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)

Two murine monoclonal antibodies, SK-930 (isotype IgG2a) and SK-117 (isotype IgG1), were produced from spleen cells of mice immunized against human pancreatic carcinoma cell lines, MIA-PaCa 2 and Panc-1. With the use of the avidin-biotin-immunoperoxidase technique, the SK-930 and SK-117 antibodies detected an antigen found in 24 and 23 formalin-fixed tissue sections, respectively, of tumors obtained from 30 different patients with pancreatic carcinoma. Reactivity was also frequently found with tumors of the gallbladder, bile duct, stomach, colon and esophagus, while a large panel of normal human tissues, including normal pancreatic tissues, displayed little reactivity. These observations suggest that SK-930 and SK-117 are of value in identifying tumor-associated antigen (TAA) expressed in pancreatic carcinoma and other carcinomas of the digestive system. SK-930 antibody immunoprecipitated a 134 kilodalton molecule from extracts of 125I- or [35S]methionine- or [3H]glucosamine-labeled tumor cells. The SK-117-defined antigen corresponds to 152/137 kilodalton molecules. Moreover, cytofluorometric analyses showed that cells treated with periodic acid exhibited greatly decreased reactivity to the two antibodies, but cells treated with neuraminidase, trypsin or pronase showed unchanged reactivity. The findings suggest that the epitopes of the novel TAA expressed on pancreatic carcinoma cells are carbohydrate moieties.
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PMID:Immunochemical and immunohistological analyses of tumor-associated antigens defined by murine monoclonal antibodies against human pancreatic carcinoma cells. 245 17

After incubation with human serum or plasma, 125I-basic fibroblast growth factor (bFGF) (molecular mass 18.5 kDa) exhibits molecular mass forms greater than 200 kDa as determined by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. These high molecular mass forms of bFGF are immunoprecipitable with antiserum raised against alpha 2-macroglobulin (alpha 2M). Purified alpha 2M and 125I-bFGF form a covalent complex in a specific, saturable manner. Excess unlabeled bFGF competes with 125I-bFGF for complex formation. Complex formation is complete after 4 h and is inhibited by pretreating alpha 2M with dithiothreitol, iodoacetamide, iodoacetic acid, and N-ethylmaleimide. The complex is resistant to acidic conditions and denaturants such as urea. Heparin, which binds bFGF, has no effect on complex formation. Methylamine, which blocks protease binding to alpha 2M, increases the amount of 125I-bFGF that can be bound 2-fold. Plasmin and trypsin treatment of alpha 2M has no effect on 125I-bFGF binding. The ability of growth factors to compete for binding is specific, as aFGF and TGF-beta compete for binding to alpha 2M, whereas platelet-derived growth factor does not. 125I-bFGF.alpha 2M complexes do not bind to low affinity bFGF binding sites and bind poorly to high affinity bFGF binding sites on BHK-21 cells. In addition, 125I-bFGF bound to alpha 2M has decreased ability to stimulate plasminogen activator production in bovine capillary epithelial cells.
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PMID:Alpha 2-macroglobulin is a binding protein for basic fibroblast growth factor. 246 67

In this paper, the characteristics of a highly stable, 34,000 molecular weight form of guinea pig (GP) acrosin are compared with those of acrosins from other mammalian species. GP acrosin, like acrosins from other species, is stable at pH 3.0, has a pH optimum of 8.0, and is inhibited by natural trypsin inhibitors and N-alpha-p-tosyl-L-lysine chloromethyl ketone. Its lack of inhibition by tosyl-phenylalanine chloromethyl ketone indicates that it has a specificity similar to trypsin but not chymotrypsin. The activity of GP acrosin was stimulated by Ca2+ below 75 mM. The enzyme was markedly inhibited by Hg2+, but only weakly inhibited by other metal cations. The disulfide reductants dithiothreitol and 2-mercaptoethanol both inhibited GP acrosin, as did the sulfhydryl reactant, iodoacetic acid. The Michaelis-Menten constant for GP testicular acrosin-catalyzed hydrolysis of the N-benzyloxycarbonyl-L-arginyl amide of 7-amino-4-trifluoromethylcoumarin at pH 8.0 was calculated from Lineweaver-Burk plots to give a value of Km = 2.0 x 10(-5) M with Vmax = 500 mumoles/min/mg protein. The corresponding lysine substrate, the N-benzyloxy-carbonyl L-lysine amide of 7-amino-4-trifluoromethyl-coumarin, had a higher Km = 4.6 x 10(-5) M and lower Vmax = 135 mumoles/min/mg protein, in accord with the substrate preference seen with other mammalian acrosins.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Guinea pig testicular proacrosin-acrosin system: further characterization of the active enzyme. 249 19

