Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A glycoprotein having a subunit weight of approximately 60,000 was isolated from rabbit liver microsomes. It is a predominant component of the hepatic microsomal membrane and reacts rapidly with diisopropylphosphorofluoridate (DFP), resulting in the loss of enzymatic activity toward artificial substrates such as acyl esters of o-nitrophenols. Automated Edman degradation of this protein together with sequence analysis of peptides provided the NH2-terminal sequence of some 70 residues as follows: His-Pro-Ser- Ala-Pro-Pro-Val-Val-Asp-Thr-Val-Lys-Gly-Lys-Val- Leu-Gly-Lys-Phe-Val-Ser-Leu-Glu-Gly-Phe-Ala-Gln- Pro-Val-Ala-Val-Phe-Leu-Gly-Val-Pro-Phe-Ala-Lys- Pro-Pro-Leu-Gly-Ser-Leu-Arg-Phe-Ala-Pro-Pro-Gln- Pro-Ala-Glu-Ser-Trp-Ser-His-Val-Lys-Asn (CHO)- Thr-Thr-Ser-Tyr-Pro-Pro-Met-Cys-Ser-Ser. A carbohydrate attachment was identified at asparaginyl residue 61. The COOH-terminal peptide of the protein was isolated from two independent enzymatic digests, and its sequence was established as Arg-Glu-Thr-Glu-His-Ile-Glu-Leu. In order to isolate the DFP binding peptide, liver microsomes were labeled with [3H]DFP and the 60-kDa protein containing covalently bound DFP isolated in pure form. Following reduction and carboxymethylation, the DFP-labeled protein was fragmented with trypsin and the digest subjected to gel filtration. Digestion of the labeled peptide preparations with chymotrypsin followed by chromatography of the digest yielded two diisopropylphosphoryl (DIP) peptides. Automated Edman degradation of these peptides provided the following amino acid sequences: Gly-Glu-DIPSer- Ala-Gly-Gly-Gln-Ser-Val-Ser-Ile-Leu-Leu-Leu-Ser- Pro and Thr-Val-Ile-Gly-Asp-DIPHis-Gly-Asp-Glu-Ile-Phe. The active site serine peptide of the 60-kDa protein shows some 70% similarity to the active center region of choline esterases. While the postulated active histidyl residue in choline esterases has not been identified, it is proposed that the DFP binding histidine of the 60-kDa protein corresponds to His-438/440 of choline esterases.
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PMID:Isolation and characterization of a 60-kilodalton glycoprotein esterase from liver microsomal membranes. 366 34

A proteolytic activity associated with the microsomal fraction of L-5178Y/Esb tumor cells has been characterized. The enzyme has a molecular weight of 80-90 kD as determined by affinity-labelling with [3H]DFP and SDS-gel electrophoresis. It cleaves ester substrates at the carboxyl position of lysine and arginine and can activate the proenzyme plasminogen. The enzyme is found to be associated with the plasma membranes of high and low metastatic tumor cell lines and is shed in high-molecular-weight form mainly by the high metastatic variant. The pH optimum for esterase and protease activities was 7.5-8.5. Although similar to trypsin in substrate specificity, the enzyme was not inhibited by lima-bean trypsin inhibitor but was inhibited by DFP, PMSF, aprotinin and leupeptin. Partially purified preparations of the protease can alone degrade 125I-labelled endothelial cell extracellular matrix, pointing at the putative role of this enzyme in tumor invasion.
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PMID:Characterization of an extracellular matrix-degrading protease derived from a highly metastatic tumor cell line. 389 58

Activation of purified urinary inactive kallikrein by an extract from the rat kidney cortex was investigated. The extract produced a dose-dependent activation of the inactive kallikrein and the optimum pH for this activation was 5.0. Marked depression of the activation was observed when the extract was pre-incubated with E-64, p-CMB and iodoacetate, but not with DFP, PMSF or pepstatin A. The molecular weight of the inactive kallikrein (Mr 44,000) was reduced to 38,000 by treatment with the extract, this molecular weight value being identical with that of urinary active kallikrein. These results indicate that the rat kidney cortex contains a protease catalyzing conversion of urinary inactive kallikrein into its active form, and that the protease has properties compatible with those of a thiol protease, but not of trypsin which has been used as a tool for the activation of urinary inactive kallikrein. The thiol protease is probably one of regulators of the kallikrein-kinin system in the kidney.
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PMID:Activation of urinary inactive kallikrein by an extract from the rat kidney cortex. 389 54

