single class of receptors with a Kd of 100 nM and a Bmax of approximately 50-60 pmol per mg of vesicle protein. In contrast, enzymes associated with the hydrolysis of hydrophilic compounds such as amylase, phospholipase A2, and deoxyribonuclease do not bind to intestinal membranes in this manner. Human pancreatic cholesterol esterase also binds specifically and saturably to cultured intestinal epithelial cells (CaCo-2), and soluble heparin significantly diminishes the cellular uptake of the resultant hydrophobic reaction products (cholesterol and free fatty acids). We conclude that a physiological role for intestinal heparin is that of a mediator to bind neutral lipolytic enzymes at the brush border and thus promote absorption of the subsequent hydrolyzed nutrients in the intestine. This mechanism may be a generalizable pathway for transport of neutral lipids into endothelial and other cells.     

Effect of aging on rat tissue peptidase activities

The process of aging is known to involve alterations in the activity of peptidases and proteases. However, the precise changes in the activity of many peptidases in aged tissues have not yet been fully characterized, and both decreases and increases in both peptidase activity and peptide levels have been reported to occur during the aging process. In the present study, we measured the activity of several peptidases in selected tissues (brain cortex, brain stem, liver, kidney, heart, and lung) of the young adult (3 months old) and aged (18 months old and 22 months old) rat. The activities of prolyl endopeptidase, pyroglutamyl peptidase I, puromycin sensitive aminopeptidase, and aminopeptidase N were assayed using beta-naphthylamine aminoacidic derivatives as substrates. The activity of the soluble fractions of prolyl endopeptidase was found to be reduced in the lungs of aged animals, while reduced activity of soluble pyroglutamyl peptidase I and also aminopeptidase N was measured in the aged kidney and heart, respectively. In contrast, increased activity of particulate prolyl endopeptidase was measured in the brain stem of older animals. Since most of these changes can be correlated with known alterations in the levels of peptides controlled by each enzyme, the results of the present study indicate that the studied peptidases may play an important role in regulating tissue peptide levels during aging.     

Product of the Pseudomonas aeruginosa gene pilD is a prepilin leader peptidase.

The related type IV pilins produced by Pseudomonas aeruginosa, Neisseria gonorrhoeae, Bacteroides nodosus, and Moraxella bovis are synthesized as precursors with short, six- or seven-amino acid N-terminal leader peptides. We have previously observed that P. aeruginosa mutations in pilD, a gene required for pilus biogenesis, result in the accumulation of unprocessed prepilin in the membrane and a general defect in the excretion of a number of extracellular enzymes. An endopeptidase activity has been detected in detergent-solubilized inner membrane of P. aeruginosa and shown to correctly cleave the prepilin of P. aeruginosa and N. gonorrhoeae. It is absent from pilD mutants, increased by pilD overexpression, and conferred on Escherichia coli by the introduction of the pilD gene. The pilD gene product, purified by immunoaffinity chromatography with antibody to a PilD-derived synthetic peptide, was identified with the endopeptidase. PilD appears to be a prototype of a class of enzymes that process not only type IV pilin precursors but also components of a protein-excretion apparatus of Gram-negative bacteria.     

Increased activity of digestive enzymes in ileal enterocytes adapting to proximal small bowel resection.

The ability of adapting ileal enterocytes to express different digestive enzymes in their brush border membranes was tested in young female Wistar rats (n = 72) receiving 60% proximal small bowel resection. In control rats with intestinal transection both neutral aminopeptidase and alpha-glucosidase activities were shown, by quantitative cytochemistry, to increase during enterocyte migration over the lower part of the villus; thereafter enzyme activities declined or remained approximately constant. Proximal enterectomy increased the amount of alpha-glucosidase but not neutral aminopeptidase activity appearing during early enterocyte development. Thymidine labelled autoradiography showed that the rate of enterocyte migration along the ileal villus nearly doubled after jejunal resection (19.3 v 11.1 microns/h). Nevertheless, the time taken for both peptidase and saccharidase activities to appear at maximal rates in the brush border membrane was diminished by about five hours. Thus ileal enterocytes adapt to proximal small bowel resection by selective increments in enzyme expression, findings that contradict the previous hypothesis of simple metabolic immaturity.     

Urinary excretion of renal brush-border enzymes in lepromatous leprosy--a preliminary investigation

Activities of the brush-border enzymes, alkaline phosphatase, maltase, leucine aminopeptidase, and gamma-glutamyl transpeptidase, were measured in urine samples of 25 lepromatous leprosy patients and an equal number of age-matched healthy controls. None of the patients were shown to be suffering from any other systematic disease. The enzymatic activities were shown to be significantly elevated in leprosy patients when compared to controls.     

Common acute lymphoblastic leukemia antigen (CALLA) is active neutral endopeptidase 24.11 ("enkephalinase"): direct evidence by cDNA transfection analysis

