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)

Carboxypeptidase M, a plasma membrane-bound enzyme, is present in many human organs and differs from other carboxypeptidase that cleave basic COOH-terminal amino acids. Cultured Madin-Darby canine kidney (MDCK) distal tubular cells contain a kininase I-type enzyme that inactivates bradykinin by releasing Arg9. We found the properties of this kininase to be identical with carboxypeptidase M. In fractionated cells, carboxypeptidase activity sediments with membranes; and detergents, trypsin, and phosphatidylinositol-specific phospholipase C solubilize it, similar to results with human placental carboxypeptidase M. Ten microM 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid and 1 mM o-phenanthroline inhibit, whereas 1.0 mM CoCl2 activates the enzyme. It has a neutral pH optimum and cleaves COOH-terminal Arg or Lys in bradykinin and in shorter peptides. The relative hydrolysis rates of peptides in the presence or absence of 1 mM CoCl2 were similar to those obtained with human carboxypeptidase M. The carboxypeptidase in MDCK cells (54 kDa) cross-reacts with antibodies to human carboxypeptidase M in Western blotting, but not with antibodies to plasma carboxypeptidase N. The enzyme is a glycoprotein; chemical deglycosylation reduced the size to 48 kDa. The presence of the enzyme on the cell membrane of MDCK cells was also shown with transmission electron microscopy using immunogold, which indicated that the enzyme is on the apical side. In addition, MDCK cells contain neutral endopeptidase 24.11 (enkephalinase) and prolylcarboxypeptidase (angiotensinase C) activities. Partitioning of solubilized carboxypeptidase M into Triton X-114 and water indicates that trypsin and phospholipase C remove a hydrophobic tail, while detergent solubilization leaves the hydrophobic moiety intact. Labeling of MDCK cells with [3H]ethanolamine resulted in the synthesis of radiolabeled carboxypeptidase M as determined by immunoprecipitation and fluorography. Thus, MDCK cells contain membrane-bound carboxypeptidase M, which is anchored to the plasma membrane via phosphatidylinositol-glycan. As a major kininase of the distal tubules, it may regulate salt and water excretion.
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PMID:Carboxypeptidase M in Madin-Darby canine kidney cells. Evidence that carboxypeptidase M has a phosphatidylinositol glycan anchor. 239 13

Actin purified from the yeast (Saccharomyces cerevisae) was polymerized faster than rabbit skeletal alpha-actin by MgCl2. The two actins polymerized at similar rates in the presence of CaCl2. Yeast actin, up to 25 microM, was not polymerized by KCl (100-300 mM); the monovalent salt also inhibited the MgCl2-induced polymerization of actin. The local structure of the subdomain-2 region in yeast actin filaments was probed by subtilisin and trypsin digestions. Loop 38-52 appeared more flexible and accessible to subtilisin in yeast than in rabbit actin. In contrast, tryptic digestions at Lys-61 and -68 occurred at the same rate for yeast and alpha-actin filaments. Modification of yeast actin by a sulfhydryl reagent CPM [7-(diethylamino)-3-(4'-maleimidophenyl)-4-methylcoumain] was specific to the Cys-374 residue; no labeling of a yeast actin mutant containing an alanine substitution for cysteine 374 was observed. The rates of Cys-374 labeling by CPM were similar for yeast and muscle actin, suggesting a similar environment for the C terminus in both polymers. In the in vitro motility assays, yeast actin required higher concentrations of heavy meromyosin (HMM) for its sliding than did the rabbit actin. At saturating concentrations of HMM, the sliding velocities of both actins were the same (3.0 microns/s). Relative forces generated by HMM with yeast and muscle actin were assessed by monitoring their in vitro motility in the presence of NEM-HMM load. The sliding of yeast actin was stopped at a level of external load (molar ratio NEM-HMM/HMM = 0.25) lower than that of muscle actin (NEM-HMM/HMM = 0.43), suggesting lower force production with yeast actin. These results are discussed in terms of the myosin cross-bridge cycle and actomyosin interactions.
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PMID:Polymerization and in vitro motility properties of yeast actin: a comparison with rabbit skeletal alpha-actin. 898 91

