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)

The synthesis of the protected duopentacontapeptide corresponding to the entire amino acid sequence I-52 of porcine pancreatic secretory trypsin inhibitor II (Kazal type) is described. The benzyloxycarbonyltetradecapeptide tert-butyloxycarbonylhydrazide (sequence 1-14) was selectively deblocked with trifluoroacetic acid and used to acylate, by the azide procedure, the peptide free base corresponding to the sequence 15-52. The isolated material was purified by ion exchange chromatography and the protecting groups were removed by successive treatments with anhydrous hydrogen fluoride, 1 M piperidine and mercuric acetate. F02M phosphate buffer, pH8. Determination of the inhibitory capacity indicated that the synthetic material is about 50% effective, at 30:1 inhibitor:trypsin molar ratio in inhibiting the tryptic hydrolysis of Nalpha-benzoyl-DL-arginine-4-nitroanilide. Full inhibition was achieved at a higher inhibitor:trypsin molar ratio. The stability constants and the standard free energy of binding of the complex between trypsin and the synthetic inhibitor have been determined.
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PMID:Studies on trypsin inhibitors. Part IX. Synthesis and trypsin inhibitory activity of the duopentacontapeptide corresponding to the amino acid sequence of porcine pancreatic secretory trypsin inhibitor II (Kazal). 4 25

All the reactive amino groups in soybean trypsin inhibitor (Kunitz) were protected by guanidination of 9 out of 10 lysyl residues with O-methylisourea and by carbamoylation of the NH2 terminal Asp with potassium cyanate. This derivative was converted to modified inhibitor (Arg63-Ile64 reactive site peptide bond hydrolyzed) by incubation with trypsin at pH 3. The NH2 terminal of Ile64 was allowed to react with phenyl isothiocyanate to produce inactive phenylthiocarbamoyl-modified inhibitor. Treatment with trifluoroacetic acid formed the anilinothiazolinone of Ile64 yielding des-Ile64-modified inhibitor. After renaturation and purification, this material coelectrophoresed with modified inhibitor but did not form a stable complex with trypsin. Incubation with tert-butyloxycarbonyl-(amino acid)-N-hydroxysuccinimide esters yielded [tert-butyloxycarbonyl-(amino acid64)]-modified inhibitor. The tert-butyloxycarbonyl protective group was removed in trifluoroacetic acid. After renaturation, active [amino acid64]-modified inhibitors were obtained for Ile64, Ala64, Leu64, and Gly64 replacements. The resynthesis of the reactive-site peptide bound by kinetic control dissociation of the trypsin-inhibitor complex yielded fully active [Ala64]-virgin inhibitor. Thus, soybean trypsin inhibitor (Kunitz) has been shown to tolerate the replacement of the P1' residue with retention of activity. The importance of P1' residues in the function of protein proteinase inhibitors is discussed.
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PMID:Chemical-enzymatic replacement of Ile64 in the reactive site of soybeen trypsin inhibitor (Kunitz). 125 49

