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)

Drosophila melanogaster contains DNA ligases I and II. The activity of DNA ligase I is especially high during early embryonic periods, but decreases rapidly afterwards. Although the activity of DNA ligase II is low, it persists throughout all developmental stages. The specific activity of DNA ligase II is high in embryos, but the total activity per body mass was highest in pupae. To characterize the properties of DNA ligase II further and to clarify its differences from DNA ligase I, DNA ligase II was prepared from pupae of D. melanogaster. The enzyme was purified about 3200-fold by ammonium sulfate fractionation (40-70% saturation), phosphocellulose (P11) and Ultrogel column chromatography. Some of the properties have been reported previously. The isoelectric point of DNA ligase II was 6.4 while those of DNA ligase I were 4.9 and 5.8. The optimum pH of DNA ligase II was 7.8-8.1 but 8.0-8.5 for DNA ligase I. The molecular masses of DNA ligase II adducts with AMP were determined as 90 and 70 kDa. These adducts were degraded to 42 and 14.4 kDa by trypsin digestion. For preparation of monoclonal antibodies, a mouse was immunized with the purified enzyme. Two clones, 10-6 and 3-3 IgM, were obtained and purified from mouse ascites. These antibodies showed both binding and neutralizing activities toward DNA ligase II from D. melanogaster, but did not react with DNA ligase I from the same origin. These results showed clearly that DNA ligases I and II have different properties and suggest they have different roles during the developmental stages of D. melanogaster.
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PMID:Purification and characterization of DNA ligase II from Drosophila melanogaster. 212 58

GM1 (II3Neu5Ac-GgOse4Cer)-oligosaccharide was prepared from the ganglioside by ozonolysis and alkaline fragmentation, reductively aminated and coupled to the heterobifunctional cross-linker succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate. The resulting derivative reacted with free sulfhydryl groups and readily cross-linked to cell surface components on rat glioma C6 cells which are GM1-deficient. Attachment of the GM1-oligosaccharide derivative, which was monitored by increased binding of 125I-cholera toxin to the cells, was both time- and concentration-dependent. Prior treatment of the cells with dithiothreitol enhanced the attachment by generating additional free sulfhydryl groups. The affinity of cholera toxin for cells treated with the GM1-oligosaccharide derivative or with GM1 was similar. The nature of the newly generated toxin receptors was determined by Western blotting. Membranes from derivatized cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resolved components were electrophoretically transferred to a nitrocellulose sheet which was overlain with 125I-cholera toxin. The toxin bound to a wide variety of membrane proteins, most of which were trypsin-sensitive. No such binding was observed using membranes from control cells. Although the GM1-neoganglioproteins newly generated on the surface of rat glioma C6 cells readily bound cholera toxin, the cells did not become more responsive to the toxin as measured by increased production of cyclic AMP or activation of adenylate cyclase. In contrast, cells exposed to GM1 became highly responsive to the toxin. Thus, neoganglioproteins on the cell surface appear to behave as nonfunctional receptors for cholera toxin.
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PMID:Generation of cell surface neoganglioproteins. GM1-neoganglioproteins are non-functional receptors for cholera toxin. 215 9

1. Various mitogens such as concanavalin A, phytohaemagglutinin, the pokeweed mitogen and trypsin were found to produce a rapid and transient activation of glycogen phosphorylase activity of lymphocytes incubated in a Krebs-Ringer-bicarbonate-glucose buffer. 2. Activation of the enzyme by these mitogens was always accompanied by an increase in the intracellular cyclic AMP concentration. 3. The presence of calcium ions in the incubation buffer was essential for obtaining the mitogen effects. Addition of ionophore A-23187 also produced an activation of glycogen phosphorylase, similar to that found in mitogen activation but without increase in intracellular cyclic AMP concentration. Dibutyril cyclic AMP also produced lymphocyte phosphorylase activation, even in the absence of extracellular calcium ions. 4. It is proposed that phosphorylase activation by mitogens occurs through a mechanism that involves the participation of both calcium ions and cyclic AMP.
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PMID:Role of calcium and cyclic AMP on the activation of lymphocyte glycogen phosphorylase by mitogens. 215 24

