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)

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

Microvillus aminopeptidase (EC 3.4.11.2) is an enzyme with a molecular weight around 300 000. Normal preparations contain three different subunits (subunit A, Mr 162 000; subunit B, Mr 123 000; subunit C, Mr 61 000). The relationship between the three subunits was studied by immunoelectrophoresis using specific antibodies against individual denatured subunits and by densitometric scanning of polyacrylamide gels after separation of the three subunits. The results suggest that microvillus aminopeptidase initially appears in the membrane as a symmetric molecule built up to two identical A subunits. These subunits are then split into equimolar amounts of subunit B and subunit C by trypsin. Subunit B cannot generate subunit C but may be further degraded. The reaction sequence described is one which occurs in vivo. Treatment of purified aminopeptidase with trypsin increases the specific activity twofold. This phenomenon does not seem to be correlated to the generation of subunit B and subunit C or to the transformation of amphiphilic form into hydrophilic form.
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PMID:Changes of the quaternary structure of microvillus aminopeptidase in the membrane. 612 Aug 37

The three brush-border enzymes--alanine aminopeptidase (EC 3.4.11.2), alkaline phosphatase (EC 3.1.3.1), and gamma-glutamyltransferase (EC 2.3.2.2)--are present in the urine of healthy persons in two variants, a particulate form and a soluble one. They can be separated by electrophoresis in agarose gel and by ultracentrifugation. The particulate forms exhibit similar electrophoretic mobility, but the soluble forms of these brush-border enzymes differ in their electrophoretic mobilities. The enzyme components of the particulate activity can be mobilized by Triton X-100 and trypsin. The electrophoretic mobility of the soluble forms of alanine aminopeptidase and gamma-glutamyltransferase is slowed by neuraminidase treatment. Both forms of gamma-glutamyltransferase are influenced in their electrophoretic mobility by treatment with n-butanol/diisopropyl ether, showing their lipid dependence. These findings enhance our knowledge of the biochemical nature of brush-border enzymes in urine.
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PMID:Electrophoretic variants of alanine aminopeptidase, alkaline phosphatase, and gamma-glutamyltransferase in urine. 614 5

The structure-function relationship of F and HN glycoproteins of HVJ were studied by proteolytic dissection. Three types of effects on the biological activity and structure of the virus particles were observed. First type of effect is preferential inactivation of biological activities related to F glycoprotein, such as hemolytic and cell fusion-inducing activities. Among enzymes which exert such effects, trypsin split F1 subunit to F1a (32,000 daltons) and F1b (19,000 daltons). By N-terminal determination, F1a was found to be derived from the N-terminal segment of F1, whereas F1b seems to correspond with the C-terminal segment of F1. Chymotrypsin and thermolysin digestion resulted in decreases in molecular weight of F1 subunit by about 3,500 daltons and 2,500 daltons, respectively. This splitting was found to occur near the N-terminus of F1, since new N-terminal amino acids were identified from the modified F1's. The second type of effect is characterized by specific splitting (for example, by a Staphylococcal proteases) of HN glycoprotein without affecting F protein. The third type has no apparent effect on the biological activities of the virion, although slight structural change of F glycoprotein was noted in some case. Exposure of the N-terminal segment of F1 to the surrounding aqueous medium despite its highly hydrophobic nature is shown by its easy splitting by aminopeptidase M, chymotrypsin and thermolysin. Based on these and previously published results, we hypothesize direct interaction of the hydrophobic segment with the lipid bilayers of the target cell membrane as an important step in fusion reactions between the viral envelope and plasma membranes.
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PMID:Structural requirements for hemolytic activity of F-glycoprotein of HVJ (Sendai virus) studied by proteolytic dissection. 630 87

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

Pig kidney microvillar proteins were extracted with octyl beta-glucoside and reconstituted in liposomes prepared from microvillar lipids of known composition. Four peptidases, namely endopeptidase (EC 3.4.24.11), aminopeptidases N (EC 3.4.11.2) and A (EC 3.4.11.7) and dipeptidyl peptidase IV (EC 3.4.14.5), were shown to be reconstituted. At lipid/protein ratios greater than 4:1, about half the detergent-solubilized protein and nearly all of the activity of the four peptidases were reconstituted. Dissolution of the liposomes with Triton X-100 did not increase the activity of any of these peptidases, a result consistent with an asymmetric, 'right-side-out', orientation of these enzymes. When purified, endopeptidase was subjected to the same procedure; the two amphipathic forms of the enzyme (the detergent form and the trypsin-treated detergent form) were fully reconstituted. The amphiphilic form, purified after toluene/trypsin treatment, failed to reconstitute. Electron microscopy of microvilli showed that the appearance of the surface particles was profoundly altered by treatment with papain. Before treatment, the microvilli were coated with particles of stalk lengths ranging from 2.5 to 9 nm. After papain treatment nearly all the particles had stalks of 2-3 nm. Reconstituted microvillar proteins in liposomes showed the same heterogeneity of stalk length. In contrast, liposomes containing reconstituted endopeptidase revealed a very homogeneous population of particles of stalk length 2 nm. Since the smallest dimension of a papain molecule is 3.7 nm, the ability of papain, and other proteinases of similar molecular size, to release microvillar enzymes is crucially affected by the length of the junctional peptide that constitutes the stalk of this type of membrane protein.
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PMID:Proteins of the kidney microvillar membrane. Reconstitution of endopeptidase in liposomes shows that it is a short-stalked protein. 634 16