The common acute lymphoblastic leukemia antigen (CALLA) is a 749-amino acid type II integral membrane protein expressed by most acute lymphoblastic leukemias, certain other lymphoid malignancies with an immature phenotype, and normal lymphoid progenitors. A computer search against the most recent GenBank release (no. 56) indicates that human CALLA cDNA encodes a protein nearly identical to the rat and rabbit neutral endopeptidase 24.11 ("enkephalinase;" EC 3.4.24.11). This zinc metalloendopeptidase, which has been shown to inactivate a variety of peptide hormones including enkephalin, chemotactic peptide, substance P, neurotensin, oxytocin, bradykinin, and angiotensins I and II, had not been identified in lymphoid cells. To determine whether CALLA cDNA derived from human acute lymphoblastic leukemia cells (Nalm-6 cell line) encodes functional neutral endopeptidase activity, we generated CALLA+ stable transfectants in the CALLA- murine myeloma cell line J558 and analyzed them for enzymatic activity in a fluorometric assay based upon cleavage of the substrate glutaryl-Ala-Ala-Phe 4-methoxy-2-naphthylamide at the Ala-Phe bond. Total lysates as well as whole-cell suspensions of the Nalm-6 line and of the CALLA+ transfectants, but not of the CALLA- J558 cells, possessed neutral endopeptidase activity. This enzymatic activity was associated with the cellular membrane fraction and was abrogated by the specific neutral endopeptidase inhibitor phosphoramidon. The unequivocal identification of CALLA as a functional neutral endopeptidase provides insight into its potential role in both normal and malignant lymphoid function.     

Localization by immunofluorescence and histochemical labeling of aminopeptidase N in relation to its biosynthesis in rabbit and pig enterocytes

Antibodies raised against highly purified rabbit intestinal brush border aminopeptidase N were found in certain rabbits or pigs to crossreact with human blood-grouplike substances present in goblet cells and at the surface of the basolateral membrane of enterocytes. To obtain antibodies strictly specific for aminopeptidase, depletion of antibodies with a water-insoluble, mucus-rich fraction or human A erythrocytes was necessary. By using ultrathin frozen sections of rabbit and pig jejunum mucosa, both the brush border and a structure at the upper pole of the nucleus were labeled by the specific antiaminopeptidase antibodies. This structure, tentatively identified as the Golgi apparatus in which aminopeptidase is glycosylated, escaped detection by histochemistry, thus suggesting that the enzyme is not yet active at this stage of its intracellular processing. By contrast, histochemistry revealed active aminopeptidase molecules in another structure located under the terminal web and in the upper part of the lateral membrane.     

Neutral endopeptidase 24.11 in human neutrophils: cleavage of chemotactic peptide.

Membrane metallo-endopeptidase (NEP; neutral endopeptidase, kidney-brush-border neutral proteinase, enkephalinase, EC 3.4.24.11) cleaves peptides at the amino side of hydrophobic amino acids. While the enzyme is known to be in organs such as kidney and brain, we found it in human neutrophils. These cells cleaved the NEP substrate glutaryl (Glut)-Ala-Ala-Phe-(4-methoxynaphthylamine) (Glut-Ala-Ala-Phe-MNA) at a rate of 9.5 nmol X hr-1 per 10(6) cells, and phosphoramidon (1 microM) inhibited the hydrolysis by 90%. Intact neutrophils from donors who smoked had NEP activities about twice that of nonsmokers. Subcellular fractionation and sucrose density gradient centrifugation of lysed neutrophils showed that most of the NEP activity was membrane bound. A washed membrane fraction from human neutrophils rapidly cleaved 0.5 mM Glut-Ala-Ala-Phe-MNA (96 nmol X min-1 X mg-1) and the hydrolysis was inhibited by phosphoramidon and by specific antiserum to human renal NEP. The washed membrane fraction also rapidly cleaved 0.1 mM bradykinin (34 nmol X min-1 mg-1) and 0.1 mM fMet-Leu-Phe (49 nmol X min-1 X mg-1). The membrane-bound enzyme cleaved the peptide substrates at the same site as the homogeneous human renal NEP, and phosphoramidon and thiorphan inhibited the hydrolysis. Kinetic studies with pure human renal NEP showed that the chemotactic peptide fMet-Leu-Phe was one of the best biologically active substrates (Km, 59 X 10(-6) M; kcat, 3654 min-1). Immunocytochemistry at the light microscopic level revealed a high concentration of NEP on the cell membrane of neutrophils. This was confirmed with electron microscopy using the immunogold technique on ultrathin cryosections. These studies indicate that NEP in neutrophils may have important functions in inflammation and chemotaxis.     

Induction and maintenance of mucosal enterokinase activity in proximal small intestine by a genetically determined response to mediated sodium transport

Mucosal enterokinase activity was established at intervals throughout the small intestine in guinea-pigs; maximum activity was present in the duodenum and proximal jejunum in new born as well as adult animals. Transposition of 5 cm lengths of small gut from the high enterokinase containing proximal region to the distal intestine and vice versa showed that mucosal enterokinase activity in the transposed segments was little changed after several weeks of healthy life. Isolation of proximal jejunal loops from luminal continuity resulted in the fall of mucosal enterokinase activity to minimal levels within 16 hours. Low levels of mucosal enterokinase activity were identified in loops of both proximal and distal jejunum 12 weeks after isolation. Luminal perfusion studies in vivo in proximal jejunal loops 24 hours after isolation showed that mucosal enterokinase activity could be restored to near normal levels within four to six hours by luminal sodium in the presence of active pancreatic endopeptidases, oligopeptides, L-amino acids, or D-glucose but not D-amino acids or D-fructose. Near normal mucosal enterokinase activity persisted in the loops for as long as luminal perfusion with 144 mM sodium and L-lysine or trypsin was maintained (24 hours). The time course of the restoration of mucosal enterokinase activity was compatible with an initial precursor activation as well as biosynthesis. The requirement for luminal sodium appeared to be absolute regardless of the co-substrate and supports the conclusion that mucosal enterokinase activity is dependent on mediated sodium transport. The ability of proximal intestinal enterocytes to respond to sodium flux with an increase in enterokinase activity is a property determined in intrauterine life: distal intestinal enterocytes may have functioning structural genes for enterokinase but appear to be unable to respond.