The carboxypeptidase activity occurring in hog intestinal mucosa is apparently due to two distinct enzymes which may be responsible for the release of basic COOH-terminal amino acids from short peptides. The plasma membrane-bound carboxypeptidase activity which occurs at neutral optimum pH levels was found to be enhanced by CoCl(2) and inhibited by guanidinoethylmercaptosuccinic acid, o-phenanthroline, ethylenediamine tetraacetic acid and cadmium acetate; whereas the soluble carboxypeptidase activity which occurs at an optimum pH level of 5.0 was not activated by CoCl(2) and only slightly inhibited by o-phenanthroline, ethylenediamine tetraacetic acid, NiCl(2) and CdCl(2). The latter activity was presumably due to lysosomal cathepsin B, which is known to be present in the soluble fraction of hog intestinal mucosa. Although the membrane-bound enzyme was evenly distributed along the small intestine, it was not anchored in the phospholipidic bilayer via a glycosyl-phosphatidylinositol moiety, as carboxypeptidase M from human placenta is. The enzyme was not solubilized by phosphatidylinositol-specific phospholipase C, but was solubilized to practically the same extent by several detergents. The purified trypsin-solubilized form is a glycoprotein with a molecular mass of 200 kDa, as determined by performing SDS-PAGE and gel filtration, which differs considerably from the molecular mass of human placental carboxypeptidase M (62 kDa). It was found to cleave lysyl bonds more rapidly than arginyl bonds, which is not so in the case of carboxypeptidase M, and immunoblotting analysis provided further evidence that hog intestinal and human placental membrane-bound carboxypeptidases do not bear much resemblance to each other. Since the latter enzyme has been called carboxypeptidase M, it is suggested that the former might be carboxypeptidase D, the recently described new member of the carboxypeptide B-type family.
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PMID:The membrane-bound basic carboxypeptidase from hog intestinal mucosa(1). 1051 94

The cystic fibrosis transmembrane conductance regulator (CFTR) is known to function as a regulated chloride channel and, when genetically impaired, to cause the disease cystic fibrosis. The novel studies reported here were undertaken to gain greater molecular insight into possible interactions among CFTR's soluble domains, which include two nucleotide binding domains (NBF1 and NBF2) and a regulatory domain (R). The NBF1+R and NBF2 regions of CFTR were highly expressed in Escherichia coli, purified to near homogeneity under denaturing conditions, and refolded. Both refolded proteins bound TNP-ATP and TNP-ADP, which could be readily replaced with ATP. Four different approaches were then used to determine whether the NBF1+R and NBF2 proteins interact. First, the purified NBF2 protein was labeled near its C-terminus with a fluorescent probe, 7-diethyl amino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM). Addition of the unlabeled NBF1+R to the CPM-labeled NBF2 caused a red-shift in lambda(max) of the CPM fluorescence, consistent with a direct interaction between the two proteins. Second, when the NBF1+R protein, the NBF2 protein, and a mixture of the two proteins were folded separately and analyzed by molecular sieve chomatography, the mixture was found to elute prior to either NBF1+R or NBF2. Third, na-tive-PAGE gel studies revealed that the mixture of the NBF1+R and NBF2 domains migrated as a single band with an R(F) value between that of NBF1+R and NBF2. Fourth, trypsin digestion of a mixture of the NBF1+R and NBF2 proteins occurred at a slower rate than that for the individual proteins. Finally, studies were carried out to determine whether an NBF1+R/NBF2 interaction could be demonstrated after expressing one of the two proteins in soluble, native form, thus avoiding the inclusion body, denaturation, and renaturation approach. Specifically, the NBF1+R protein was overexpressed in E. coli in fusion with glutathione-S-transferase near a thrombin cleavage site. Following binding of the GST-(NBF1+R) fusion protein to a GST Sepharose affinity column, added NBF2 was shown to bind and then to coelute with NBF1+R upon addition of glutathione or thrombin. Collectively, these experiments demonstrate that CFTR's NBF1+R region and its NBF2 domain, after folding separately as distinct units, have a strong propensity to interact and that this interaction is stable in the absence of added nucleotides or exogenously induced phosphorylation. These findings, together with the additional observation that the NBF1+R/NBF2 interaction induces a change in the C-terminus of NBF2, which resides within the C-terminal region of CFTR, may have important implications not only for the function of CFTR per se, but its interaction with other proteins.
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PMID:Cystic fibrosis transmembrane conductance regulator: the purified NBF1+R protein interacts with the purified NBF2 domain to form a stable NBF1+R/NBF2 complex while inducing a conformational change transmitted to the C-terminal region. 1068 44