The unknown enzymatic mechanism of enhanced protein breakdown in steroid myopathy was studied in functionally and biochemically different muscles of rabbits treated with dexamethasone for three weeks. After glucocorticoid administration the fast-twitch glycolytic semimembraneous muscle of treated animals was atrophied, whereas the weight of the slow-twitch oxidative soleus muscle was not altered. The specific activity of the lysosomal endo- and exopeptidases (cathepsin D, E, B and L, lysosomal carboxypeptidase A and dipeptidylpeptidase I) was increased about 2-fold in the atrophied white muscle. The activity of the cytosol enzyme Ca++-activated neutral proteinase was also elevated, whereas that of the other cytosol endopeptidase, chymotrypsin-like enzyme, was unaltered. The level of alanine aminopeptidase was only slightly increased. On the other hand, there were no unequivocal changes in protease activity in the soleus muscle. These findings are in agreement with the known differences in glucocorticoid-sensitivity of the various muscles. Our results suggest that the lysosomal proteolytic system and the Ca++-activated neutral proteinase may play an important role in the glucocorticoid-induced intracellular protein catabolism in muscle. The inhibitor capacities of cathepsin B and trypsin detectable in muscle cytosol were not altered after steroid treatment. Consequently, the increase in cathepsin B activity was not due to the loss of its inhibitor.
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PMID:Proteases and proteinase inhibitors in experimental glucocorticosteroid myopathy. 676 81

Membrane-bound arylamidases (EC 3.4.11.2) from human placenta and kidney were purified. The enzymes were solubilized from membrane fractions using trypsin. Purification procedures included DEAE-cellulose column chromatography, concanavalin-A--Sepharose 4B column chromatography, hydroxyapatite column chromatography and Sephadex G-200 gel filtration. The final recoveries of placental and kidney enzymes were 22.0% and 20.8% respectively. Polyacrylamide gel electrophoresis, analytical isoelectric focusing, immunoelectrophoresis and ultracentrifugation indicated the homogeneity of both purified enzymes. Equilibrium ultracentrifugation showed molecular weights of 193000 for placental enzyme and 211000 for kidney enzyme. Electrophoresis of the enzymes under denaturing conditions on dodecylsulfate/polyacrylamide gel indicated that each enzyme was a dimer consisting of two identical subunits. Isoelectric points of placental and kidney enzymes were 4.20 and 4.32 at 4 degrees C, respectively. The amino acid compositions of the two membrane-bound arylamidases were similar. The carbohydrate accounted for 18.2% of placental enzyme and 17.4% of kidney enzyme. The purified placental enzyme had a specific activity of 60.1 mumol min-1 (mg protein)-1 and the kidney enzyme had a specific activity of 96.4 mumol min-1 (mg protein)-1. Kcat values of placental and kidney enzymes were 187.8 s-1 and 328.6 s-1, respectively. The hydrolytic coefficient (Kcat/Km) of placental enzyme for L-alanyl-beta-naphthylamide was 2159 mM-1 s-1 and that of kidney enzyme was 3778 mM-1 s-1. Rabbit antibodies against placental and kidney membrane-bound arylamidases inhibited the activities of the corresponding antigens, but the inhibitions were never complete. Three membrane-bound arylamidases from placenta, kidney and renal cell carcinoma were immunochemically indistinguishable.
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PMID:Isolation and characterization of membrane-bound arylamidases from human placenta and kidney. 676 48

Aminopeptidase A (aspartate aminopeptidase, EC 3.4.11.7) was purified 2000-fold from pig kidney cortex. The essential step in the purification was chromatography on an immunoadsorbent column prepared from a rabbit antiserum raised against pig intestinal aminopeptidase A. Glutamyl and aspartyl substrate were attacked most rapidly and their hydrolyses were stimulated by Ca2+. The 2-naphthylamide derivatives of neutral and basic amino acids were also hydrolysed by aminopeptidase A, but at rates about two orders of magnitude lower, and Ca2+ was inhibitory. The possibility that these atypical substrates were hydrolysed by traces of aminopeptidase M (EC 3.4.11.2) contaminating the preparation could be excluded on several grounds. Aminopeptidase A was sensitive to inhibition by chelating agents and the inactive enzyme could be reactivated by Ca2+ or Mn2+. Atomic absorption spectrophotometry revealed 1 g-atom of Ca/143000 g of protein. Two forms of the enzyme were purified: an amphipathic form solubilized from the membrane by Triton X-100 (detergent form) and a hydrophilic form released by incubation with trypsin (proteinase form). The detergent form exhibited charge-shift in crossed immunoelectrophoresis when anionic or cationic detergents were present. On gel filtration, mol.wts. of 350000--400000 and 270000 were calculated for the detergent and proteinase forms. Electron microscopy after negative staining of the proteinase form revealed a dimeric structure. Electrophoresis of either form in the presence of sodium dodecyl sulphate revealed four polypeptides with mobilities corresponding to apparent mol.wts. of 155000, 110000, 90000 and 45000. All four bands stained positively for carbohydrate. Pig serum possesses weak aminopeptidase A activity; immunological experiments showed it to be a similar protein.
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PMID:Proteins of the kidney microvillar membrane. Aspartate aminopeptidase: purification by immunoadsorbent chromatography and properties of the detergent- and proteinase-solubilized forms. 701 18


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