Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Angiotensin-converting enzyme (ACE) was isolated from detergent-derived extracts of human intestinal brush-border membranes (BBMs) by immunoprecipitation using a monoclonal antibody. Analysis of the immunoprecipitates by SDS/PAGE revealed a polypeptide of apparent M(r) 184,000 under reducing and non-reducing conditions, indicating that ACE does not contain intermolecular disulphide bridges. The quaternary structure of ACE was examined using cross-linking experiments with dithiobis[succinimidylpropionate] (DSP) and density gradient centrifugation on sucrose gradients. Both approaches demonstrated that ACE is assembled in the membrane as a monomer. By contrast, the control glycoprotein aminopeptidase N (ApN) exists as a dimer. Biosynthetic labelling experiments in intestinal tissue explants demonstrated that the 184,000-M(r) protein is generated from a single-polypeptide, mannose-rich precursor of ACE (M(r) 175,000) by modification of the carbohydrate side-chains in the Golgi apparatus. The mode of association of the mature form of the enzyme with BBMs was investigated by hydrophobic labelling of right-side-out brush-border vesicles with the photoactivatable carbene-generating reagent 125I-labelled 3-(trifluoromethyl)-3-(m[formylamino]phenyl)diazirine (125I-labelled TID), followed by treatment with trypsin at dilutions that do not cause substantial degradation of ACE. These studies demonstrated that ACE is associated with the membrane via a hydrophobic segment. Furthermore, treatment of 35S-labelled inside-out membrane vesicles with trypsin revealed that ACE possesses a cytoplasmic tail, and therefore has a transmembraneous orientation.
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PMID:Angiotensin-converting enzyme of the human small intestine. Subunit and quaternary structure, biosynthesis and membrane association. 132 43

1. The membrane anchor of aminopeptidase N associated with larval midgut cell membranes of the silkworm, Bombyx mori, was investigated by using phosphatidylinositol-specific phospholipase C (PIPLC) and proteases. 2. Aminopeptidase N, which was virtually all localized in the brush border membrane, was solubilized by PIPLC but not by papain or trypsin. 3. Detergent-solubilized amphiphilic aminopeptidase N was converted into a hydrophilic form by PIPLC but not by papain. 4. Either of these effects of PIPLC on aminopeptidase N was maximally 40%. 5. These results suggest that in larval midgut cells of the silkworm, B. mori, at least 40% aminopeptidase N is anchored in the brush border membrane via glycosyl-phosphatidylinositol.
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PMID:Partial release of aminopeptidase N from larval midgut cell membranes of the silkworm, Bombyx mori, by phosphatidylinositol-specific phospholipase C. 135 82

Membrane vesicle preparations are very appropriate material for studying the topology of glycoproteins integrated into specialized plasma membrane domains of polarized cells. Here we show that the flow cytometric measurement of fluorescence energy transfer used previously to study the relationship between surface components of isolated cells can be applied to membrane vesicles. The fluorescein and rhodamine derivatives of a monoclonal antibody (4H7.1) that recognized one common epitope of the rabbit and pig aminopeptidase N were used for probing the oligomerization and conformational states of the enzyme integrated into the brush border and basolateral membrane vesicles prepared from rabbit and pig enterocytes. The high fluorescent energy transfer observed in the case of pig enzyme integrated into both types of vesicles and in the case of the rabbit enzyme integrated into basolateral membrane vesicles agreed very well with the existence of a dimeric organization, which was directly demonstrated by cross-linking experiments. Although with the latter technique we observed that the rabbit aminopeptidase was also dimerized in the brush border membrane, no energy transfer was detected with the corresponding vesicles. This indicates that the relative positions of two associated monomers differ depending on whether the rabbit aminopeptidase is transiently integrated into the basolateral membrane or permanently integrated into the brush border membrane. Cross-linking of aminopeptidases solubilized by detergent and of their ectodomains liberated by trypsin showed that only interactions between anchor domains maintained the dimeric structure of rabbit enzyme whereas interactions between ectodomains also exist in the pig enzyme. This might explain why the noticeable change in the organization of the two ectodomains observed in the case of rabbit aminopeptidase N does not occur in the case of pig enzyme.
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PMID:Conformational change of rabbit aminopeptidase N into enterocyte plasma membrane domains analyzed by flow cytometry fluorescence energy transfer. 247 1