Bradykinin (BK) and kallidin (Lys-BK), liberated from kininogens by kallikreins, are ligands of the BK B(2) receptor. We investigated whether kallikreins, besides releasing peptide agonist, could also activate the receptor directly. We studied the effect of porcine and human recombinant tissue kallikrein and plasma kallikrein on [Ca(2+)](i) mobilization and [(3)H]arachidonic acid release from cultured cells stably transfected to express human BK B(2) receptor (CHO/B(2), MDCK/B(2), HEK/B(2)), and endothelial cells were used as control cells. As with BK, the actions of kallikrein were blocked by the B(2) antagonist, HOE 140. Kallikrein was inactive on cells lacking B(2) receptor. Kallikrein and BK desensitized the receptor homologously but there was no cross-desensitization. Furthermore, 50 nM human cathepsin G and 50 nM trypsin also activated the receptor; this also was blocked by HOE 140. Experiments excluded a putative kinin release by proteases. [(3)H]AA release by BK was reduced by 40% by added kininase I (carboxypeptidase M); however, receptor activation by tissue kallikrein, trypsin, or cathepsin G was not affected. Prokallikrein and inhibited kallikrein were inactive, suggesting cleavage of a peptide bond in the receptor. Kallikreins were active on mutated B(2) receptor missing the 19 N-terminal amino acids, suggesting a type of activation different from that of thrombin receptor. Paradoxically, tissue kallikreins decreased the [(3)H]BK binding to the receptor with a low K(D) (3 nM) and inhibited it 78%. Thus, kallikreins and some other proteases activate human BK B(2) receptor directly, independent of BK release. The BK B(2) receptor may belong to a new group of serine protease-activated receptors.
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PMID:Human bradykinin B(2) receptor is activated by kallikrein and other serine proteases. 1099 54

Human carboxypeptidase (CP) M was expressed in baculovirus-infected insect cells in a glycosylphosphatidylinositol-anchored form, whereas a truncated form, lacking the putative signal sequence for glycosylphosphatidylinositol anchoring, was secreted at high levels into the medium. Both forms had lower molecular masses (50 kDa) than native placental CPM (62 kDa), indicating minimal glycosylation. The predicted glycosylphosphatidylinositol-anchor attachment site was investigated by mutation of Ser(406) to Ala, Thr or Pro and expression in HEK-293 and COS-7 cells. The wild-type and S406A and S406T mutants were expressed on the plasma membrane in glycosylphosphatidylinositol-anchored form, but the S406P mutant was not and was retained in a perinuclear location. The roles of Glu(260) and Glu(264) in CPM were investigated by site-directed mutagenesis. Mutation of Glu(260) to Gln had minimal effects on kinetic parameters, but decreased heat stability, whereas mutation to Ala reduced the k(cat)/ K(m) by 104-fold and further decreased stability. In contrast, mutation of Glu(264) to Gln resulted in a 10000-fold decrease in activity, but the enzyme still bound to p-aminobenzoylarginine-Sepharose and was resistant to trypsin treatment, indicating that the protein was folded properly. These results show that Glu(264) is the critical catalytic glutamic acid and that Glu(260) probably stabilizes the conformation of the active site.
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PMID:Effect of mutation of two critical glutamic acid residues on the activity and stability of human carboxypeptidase M and characterization of its signal for glycosylphosphatidylinositol anchoring. 1245 62

Phosphorylated kininogen and some of its fragments containing serine phosphorylated bradykinin ([pS(6)]-Bk) were identified in human serum and plasma by a phosphoproteomic approach. We report the kininogenase ability of human tissue and plasma kallikreins and tryptase to generate [pS(6)]-Bk or Lys-[pS(6)]-Bk having as substrate the synthetic human kininogen fluorescent fragment Abz-MISLMKRPPGF[pS(386)]PFRSSRI-NH2. The pharmacological assays of [pS(6)]-Bk showed it as a full B2 bradykinin receptor agonist in smooth muscle, it produces a portal liver hypertensive response in rat and mouse paw edema that lasts longer than Bk. The rat hypotensive response to infusions of Bk is greater than that of [pS(6)]Bk, both if injected through femoral vein or aorta. [pS(6)]-Bk was more resistant than Bk to kininase digestion performed with angiotensin converting enzyme, neprilysin, thimet oligopeptidase, aminopeptidase P and carboxypeptidase M. (1)H-NMR experiments indicated that [pS(6)]-Bk has lower flexibility, with the pS(6)-P(7) bond restricted to the trans conformation, and can explain [pS(6)]-Bk resistance to hydrolysis. In conclusion, [pS(6)]-Bk presenting lower activity than Bk, with longer lasting effects and being slowly released by kininogenases from synthetic Abz-MISLMKRPPGF[pS(386)]PFRSSRI-NH2, suggests that phosphorylation of the kininogens can be an efficient kallikrein-kinin system regulator.
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PMID:Pharmacological Activities and Hydrolysis by Peptidases of [Phospho-Ser(6)]-Bradykinin (pS(6)-BK). 2623 42