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)

In an aqueous ultrafiltrate (10 000--50 000) prepared from Ehrlich ascites mammary carcinoma (EAC) cells, ascites fluid and bovine mammary gland, a new factor was obtained which is involved in the growth regulating system of EAC cells as shown by the following facts: 1) It reversibly inhibits the proliferation of EAC cells in a 24 h suspension culture, depending on their proliferative state. Thus, stationary cells from the plateau phase of growth in vivo are prevented from resuming growth in vitro, while cells taken from the active phase of in vivo growth are not inhibited under the same conditions. Likewise, stationary cells do not respond when incubated in serum before the addition of the factor (depending on serum concentrations and time). The dose-response curve levels off at higher factor concentrations so that the maximal inhibition is about 50--65% (explained with different sensitivities of the cells in the assay system). The time course of the inhibition as well as preliminary data from flow cytophotometry and labeling with tritiated thymidine indicate an interference with the progression of G1-phase cells into the S-phase. 2) The activity of the factor is counteracted by insulin and proinsulin, both known as growth factors. The insulin concentration needed is dependent on the factor concentration; an almost maximal inhibition can be prevented by physiological concentrations of insulin. The activity can be destroyed by heat and trypsin and differs also in other properties from that of polyamines. The factor could not be detected in lung, liver, spleen, kidney, heart and L 1210 ascites fluid of the mouse or in bovine malignant lymph nodes, thymus, kidney or liver. The factor was purified from a homogenate of bovine mammary gland by ultrafiltration and affinity chromatography on sepharose-bound wheat lectin. Polyacrylamide electrophoresis showed about 5 bands of which one contained the activity (in some experiments overlapping with a second one). In this way a 800fold purification (starting from the ultrafiltrate) could be obtained. The overall purification (relative to the centrifuged homogenate) can be calculated to be more than 100 000 fold.
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PMID:On a "chalone"-like factor for Ehrlich ascites mammary carcinoma. 644 29

The present work concerns our studies to search for factor(s) which may influence the hemostatic process in or around metastasis of tumours. We studied the platelet aggregating property of a methyl cholanthrene induced experimental tumour. Platelet aggregating material was found to be different from the known aggregating agents like thrombin, ADP, collagen, thromboxane A2 and trypsin. It depends on a critical level of calcium for its action. PAM is a high molecular weight substance which contains sialic acid. It is trypsin and plasmin insensitive. The activity of this substance is not being destroyed by phospholipase-C. Metabolic study indicates that PAM acts by mitochondrial energy metabolic pathway of the platelets.
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PMID:A new platelet aggregating material (PAM) in an experimentally induced rat fibrosarcoma. 674 May 52

This study extends our earlier work (Abumrad, N. A., Perkins, R.C., Park, J.H., and Park, C.R. J. Biol. Chem. 256, 9183-9191) which showed that oleate permeates the plasma membrane of the rat adipocyte principally by a transport process with the characteristics of facilitated diffusion. In the present study, fatty acid (FA) transport is characterized with regard to its specificity and susceptibility to inhibition by protein modifiers. The kinetics of competitive inhibition for transport of oleate and stearate are shown under conditions where complications due to competition for binding of FAs to the albumin in the medium are minimized. Stearate inhibits influx of tracer oleate with a Ki that closely approximates its Km and, conversely, oleate inhibits similarly the influx of tracer stearate. Specificity of the FA transport system is shown in studies using a variety of natural FAs of different chain length, or FA analogues. Oleate (Km = 0.06 microM), stearate (Km = 0.16 microM), linoleate (Km = 0.22 microM), palmitate, (Km = 0.2 microM), and laurate (Km = 1.5 microM) are good substrates, but octanoate is not transported. An oxazolidine ring on C-5 but not on C-16 of stearate blocks binding to the transporter. Methylation of the carboxyl function but not alpha-bromination inhibits transport. These studies suggest that a FA must have a hydrocarbon chain of at least nine carbons and a free carboxyl function to be recognized by the transporter. FA transport does not require Na or ATP. Pronase but not trypsin treatment of intact cells reduces fatty acid influx. Transport is insensitive to maleimides. It is strongly and irreversibly blocked by pretreatment of the cells with the stilbene compounds, 4,4'-diisothiocyanostilbene-2,2'-disulfonate and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, but only slightly inhibited by dipyridamole. Polyacrylamide gel electrophoresis of plasma membrane proteins from cells treated with [3H] 4,4'-diisothiocyanostilbene-2,2'-disulfonate shows a peak of radioactivity at about Mr = 85,000. When cells are incubated in various concentrations of this agent, the counts recovered in the peak reach a maximum coincident with maximum inhibition of transport. We conclude that permeation of the plasma membrane of the adipocyte by long-chain FAs at physiological concentrations is mediated by a protein transporter with distinct specificity requirements.
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PMID:Permeation of long-chain fatty acid into adipocytes. Kinetics, specificity, and evidence for involvement of a membrane protein. 674 32