A biologically active neutral peptide mediator is cleaved from a plasma protein substrate by an alpha-1-antitrypsin-inhibitable serine protease apparently residing on the membrane of the human neutrophil. The peptide mediator has an approximate mol wt of 1,000, and is distinguished from the kinin peptides by a neutral isoelectric point, susceptibility to inactivation by trypsin as well as chymotrypsin and activity on the isolated, atropinized, and antihistamine-treated guinea pig ileum with relatively little action on the estrous rat uterus. The neutrophil protease is fully inhibitable by DFP, trypsin inhibitors from lima or soy bean, and alpha-1-antitrypsin and is associated with the high mol wt fragments of the neutrophil and not the nuclear, lysosomal, or cytoplasmic subcellular fraction. The substrate has an approximate mol wt of 90,000 and is chromatographically separable from kininogen. The exquisite sensitivity of the neutrophil protease to alpha-1-antitrypsin was established both by inhibition with highly purified alpha-1-antitrypsin and by the inability of the protease to generate detectable neutral peptide in a homozygous (ZZ) alpha-1-antitrypsin-deficient patient without heat inactivation of the residual inhibitor. On the other hand, plasma from a (null) alpha-1-antitrypsin-deficient patient supported neutral peptide generation and revealed an additional factor which inactivated neutral peptide.
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PMID:A neutrophil-dependent pathway for the generation of a neutral peptide mediator: partial characterization of components and control by alpha-1-antitrypsin. 454 25

Unactivated partial thromboplastin antecedent (PTA) has been purified by sequential chromatography of plasma on quaternary aminoethyl Sephadex, sulphoprophyl Sephadex, Sephadex G-150, and passage over an anti-IgG immunoadsorbant. The preparation gave a single band after alkaline disc gel electrophoresis, sodium dodecyl sulfate (SDS) gel electrophoresis and isoelectric focusing in acrylamide gels and was found to have a mol wt of 175,000 by gel filtration, 163,000 by SDS gel electrophoresis, and an isoelectric point of 8.8-9.4 (peak 9.0-9.1). Pre-PTA was activated directly by activated Hageman factor or by Hageman factor prealbumin fragments. Its coagulant activity was inhibited by DFP, soybean trypsin inhibitor and trasylol but not by lima bean trypsin inhibitor or ovomucoid trypsin inhibitor indicating that activated PTA possesses the same inhibition profile utilizing these reagents as does plasma kallikrein. A major plasma inhibitor of activated PTA was found to be a 65,000 mol wt alpha-globulin which was isolated free of alpha(1)-chymotrypsin inhibitor, inter alpha-trypsin inhibitor, alpha(2)-macroglobulin, and the other known inhibitors of activated PTA, the activated first component of complement (C1 INH), and antithrombin III. Its physicochemical properties were identical to alpha(1)-antitrypsin, and it was absent in alpha(1)-antitrypsin-deficient plasma thereby identifying this PTA inhibitor as alpha(1)-antitrypsin.
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PMID:Substrates of Hageman factor. I. Isolation and characterization of human factor XI (PTA) and inhibition of the activated enzyme by alpha 1-antitrypsin. 454 83

The activity of properdin factor D was measured by the generation of the hemolytically active cellular intermediate, EAC43B(D), bearing the C3b-dependent alternate pathway C3 convertase. Treatment of factor D with DFP prevented formation of EAC43B(D); thus, a serine esterase is essential for the generation of the alternate pathway C3 convertase, a situation analogous to the role of C1 in the formation of the classical C3 convertase, C42. The definition of factor D as a serine esterase prompted a search for its proenzyme form, and resulted in the chromatographic isolation from plasma of a single peak of trypsin-inducible factor D activity, distinct from activated factor D. Analytical gel filtration indicated an apparent mol wt of 25,000. This protein from which trypsin elaborated factor D activity, as assessed by the formation of EAC43B(D), the generation of the CoVF-dependent C3 convertase, and the cleavage of factor B in the presence of C3b, was designated "precursor factor D." The DFP resistance of precursor factor D, and the susceptibility of its trypsin-activated form to inactivation by DFP is analogous to the behavior of other plasma serine esterases, including C1.
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PMID:Properdin factor D: characterization of its active site and isolation of the precursor form. 485 53

An insoluble fibrinolytic enzyme with a molecular weight of approximately 30,000, was purified from the human spleen. A single protein band possessing fibrinolytic activity was obtained on polyacrylamide gel disk electrophoresis at pH 4.5. The enzyme, tentatively termed spleen fibrinolytic proteinase (SFP), degraded fibrinogen at neutral pH following Michaelis-Menten kinetics. The fibrinogenolytic activity was not inhibited by t-AMCHA, a specific plasmin inhibitor. SFP barely degraded certain synthetic ester or polypeptide substrates for trypsin, chymotrypsin, plasma, Xa, elastase and collagenase. These results indicate a different nature for SFP compared to other enzymes examined. SFP was found to digest no elastin and its fibrinogenolytic activity was strongly inhibited by STI, indicating that it was not an elastase. SFP required neither Zn++ nor CA++ for its fibrinogenolytic activity, indicating that it differed from metal-dependent proteinases such as collagenase. SFP was inhibited by DFP but not by TLCK, suggesting that it contains an active serine residue, but no trypsin type histidine at its active center. These results appear to show that SFP is a unique proteinase in the spleen, which is capable of degrading fibrin and fibrinogen at neutral pH.
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PMID:Human spleen insoluble fibrinolytic proteinase acting at neutral pH: its partial purification and characterization. 626 69