Modified (Arg63-Ile64 reactive-site peptide bond hydrolyzed) soybean trypsin inhibitor (Kunitz) with all reactive amino groups, except that of Ile64, protected was described in the preceding paper (Kowalski, D., and Laskowski, M., Jr. (1976), Biochemistry, preceding paper in this issue). Treatment of this inhibitor with tert-butyloxycarbonyl-Ala- and tert-butyloxycarbonyl-Ile-N-hydroxy-succinimide esters yields inactive endo-tert-butyloxycarbonyl-Ala63A-and endo-tert-butyloxycarbonyl-Ile63A-modified inhibitors. The tert-butyloxycarbonyl groups were removed by treatment of the proteins with trifluoroacetic acid. After renaturation and purification, the resultant endo-Ala63A- and endo-Ile63A-modified inhibitors co-electrophorese with modified inhibitor both on disc gels (pH 9.4) and sodium dodecyl sulfate gels (after reduction of disulfide bonds) and show end groups corresponding to the 63A residue. These derivatives fail to form stable complexes with trypsin, extending the previous observation (Kowalski, D., and Laskowski, M., Jr. (1972), Biochemistry 11, 3451) that acylation of the P1' residue in modified inhibitors leads to inactivation. However, the incubation of endo-Ala63A- and endo-Ile63A-modified inhibitors with trypsin at pH 6.5 leads to the synthesis of the Arg63-Ala63A and Arg63-Ile63A peptide bonds in 4% yield. This is very close to the yield anticipated from a semiquantitative theory for the value of the equilibrium constant for reactive-site peptide bond. An alternative chemical method of insertion is also described. Controlled treatment of modified inhibitor with the N-carboxyanhydride of Glu produced inactive endo-Glu63A-modified inhibitor. Incubation of this inactive derivative with trypsin at pH 6.5 leads to 16% synthesis of the Arg63-Glu63A peptide bond. The higher yield of single chain protein in this case is attributed to the influence of the negative charge of the Glu63A side chain. Thus, the insertion of an amino acid residue between the P1 and P1' residues in soybean trypsin inhibitor (Kunitz) converts a trypsin inhibitor into a trypsin substrate.
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PMID:Chemical-enzymatic insertion of an amino acid residue in the reactive site of soybean trypsin inhibitor (Kunitz). 125 50

Reversed-phase liquid chromatography was used as an alternative method for the characterization of the precursor and activated forms of porcine and human pancreatic colipase. Using a Beckman Ultrasphere column with an increasing acetonitrile gradient in 0.1% trifluoroacetic acid, it was possible to obtain well-resolved separation of the precursor form of colipase (procolipase) from its trypsin-activated derivative. This protocol was used (1) to study the activation of porcine procolipase by trypsin or thrombin in vitro, (2) to assess the homogeneity of porcine colipase preparations used in tridimensional structure studies and in combination with immunoaffinity chromatography, (3) to identify the form of colipase present in samples of human pancreatic juice.
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PMID:Separation and characterization of the precursor and activated forms of porcine and human pancreatic colipase by reversed-phase liquid chromatography. 147 89

Human lung cancer cell line, T3M-30, has been shown to produce a growth factor that stimulates proliferation of peripheral blood monocytes. In the presence of this factor, human circulating monocytes were able to proliferate in vitro. Gel exclusion chromatography of the conditioned medium revealed a single peak of monocyte growth-promoting activity at an apparent molecular weight of 16,000. The growth-promoting activity was adsorbed to an anion-exchange column, Mono Q, and eluted with a salt gradient as a single peak of bioactivity at 300 mM NaCl. When the sample was applied to a Vydac C4 column, a reverse-phase high-performance liquid chromatography column, a single peak of activity was observed at a concentration of 76% acetonitrile in 0.1% trifluoroacetic acid. The monocyte growth-promoting activity was heat stable at 56 degrees C. It was partially destroyed by trypsin. The activity was lost after treatment with 2-mercaptoethanol.
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PMID:A human monocyte growth factor produced by lung cancer cells. 216 45