Pig small intestinal mucosal explants, labelled with [35S]-methionine, were fractionated into Mg2+-precipitated (intracellular and basolateral) and microvillar membranes, and the orientation of newly synthesized aminopeptidase N (EC 3.4.11.2) in vesicles from the two fractions was studied by its accessibility to proteolytic cleavage. The mature polypeptide of Mr 166 000 from the latter fraction was cleaved by trypsin, proteinase K and papain, consistent with an extracellular location of the enzyme at its site of function. In contrast, both the mature form and the transient form of Mr 140 000 from the Mg2+-precipitated fraction were equally well protected from proteolytic cleavage (in the absence of Triton X-100). This indicates that the basolateral plasma membrane is unlikely to be involved in the post-Golgi transport of newly synthesized aminopeptidase N and suggests instead a direct delivery of the enzyme to the apical plasma membrane. A crude membrane preparation from labelled explants was used in immunoelectrophoretic purification of membranes to determine at what stage during intracellular transport newly synthesized microvillar enzymes are sorted, i.e., accumulated in areas of the membrane from where other proteins are excluded. The transient form of aminopeptidase N was only moderately enriched by immunopurification, using antibodies against different microvillar enzymes, but the mature form was enriched approximately 30-fold from explants, labelled for 30 min. This suggests that for microvillar enzymes, the aspects of sorting studied take place in, or shortly after exit from, the Golgi complex.
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PMID:Biosynthesis of intestinal microvillar proteins. Evidence for an intracellular sorting taking place in, or shortly after, exit from the Golgi complex. 286 38

Procedures have been validated for the investigation of the physical properties of canine microvillar membrane proteins by SDS-polyacrylamide gel electrophoresis. These have been used to examine mucosal samples from eight control dogs and from five dogs with naturally occurring exocrine pancreatic insufficiency (EPI) in order to evaluate the potential role of the pancreas in the normal turnover of microvillar membrane proteins in the dog. Gel scanning showed that the proportion of total membrane protein in bands corresponding to a molecular mass greater than 200 kDa was up to 20-times higher in dogs with EPI than in control dogs. In particular, a band of apparent molecular mass 218 kDa represented between 8 and 28% of membrane protein in all affected dogs, compared with only 0.5 to 1.8% in controls, and is most likely to contain single chains of both pro-maltase-glucoamylase and pro-sucrase-isomaltase. Incubation of microvillar membranes in vitro with either trypsin or canine pancreatic juice resulted in degradation of this high molecular mass band and a corresponding increase in the amount of protein in three bands representing molecular masses of 150, 133 and 106 kDa. In samples from control dogs aminopeptidase N was identified in the 133 kDa band by Western blotting and incubation with monospecific antiserum. These findings suggest that pancreatic enzymes play a major role in the normal post-translational processing of intestinal microvillar membrane proteins in the dog.
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PMID:Investigation of the physical properties of dog intestinal microvillar membrane proteins by polyacrylamide gel electrophoresis: a comparison between normal dogs and dogs with exocrine pancreatic insufficiency. 340 88

The aminopeptidase A of the porcine intestinal brush-border membrane has been purified following solubilization by trypsin (p-form) or Emulphogen (d-form). Full purification of d-amino-peptidase A required the use of anti-impurities immunoabsorbant chromatography. The d-amino-peptidase A constitutes about 4% of the total proteins of the membrane, compared to 8-12% for another, already characterized, brush-border aminopeptidase N. Both d-form and p-form of aminopeptidase A have been clearly shown to be dimeric. Experimental evidence is presented favoring the view that they are symmetrical dimers, with the consequence that each of the two subunits of the d-form possesses an hydrophobic anchor holding them at the membrane surface. As already demonstrated for several other brush border hydrolases, the hydrophobic anchor is N-terminal in porcine intestinal aminopeptidase A. The molecular weight of the peptide including the anchor liberated by trypsin during the conversion of the d-form into the p-form has been estimated by an isotopic dilution method to be about 4500 (42 residues). This value which compares well with those recently obtained in the case of rabbit aminopeptidase N (3700-3800; 36-38 residues), indicates that the anchor is much shorter than believed earlier. A preliminary survey of the specificity of both aminopeptidases A and N towards four synthetic amino acid p-nitroanilides confirms that aminopeptidase A mostly cleaves acidic residues. Its activity towards neutral residues is much lower, but probably significant in certain cases.
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PMID:Purification and characterization of an aminopeptidase A from hog intestinal brush-border membrane. 610 77

The presence of human blood group A determinants has been shown on the A+ rabbit intestinal brush border glycoproteins, particularly hydrolases. Sugar compositions of aminopeptidases N from A+ and A- rabbits were compatible with the presence in these molecules of eight N-linked glycans and of two O-linked glycans bearing the A determinants in A+ animals. The exact relative molecular masses of hydrophobic domain(s) of aminopeptidases N and A from pig and rabbit intestinal brush border have been determined by an isotopic dilution technique. The values obtained were compatible with the anchorage in the membrane of the monomeric rabbit enzymes, or of each subunit of the dimeric pig enzymes, by their N-terminal sequences, composed of 20-25 hydrophobic amino acids. This N-terminal hydrophobic sequence (14 residues) has been determined for rabbit aminopeptidase N. Short peptides containing approximately 60% hydrophobic amino acids have been extracted by chloroform-methanol from purified brush border and basolateral membranes of pig enterocytes. Their molecular properties were very similar to those of the aminopeptidase anchors released by trypsin treatment of detergent-extracted enzymes. However, several lines of evidence failed to support the assumption that these free hydrophobic peptides can be identified with anchors left inside the bilayer after proteolytic cleavage of surface hydrolases.
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PMID:Aminopeptidases and proteolipids of intestinal brush border. 634 98