Human glandular kallikrein was purified from urine and subjected to detailed structural characterization. The protein was carboxymethylated with iodoacetic acid and digested with TPCK-trypsin, Staphylococcal aureus V-8 protease and endo LysC peptidase. The resulting peptide fragments were separated by reverse-phase HPLC using C-4 columns and acetonitrile-trifluoroacetic acid gradient elution. The complete amino acid sequence of the carboxymethylated derivative was elucidated by sequence analysis and alignment of peptides derived from different proteolytic cleavages. A procedure using in situ CNBr cleavage of a large endo LysC peptidase-derived peptide followed by direct sequencing was carried out to provide overlap for two glycosylation sites at residues 78 and 84. Three Asn-linked glycosylation sites were confirmed by the direct sequence analysis of the isolated glycopeptides. However, the third glycosylation at Asn-144 occurs only in 60% of kallikrein molecules. Reverse-phase HPLC effectively separates two species of HUK which correspond to molecules glycosylated and non-glycosylated at Asn-144, respectively. The human urinary kallikrein contains 238 amino acid residues with Ile and Ser as N- and C-terminal amino acids, respectively. The primary structure is completely identical to that deduced from a human genomic DNA sequence (F.K. Lin et al., manuscript in preparation) and is different in one amino acid (Lys-162 vs. Glu-162) from that deduced from pancreatic or kidney cDNA sequence.
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PMID:Human urinary kallikrein. Complete amino acid sequence and sites of glycosylation. 266 27

Chloroplast fructose-1,6-bisphosphatase is an essential enzyme in the photosynthetic pathway of carbon dioxide fixation into sugars and the properties of this enzyme are clearly distinct from cytosolic gluconeogenic fructose-1,6-bisphosphatase. Light-dependent activation via a ferredoxin/thioredoxin system and insensitivity to inhibition by AMP are unique characteristics of the chloroplast enzyme. In the present study, purified spinach chloroplast fructose-1,6-bisphosphatase was reduced, S-carboxymethylated with iodoacetic acid, and cleaved with either cyanogen bromide or trypsin. The resulting peptides were purified by reversed-phase high performance liquid chromatography. Automated Edman degradation of some of the purified peptides showed amino acid sequences highly homologous to residues 72-86, 180-199, and 277-319 of pig kidney fructose-1,6-bisphosphatase. These findings suggest a common evolutionary origin for mammalian gluconeogenic and chloroplast fructose-1,6-bisphosphatase, enzymes catalyzing the same reaction but having different functions and modes of regulation.
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PMID:Amino acid sequence similarity between spinach chloroplast and mammalian gluconeogenic fructose-1,6-bisphosphatase. 300 49

A trypsin-like protease (named RSP-V) was purified to homogeneity from rat submandibular glands by isoelectric focusing and high-performance liquid chromatography. The purified enzyme had an isoelectric point of 5.3 and an apparent molecular weight of 25,000, and consisted of two subunits with molecular weights of 19,500 and 6,000. RSP-V hydrolyzed BAEE, BAPA, and TAME, but not ATEE or BTPA. It had an optimum pH at around 10.0. RSP-V was strongly inhibited by soybean trypsin inhibitor, aprotinin, leupeptin, antipain, and benzamidine, but not by ovomucoid trypsin inhibitor, p-CMB, or iodoacetic acid. This enzyme partly resembled, but was not identical with, tonin. It was also different from kallikrein, salivain, and glandulain in rat submandibular gland. Although the physiological role of RSP-V has not yet been clarified, this enzyme inactivated dopa decarboxylase alone among catecholamine-synthesizing enzymes.
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PMID:Purification and characterization of a trypsin-like protease in the submandibular gland of rats. 301 67

Colony-stimulating factor 1 (CSF-1) was purified from the serum-free conditioned medium of a human pancreatic carcinoma cell line (MIA PaCa-2) by a combination of conventional chromatography and high-performance liquid chromatography. The purity of human CSF-1 was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a diffuse single band of Mr 42,000-50,000 and by N-terminal amino acid analysis of glutamate residue. The CSF-1 was stable at 50 degrees C for 30 min. It is sensitive to treatment with trypsin, chymotrypsin, and subtilisin but less sensitive to papain digestion. Treatment of CSF-1 with different glycosidases did not affect the biological activity. Sulfhydryl reagents such as dithiothreitol (DTT), iodoacetic acid, and N-ethylmaleimide did not affect the biological activity at the concentration of 1 mM. However, CSF-1 activity was inhibited totally by the combination of 10 mM DTT and 1 mM SDS. Under denaturing and reducing conditions, CSF-1 appeared on SDS-PAGE as a single protein band of Mr 21,000-25,000 and concurrently lost its activity, indicating that human CSF-1 possibly consists of two similar subunits and that the intact quaternary structure is essential for the biological activity. When treated with neuraminidase and endo-beta-D-N-acetylglucosaminidase D, the molecular weight of CSF-1 was reduced to 36,000-40,000, and to 18,000-20,000 in the presence of mercaptoethanol. Because of the specificity of endo-beta-D-N-acetylglucosaminidase D, it is suggested that the carbohydrate moieties are Asn-linked "complex-type" units.
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PMID:Purification and characterization of human colony-stimulating factor 1 from human pancreatic carcinoma (MIA PaCa-2) cells. 354 83