The affinity label 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-triphosphate (8-BDB-TA-5'-TP) reacts covalently with rabbit muscle pyruvate kinase, incorporating 2 mol of reagent/mol of enzyme subunit upon complete inactivation. Protection against inactivation is provided by phosphoenolpyruvate, K+, and Mn2+ and only 1 mol of reagent/mol of subunit is incorporated [DeCamp, D.L., Lim, S., & Colman, R.F. (1988) Biochemistry 27, 7651-7658]. We have now identified the resultant modified residues. After reaction with 8-BDB-TA-5'-TP at pH 7.0, modified enzyme was incubated with [3H]NaBH4 to reduce the carbonyl groups of enzyme-bound 8-BDB-TA-5'-TP and to introduce a radioactive tracer into the modified residues. Following carboxymethylation and digestion with trypsin, the radioactive peptides were separated on a phenylboronate agarose column followed by reverse-phase high-performance liquid chromatography in 0.1% trifluoroacetic acid with an acetonitrile gradient. Gas-phase sequencing gave the cysteine-modified peptides Asn162-Ile-Cys-Lys165 and Cys151-Asp-Glu-Asn-Ile-Leu-Trp-Leu-Asp-Tyr-Lys161, with a smaller amount of Asn43-Thr-Gly-Ile-Ile-Cys-Thr-Ile-Gly-Pro-Ala-Ser-Arg55. Reaction in the presence of the protectants phosphoenolpyruvate, K+, and Mn2+ yielded Asn-Ile-Cys-Lys as the only labeled peptide, indicating that inactivation is caused by modification of Cys151 and Cys48.
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PMID:Cysteinyl peptides of rabbit muscle pyruvate kinase labeled by the affinity label 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-triphosphate. 233 78

Exocrine proteins contained in human pancreatic juice were analyzed by reversed-phase high performance liquid chromatography (RP-HPLC). Pancreatic juice was saved by endoscopic retrograde cannulation of the main pancreatic duct in 17 persons: 12 without pancreatic disease, 3 patients suffering from recurrent acute pancreatitis probably due to pancreas divisum, 1 patient with a carcinoma of the pancreas, and 1 patient with chronic calcified pancreatitis. The juice proteins were separated on a silica column (Nucleosil 300-7 RP) by use of a multistep acetonitrile/water gradient (+0.1% trifluoroacetic acid). Up to 18 individual peaks could be separated by one analytical run (60 min). Molecular weight analysis by sodium dodecyl sulfate-gel electrophoresis indicated the presence of enzymes such as amylase, prophospholipase A2, procarboxypeptidases, trypsinogens, and chymotrypsinogens in certain peaks. Small residual enzymatic activities correlating with certain peaks were detected for amylase and chymotrypsin, and high residual activities were found for phospholipase A (recovery of enzymatic activity compared with the original sample amounted to 65%). Significant amounts of cathodic trypsin-like immunoreactivity were found in two certain peaks. By always loading 350 micrograms of protein/injection on the column the profiles of various samples showed similar patterns. Repeated injections of aliquots revealed highly reproducible profiles. RP-HPLC offers precise, reproducible, and rapid separation of the major proteins of human pancreatic juice.
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PMID:Resolution of human exocrine pancreatic juice proteins by reversed-phase high performance liquid chromatography (HPLC). 234 40

Two high-resolution tryptic maps have been developed for recombinant tissue plasminogen activator (rt-PA) that separate the expected 51 tryptic peptides. The trypsin digestion was performed after reduction and S-carboxymethylation of the protein. The high-performance liquid chromatographic separation of the tryptic peptides used a Nova-Pak C18 (5 microns) column with a mobile phase that contained 0.1% aqueous trifluoroacetic acid (TFA) or 50 mM sodium phosphate (pH 2.85) and a linear gradient of acetonitrile. A TFA solvent system was also used for re-purification and for characterization of the peptides isolated from the phosphate-based separation. All of the isolated peptides had compositions consistent with the sequence proposed for rt-PA. The identities of the glycopeptides were confirmed by lectin chromatography on concanavalin A-Sepharose. The mixture of tryptic peptides was also treated with endo-beta-N-acetylglucosaminidase H and peptide:N-glycosidase F to locate the position of either high mannose or complex oligosaccharides. These studies demonstrated that a high mannose oligosaccharide is attached to Asn-117 while complex carbohydrate side-chains are attached to Asn-184 and Asn-448. The residue Asn-184 is the site of optional glycosylation that results in the formation of two rt-PA variants that contain either two or three oligosaccharides.
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PMID:Study of the primary structure of recombinant tissue plasminogen activator by reversed-phase high-performance liquid chromatographic tryptic mapping. 249 37