The purification of detergent-solubilized kidney microvillar endopeptidase (EC 3.4.24.11) by immuno-adsorbent chromatography is described. The product (the d-form) was 270-fold purified compared with the homogenate of kidney cortex and was obtained in a yield of 5%. It was free of other peptidase activities and homogeneous by electrophoretic analyses. It contained about 15% carbohydrate and one Zn atom/subunit. Two trypsin-treated forms were also characterized. One (dt-form) was obtained by treatment of the d-form. The other (tt-form) was the result of solubilizing the membrane by treatment with toluene and trypsin. All three forms had apparent subunit Mr values of approx. 89 000, but the d-form appeared to be slightly larger than the other two. Estimates of Mr by gel filtration showed that of the tt-form to be 216 000 whereas those of the other forms were 320 000. An estimate of the detergent (Triton X-100) bound to the d- and dt-forms accounted for this difference. By several criteria, including charge-shift crossed immunoelectrophoresis and hydrophobic chromatography, the d- and dt-forms were shown to be amphipathic molecules. In contrast, the tt-form was hydrophilic in its properties. Differences in ionic properties were also noted, consistent with the loss, in the case of the dt-form, of a positively charged peptide. The results indicate that the native endopeptidase is a dimeric molecule, each subunit being anchored in the membrane by a relatively small region of the polypeptide close to one or other terminus. The d- and dt-forms had similar enzyme activity when assayed by the hydrolysis of 125I-insulin B-chain. Chelating agents and phosphoramidon inhibited the endopeptidase. The kinetic constants were determined by a new two-stage fluorimetric assay using glutarylglycylglycylphenylalanine 2-naphthylamide as substrate and aminopeptidase N (EC 3.4.11.2) to hydrolyse phenylalanine 2-naphthylamide. The Km was 68 microM and Vmax. 484nmol X min-1 X (mg of protein)-1.
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PMID:Proteins of the kidney microvillar membrane. The amphipathic forms of endopeptidase purified from pig kidneys. 634 15

A short treatment of dog renal brush-border membrane vesicles (BBMV) with sodium cholate, followed by dialysis of the detergent, reorients the polarity of H(+)-ATPase in the membrane and exposes its ATP binding sites to the extravesicular space, as previously shown with pig BBMV. In cholate-pretreated vesicles, the H(+)-ATPase remains fully active, but is inserted under the reversed polarity in sealed vesicles. A large spontaneous N-ethylmaleimide-sensitive ATPase activity is thus observed, as well as a steep intravesicular acidification upon external ATP addition, two findings absent in native vesicles. The ability of nitrate plus ATP to dissociate the hydrolytic subunits ot the proton pump in cholate-pretreated vesicles, but not in native vesicles, demonstrates that most of the ATP binding subunits are accessible to ATP following cholate treatment. The sensitivity of the cytoplasmic domain of the H(+)-ATP activity to trypsin also confirms the reorientation of the enzyme in cholate-pretreated vesicles. The H(+)-ATPase and alkaline phosphatase remain largely associated with the membranes after the treatment with cholate, but gamma-glutamyltranspeptidase, aminopeptidase N, and neutral endopeptidase are largely solubilized. Upon dialysis of cholate, all these enzymes are in part reinserted in the membrane according to their original polarity. The reorientation process is however specific for the H(+)-ATPase. Cholate treatment does not increase the formation of inside-out vesicles. Thus the treatment with cholate really reorients the polarity of the H(+)-ATPase in vesicles and allows for study of the proton pumping capacity of vacuolar H(+)-ATPase of proximal tubules.
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PMID:Effect of cholate on H(+)-ATPase and other proteins of dog renal brush-border membrane. 812 55

The serum level of placental leucine aminopeptidase (P-LAP) increases during pregnancy. P-LAP degrades several peptide hormones such as oxytocin and vasopresin, suggesting a role in maintaining homeostasis during pregnancy. In the study reported here, we have isolated a cDNA clone with 4084 base pairs encoding P-LAP from a human placental cDNA library. The amino acid sequence deduced from the cDNA contained all of the sequences of the peptide fragments obtained by digestion of the purified protein with trypsin. The predicted P-LAP contains the HEXXH consensus sequence of zinc metallopeptidases, indicating that the enzyme belongs to this family, which includes aminopeptidase N and aminopeptidase A. The deduced sequence also contains a hydrophobic region near the N terminus, suggesting that the enzyme is a type II integral membrane protein. Northern blot analysis revealed that P-LAP was expressed in several tissues, some of which expressed two forms of mRNAs. These results suggest that the enzyme is synthesized as an integral membrane protein and is released into blood under some physiological conditions.
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PMID:Human placental leucine aminopeptidase/oxytocinase. A new member of type II membrane-spanning zinc metallopeptidase family. 855 Jun 19


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