6-[(4-Bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate (6-BDB-TADP) has been shown to react at the reduced diphosphopyridine nucleotide (DPNH) inhibitory site of bovine liver glutamate dehydrogenase with incorporation of 1 mol of reagent/mol of enzyme subunit [Batra, S. P., & Colman, R. F. (1984) Biochemistry 23, 4940-4946]. The modified enzyme had lost one of the six free sulfhydryl groups per enzyme subunit as detected by 5,5'-dithiobis(2-nitrobenzoate). In the unmodified enzyme digested with trypsin, six cysteinyl peptides labeled with [14C]iodoacetic acid were detected by high-performance liquid chromatography (HPLC), whereas only five were observed in the 6-BDB-TADP-modified enzyme. A cysteinyl peptide has been isolated from modified enzyme digested with trypsin and chymotrypsin. Purification of the nucleotidyl peptide was accomplished by chromatography on phenyl boronate-agarose, followed by gel filtration on Sephadex G-25 and Bio-Gel P-4 in 50 mM ammonium bicarbonate, pH 8.0. The modified peptides were finally purified by HPLC on a C18 column using 0.1% trifluoroacetic acid with an acetonitrile gradient. By comparison of the amino acid composition and N-terminal residue of the isolated peptide with the known amino acid sequence of the enzyme, the peptide in the DPNH inhibitory site labeled by 6-BDB-TADP has been identified as the 19-membered fragment from Glu-311 to Lys-329. A unique residue, Cys-319, was identified as the reactive amino acid within the DPNH inhibitory site.
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PMID:Isolation and identification of cysteinyl peptide labeled by 6- [( 4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate in the reduced diphosphopyridine nucleotide inhibitory site of glutamate dehydrogenase. 371 40

Selective cleavage of the interchain disulfide bonds present in the two IgG1-kappa monoclonal cryoglobulins Ger and Muk results in a partial loss of cryoprecipitability of the parent proteins at 0 degree C. The progressive loss of cryoprecipitability which occurs as a function of increasing reductant concentration parallels the successive cleavage of interheavy-light and interheavy-heavy chain disulfides. Circular dichroism shows that reduction and alkylation of hinge region disulfides induces small conformational changes in the IgG molecules that could alter cryoprecipitability. The N-terminal amino acid sequence of the Fc component derived by restricted proteolysis with trypsin of protein Muk was found to be completely homologous with N-terminal Fc sequences of noncryoglobulin IgG reference proteins, indicating identical hinge regions. Reduction and alkylation of two monoclonal IgM cryoglobulins also reduces cryoprecipitability. After reduction and alkylation of either the monoclonal IgM rheumatoid factor or the polyclonal IgG component of two mixed cryoglobulins recombination results in decreased cryoprecipitation of the intact cryoglobulin complex. In all cases inhibition of cryoprecipitation is greater when iodoacetic acid rather than iodoacetamide is employed as the S-alkylating group. These results do not support a direct role for the hinge region in the precipitation of cryoimmunoglobulins.
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PMID:The effect of interchain disulfide bond cleavage on the cold induced precipitation of cryoimmunoglobulins. 378 75

We have compared the immunoreactive oxytocin neurophysin from the human pineal gland with human neurohypophyseal neurophysin. The two proteins were reduced and carboxymethylated with [14C]-iodoacetic acid. Since the pineal protein was available only in a very small amount, we were obliged to use [12C]-carboxymethyl apomyoglobin as a nonalkylatable carrier. The trypsin and subtilisin digests of the two labelled proteins were compared by high-voltage electrophoresis on paper, partition paper chromatography, and high-performance liquid chromatography. The distribution of radioactivity in the above peptide separations suggests a great similarity between the two proteins and few significant differences. As far as we are aware, this study is the first attempt to analyze an extrahypophyseal neurophysin at the molecular level.
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PMID:Similarity of pituitary and pineal human oxytocin neurophysins indicated by peptide mapping after radioactive alkylation and proteolysis. 390 Jul 91


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