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

A sensitive radioimmunoassay technique was developed to quantitate the level of human breast celltype specific antigens on cells from normal breast and from various established cell lines of breast and nonbreast origins. Polyacrylamide gel electrophoresis revealed four major proteinaceous components (150,000; 75,000; 60,000; and 48,000) in human milk fat globule membranes that were used to immunize rabbits in order to elicit antimammary epithelial cell antibody. Antisera obtained were rendered specific by absorptions and were able to recognize three specific mammary epithelial components of the breast epithelial cell. Human mammary epithelial (HME) antigen expression was highest (1290 ng/10(6) cells) in normal breast epithelial cells from primary cultures of normal breasts. Lower levels (range: 955 to 330 ng/10(6) cells) were found in breast epithelial cells from cell lines established from cancerous breast tissue. Cells of nonbreast origins as well as fibroblasts from breast gave much lower values (less than 30 ng/10(6) cells). On treatment, with trypsin, of two breast epithelial cell lines (MDA-MB-157 and MCF-7) 80 to 85% of their HME antigen expression was lost, suggesting that a majority of these breast antigens reside on the cell surface.
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PMID:Quantitation of human mammary epithelial antigens in cells cultured from normal and cancerous breast tissues. 727 41

The adhesive glycoprotein fibronectin has been isolated from fresh hamster plasma by affinity chromatography on gelatin coupled to Sepharose beads by the method of Engvall & Ruoslahti [Int. J. Cancer (1979) 20, 1-5]. Polyacrylamide-gel electrophoresis of material heated in sodium dodecyl sulphate and 2-mercaptoethanol shows two prominent polypeptide subunits of approx. mol.wts. 215 000 and 200 000, with variable amounts of lower-molecular-weight fragments. The unexpected polypeptide heterogeneity of different preparations of hamster fibronectins and bovine serum fibronectin is shown to be partly an artefact and is generated during isolation and storage of purified fibronectin. Treatment of each hamster fibronectin subunit or a smaller fragment of approx. mol.wt. 140 000 with thermolysin or trypsin after radioiodination produces similar patterns of tyrpsine-containing peptides, indicating similar primary amino-acid sequences. Antibodies raised against the major subunits of hamster plasma fibronectin were coupled to Sepharose beads and used in conjunction with gelatin affinity chromatography to isolate fibronectins extracted with urea from baby-hamster kidney (BHK) cells and present in the long-term culture medium of these cells. The cell and medium fibronectins are similar to hamster plasma fibronectin in amino-acid and carbohydrate composition and also produce very similar peptide 'maps'. We conclude that the various forms of hamster fibronectins are structurally analogous in agreement with indistinguishable biological properties in mediating the substance adhesion of BKH cells [Pena & Hughes (1978) Cell Biol. Int. Rep. 3, 339-344].
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PMID:Polypeptide heterogeneity of hamster and calf fibronectins. 745 16

Polyacrylamide gel electrophoresis and high performance liquid chromatography of multicatalytic proteinase complexes (MPC) isolated from bovine pituitary, lung, and liver showed marked differences in the pattern of subunits. The concentrations of LMP7 in the lung and liver were 10 and 5 times, respectively, greater than those in the pituitary, whereas the chymotrypsin-like activity and the amount of a subunit (BO2), necessary for its expression, were markedly decreased in the lung and moderately decreased in the liver. Lower trypsin-like, small neutral amino acid preferring, and peptidyl-glutamyl-peptide hydrolyzing activities were also found in the lung and liver. The activity of the branched chain amino acid preferring component (BrAAP), predominantly latent in the pituitary, was highly activated in the lung and liver, as evidenced by a greatly decreased Km and a 20-fold increase of the specificity constant Vmax/Km, indicating facilitated substrate access to its active site and increased affinity toward substrates with branched chain amino acids in the P1 position. It is suggested that overexpression of LMP7 in the lung is related to increased exposure of the airways to foreign antigens. The possible association between amounts of LMP7 and the activation of the BrAAP component needs further examination.
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PMID:Differences in catalytic activities and subunit pattern of multicatalytic proteinase complexes (proteasomes) isolated from bovine pituitary, lung, and liver. Changes in LMP7 and the component necessary for expression of the chymotrypsin-like activity. 767 55