Incubation of eosinophils (EOS) with alveolar macrophage (AM) supernatants isolated from asthmatic subjects followed by stimulation with the calcium ionophore A23187 resulted in enhancement of the capacity of EOS to elaborate leukotriene C4 (LTC4) (mean enhancement 169 +/- 37%, n = 31). Pretreatment of EOS with AM supernatants derived from normal individuals did not enhance LTC4 generation as compared with control medium. Enhancement was maximal when EOS were preincubated with a 1:6 dilution of AM supernatants for 5 min at 37 degrees C and were then stimulated with 5 microM A23187 for 15 min. Separation of AM supernatants by size-exclusion HPLC using a TSK G3000 SW column resulted in a peak of enhancing activity with an estimated molecular mass of approximately 30,000 D. Further purification by anion exchange HPLC using a TSK DEAE 5PW column (pH 7.4) resolved the activity into a minor peak at 0.17 M NaCl and a major peak at 0.2 M NaCl. The activities were distinct from interleukin-1 and tumor necrosis factor. Resolution of the major peak of activity by reverse-phase HPLC using a C18 spherisorb ODS column and a slope gradient of 0 to 100% acetonitrile/0.1% trifluoroacetic acid demonstrated a single peak of activity that eluted at 41% acetonitrile. The enhancing activity was sensitive to trypsin and heat and was neutralized by a specific antibody to granulocyte-macrophage colony-stimulating factor (GM-CSF). Pretreatment of EOS with recombinant GM-CSF primed the cells for enhanced LTC4 generation following subsequent stimulation with A23187. GM-CSF may play a role in the amplification of the eosinophilic inflammation in asthmatic airways.
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PMID:Identification of an alveolar macrophage-derived activity in bronchial asthma that enhances leukotriene C4 generation by human eosinophils stimulated by ionophore A23187 as a granulocyte-macrophage colony-stimulating factor. 251 May 65

Although the total concentration of cGMP in rod outer segments is thought to be substantially greater than the free concentration, no quantitatively relevant site for the bound cGMP has been described in mammalian photoreceptors. We have found that preparations of purified bovine rod photoreceptor cyclic nucleotide phosphodiesterase (PDE) contain 1.8 +/- 0.3 mol of tightly bound cGMP per mol of PDE. When subunits of the purified PDE were separated by reverse-phase HPLC in 0.1% trifluoroacetic acid and acetonitrile, a peak of material having spectral properties characteristic of a guanine ring was seen. This material was identified as cGMP by comigration with authentic cGMP on HPLC, conversion to 5-GMP by trypsin-activated rod PDE, and conversion to guanosine by a combination of trypsin-activated PDE and 5'-nucleotidase-containing snake venom. When incubated with 1 microM [3H]cGMP, only 0.1 mol of [3H]cGMP bound per mol of purified PDE, presumably because nearly all binding sites were occupied by tightly bound endogenous cGMP carried through the purification. Scatchard plots of [3H]cGMP binding have indicated that two classes of binding sites are present on the rod PDE. The off-rate of cGMP from the slowly dissociating site is extremely slow; it has a t1/2 of approximately 4 hr at 37 degrees C. At lower temperatures, very little cGMP dissociates; the amount of [3H]cGMP bound to rod PDE after 2 hr at 4 degrees C was essentially the same as at the beginning of the incubation. The observation that stoichiometric amounts of cGMP are tightly bound to PDE accounts for the inability to purify the bovine rod PDE on cGMP affinity columns or to demonstrate stoichiometric high-affinity binding sites with [3H]cGMP. More significantly, the tightly bound cGMP may resolve the apparent discrepancy between the free and total cGMP concentrations of photoreceptor outer segments.
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PMID:cGMP is tightly bound to bovine retinal rod phosphodiesterase. 254 68


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