Succinyltrialanine p-nitroanilide(STANA)-hydrolytic enzyme was purified 5,200-fold from porcine liver soluble fraction with a yield of 75% by ammonium sulfate fractionation and chromatographies on DEAE-Sephacel, Sephadex G-150, and hydroxylapatite columns. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate (SDS). The pI of the enzyme was 4.9 by dis gel electrofocusing and the molecular weight was calculated to be 72,000 by gel filtration on a Sephadex G-150 column and 74,000 by SDS-polyacrylamide gel electrophoresis. Acidic amino acids amounted to 17.2% of the total amino acid residues, and the basic ones, 12.9%. No hexosamine was detected. The STANA-hydrolytic enzyme showed maximal activity at pH 7.4 against succinyltrialanine p-nitroanilide and at pH 6.5 against succinyl-Gly-Pro-4-methylcoumaryl 7-amide (MCA), and was stable between pH 6 and 7 in the presence of dithiothreitol. This enzyme hydrolyzed succinyl-Gly-Pro-Leu-Gly-Pro-MCA, succinyl-Gly-Pro-MCA, succinyl-Ala-Pro-Ala-MCA, and several proline-containing natural peptides in addition to succinyltrialanine p-nitroanilide, but was unable to hydrolyze the substrates of aminopeptidases, dipeptidylaminopeptidase IV, trypsin, and chymotrypsin. Elastatinal and chymostatin were effective inhibitors and their IC50 values were 8.7 micrograms/ml and 18.2 micrograms/ml, respectively. The enzyme was completely inhibited by 10(-7) M p-chloromercuribenzoic acid (pCMB), 10(-7) M p-chloromercuriphenylsulfonic acid (pCMPS), and 10(-4) M diisopropyl phosphofluoridate (DFP), but not by 1 mM E-64, which is known as an inhibitor specific to thiol proteinase. The enzyme was easily inactivated by agitation in a Vortex mixer, and its activity was recovered by the addition of thiol compounds such as dithiothreitol, 2-mercaptoethanol and cysteine. The effects of inhibitors and thiol compounds were substantially identical when the enzyme activity was measured with either succinyltrialanine p-nitroanilide or succinyl-Gly-Pro-MCA as a substrate. These results indicate that the STANA-hydrolytic enzyme in the liver soluble fraction is a post-proline cleaving enzyme [EC 3.4.21.26].
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PMID:Porcine liver succinyltrialanine p-nitroanilide hydrolytic enzyme. Its purification and characterization as a post-proline cleaving enzyme. 636 Oct 13

A clotting enzyme, associated with the endotoxin-mediated activation of the cellularly based coagulation system of the American horseshoe crab (Limulus polyphemus), was considerably purified by a modification of the method employed to purify the corresponding enzyme from the Japanese horseshoe crab (Tachypleus tridentatus) (Nakamura, et al., 1982). This enzyme was inhibited by DFP, benzamidine, p-aminobenzamidine, antithrombin III, soybean trypsin inhibitor, and antipain, suggesting that it is a trypsin-type serine protease. The enzyme demonstrated amidolytic activity to Ac-Ile-Glu-Gly-Arg-pNA (S-2423) and related synthetic substrates (S-2222, S-2422, S-2337, and Boc-Leu-Gly-Arg-pNA) but not to other substrates (S-2160, S-2238, S-2251, S-2444, S-2266, and S-2302), indicating specificity similar to mammalian blood coagulation Factor Xa. These properties of the Limulus enzyme were identical with those of the corresponding Tachypleus enzyme. The structure and function of the enzymes in these two species probably have been highly conserved during the past few hundred million years of their evolution.
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PMID:Properties of the clotting enzyme responsible for endotoxin-mediated Limulus coagulation. 637 72

Rabbit prekallikrein (RPK) was purified from rabbit plasma by ion exchange and lectin column chromatography and preparative polyacrylamide gel electrophoresis. A 1500-fold purification was routinely achieved with a final yield of 5-10%. The purified RPK was found to be a glycoprotein with an apparent molecular weight of 88,000. Activation of RPK with either trypsin or rabbit Hageman factor (active) occurs by limited proteolytic cleavage, producing two disulfide-linked polypeptide chains with molecular weights of 55,000 and 35,000. Both chains contain carbohyrate and the 35,000-molecular-weight polypeptide was shown to incorporate [3H]DFP. Activation of RPK in kaolin-treated plasma was shown to proceed by an analogous mechanism yielding 55,000- and 35,000-molecular-weight polypeptide chains.
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PMID:Rabbit prekallikrein. Purification, biochemical characterization, and mechanism of activation. 690 16


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