Proteins encompassing the two catalytic domains (monooxygenase and lyase) and the COOH-terminal domain of rat peptidylglycine alpha-amidating monooxygenase (rPAM)3 were purified from recombinant Escherichia coli overexpressing each domain and used to raise domain-specific polyclonal antibodies. Four alternatively spliced forms of PAM RNA (PAM-1, -2, -3, and -4) were transcribed in vitro and used to synthesize PAM proteins in a cell-free translation system. The orientation of the proteins in microsomal membrane vesicles was analyzed using trypsin protection assays and immunoprecipitation with the domain-specific antibodies. Only one of the two potential N-glycosylation sites (Asn765-Phe-Ser) in PAM-1 was efficiently utilized by microsomal membranes. PAM-1 and PAM-2 were shown to be type Ia membrane proteins with their two catalytic domains residing within microsomal vesicles and their COOH-terminal domains exposed to the cytosol. In contrast, PAM-3 and PAM-4 were shown to be soluble proteins contained entirely within vesicles. Thus, the COOH-terminal domain underwent topological switching between the cytosolic (PAM-1 and -2) and luminal (PAM-3) compartments as a function of alternative splicing of exons Ba/Bb. Computer analyses of the PAM protein sequence correlated the exons encoding PAM-1 with a model for the structural and functional domains of the PAM protein. The dual topologies of the PAM proteins confer an important means of functional regulation to this secretory granule associated neuropeptide processing enzyme.
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PMID:Topological switching of the COOH-terminal domain of peptidylglycine alpha-amidating monooxygenase by alternative RNA splicing. 768 Jan 92

Mutant forms of Escherichia coli succinyl-CoA synthetase, W76F (Trp beta 76 replaced by Phe) (Nishimura, J. S., Mann, C. J., Ybarra, J., Mitchell, T., and Horowitz, P. M. (1990) Biochemistry 29, 862-865), and W43,76,248F (all three Trp replaced by Phe) were found to be more sensitive to proteolysis by clostripain than the wild-type enzyme or other Trp mutant proteins. Like wild-type enzyme, sensitivity to trypsin was apparent when the enzyme forms were in the dephosphorylated state. Sensitivity to clostripain was the same, whether mutant or wild-type forms were in the phosphorylated or dephosphorylated state. The substrates ADP and ATP both protected the enzymes against inactivation by clostripain, with dissociation constants for protection of W76F of 33 and 125 microM, respectively. Polyacrylamide gel electrophoresis of clostripain digests revealed preferential digestion of the beta-subunit and the appearance of 40- and 31-kDa species, with amino termini corresponding to residues 15 and 81, respectively, of the beta-subunit. Mutagenic replacement of Arg beta 80, but not Arg beta 14, with Lys resulted in an enzyme that was as resistant to clostripain as wild-type enzyme. These results suggest that Arg beta 80 is the principal site of inactivation by clostripain and may be involved in the binding of ADP and ATP to succinyl-CoA synthetase.
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PMID:Sensitivity of Escherichia coli succinyl-CoA mutants at Trp beta 76 to clostripain and to trypsin. ADP and ATP protect against cleavage by clostripain at Arg beta 80. 851 3

Gaseous CO2 was used as an antisolvent to induce the fractional precipitation of alkaline phosphatase, insulin, lysozyme, ribonuclease, trypsin, and their mixtures from dimethylsulfoxide (DMSO). Compressed CO2 was added continuously and isothermally to stationary DMSO solutions (gaseous antisolvent, GAS). Dissolution of CO2 was accompanied by a pronounced, pressure-dependent volumetric expansion of DMSO and a consequent reduction in solvent strength of DMSO towards dissolved proteins. View cell experiments were conducted to determine the pressures at which various proteins precipitate from DMSO. The solubility of each protein in CO2-expanded DMSO was different, illustrating the potential to separate and purify proteins using gaseous antisolvents. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) was used to quantify the separation of lysozyme from ribonuclease, alkaline phosphatase from insulin, and trypsin from catalase. Lysozyme biological activity assays were also performed to determine the composition of precipitates from DMSO initially containing lysozyme and ribonuclease. SDS-PAGE characterizations suggest that the composition and purity of solid-phase precipitated from a solution containing multiple proteins may be accurately controlled through the antisolvent's pressure. Insulin, lysozyme, ribonuclease, and trypsin precipitates recovered substantial amounts of biological activity upon redissolution in aqueous media. Alkaline phosphatase, however, was irreversibly denaturated. Vapor-phase antisolvents, which are easily separated and recovered from proteins and liquid solvents upon depressurization, appear to be a reliable and effective means of selectively precipitating proteins.
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PMID:Protein purification with vapor-phase carbon dioxide. 1009 36


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