The role of cyclic AMP in the regulation of lutropin (luteinizing hormone, LH) receptors has been investigated in cultured mouse tumour (MA10) Leydig cells. The LH receptors were quantified by measuring the binding of 125I-labelled human chorionic gonadotropin (hCG). LH (0.03 nM) in the presence of 1 mM-dibutyryl-cyclic AMP [(Bu)2cAMP] caused a 3-8-fold increase in subsequent 125I-hCG binding. (Bu)2cAMP (1 mM), cholera toxin (11.9 nM) and forskolin (1 microM) each caused a 2-4-fold increase in binding. In the presence of translation (cycloheximide) and transcription (actinomycin D) inhibitors, there was a loss of detectable binding sites. (Bu)2cAMP increased the rate of recovery of binding sites after trypsin treatment of MA10 cells, with a concomitant 2-fold increase in the level of binding sites. Under conditions where receptor levels were increased by 3-8-fold there was also a significant increase in pregnenolone production. It is concluded that LH and cyclic AMP have positive regulatory effects on LH receptors in MA10 cells by inducing the synthesis of new receptors. These induced receptors are functionally coupled to steroidogenesis.
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PMID:Induction of lutropin receptors by lutropin and cyclic AMP in cultured mouse tumour (MA10) Leydig cells. 216 44

In human erythrocytes, dibutyryl cyclic AMP induces the phosphorylation of protein 4.1 on sites within the adjacent 16 kDa and 10 kDa chymotryptic domains (Horne, W.C., Leto, T.L. and Marchesi, V.T. (1985) J. Biol. Chem. 260, 9073-9076). The 10 kDa domain also contains the spectrin/actin-binding site (Correas, I., Leto, T.L., Speicher, D.W. and Marchesi, V.T. (1986) J. Biol. Chem. 261, 3310-3315) and it has been shown that phosphorylation of protein 4.1 by cyclic AMP-dependent protein kinase inhibits the binding of protein 4.1 to spectrin and actin (Ling, E., Danilov, Y.N. and Cohen, C.M. (1988) J. Biol. Chem. 263, 2209-2216). In this study, we have identified two sites on protein 4.1 which account for 80% of the phosphate incorporated into protein 4.1 during metabolic labelling of erythrocytes in the presence of dibutyryl cyclic AMP. More than 95% of the 32P incorporated into protein 4.1 was in the form of phosphoserine. Reverse-phase HPLC of the peptides generated by digestion of the isolated protein with trypsin or endoproteinase lysine C produced two major radioactive peaks. The phosphorylation sites, identified by gas phase sequencing of the purified phosphopeptides and confirmed by determining the residues converted to S-ethylcysteine by reacting the phosphopeptides with ethanethiol under alkaline conditions, were Ser-331, in the 16 kDa domain and Ser-467, in the 10 kDa domain.
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PMID:Identification of two cAMP-dependent phosphorylation sites on erythrocyte protein 4.1. 217 79

DNA ligases play obligatory roles during replication, repair, and recombination. Multiple forms of DNA ligase have been reported in mammalian cells including DNA ligase I, the high molecular mass species which functions during replication, and DNA ligase II, the low molecular mass species which is associated with repair. In addition, alterations in DNA ligase activities have been reported in acute lymphocytic leukemia cells, Bloom's syndrome cells, and cells undergoing differentiation and development. To better distinguish the biochemical and molecular properties of the various DNA ligases from human cells, we have developed a method of purifying multiple species of DNA ligase from HeLa cells by chromatography through DEAE-Bio-Gel, CM-Bio-Gel, hydroxylapatite, Sephacryl S-300, Mono P, and DNA-cellulose. DNA-cellulose chromatography of the partially purified enzymes resolved multiple species of DNA ligase after labeling the enzyme with [alpha-32P]ATP to form the ligase-[32P]AMP adduct. The early eluting enzyme activity (0.25 M NaCl) contained a major 67-kDa-labeled protein, while the late eluting activity (0.48 M NaCl) contained two major labeled proteins of 90 and 78 kDa. Neutralization experiments with antiligase I antibodies indicated that the early and late eluting activity peaks were DNA ligase II and I, respectively. The three major ligase-[32P]AMP polypeptides (90, 78, and 67 kDa) were subsequently purified to near homogeneity by elution from preparative sodium dodecyl sulfate-polyacrylamide gels. All three polypeptides retained DNA ligase activities after gel elution and renaturation. To further reveal the relationship between these enzymes, partial digestion by V8-protease was performed. All three purified polypeptides gave rise to a common 22-kDa-labeled fragment for their AMP-binding domains, indicating that the catalytic sites of ligase I and II are quite similar, if not identical. Similar findings were obtained from the two-dimensional gel electrophoresis of their AMP-binding domains in the trypsin-digested protein fragments. The results also suggested that these isozymes have been derived from the same primordial DNA sequence or from the same precursor protein. The purification scheme and the data obtained will be instrumental for the further elucidation of the biological roles of various DNA ligases from human cells.
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PMID:Fingerprinting of near-homogeneous DNA ligase I and II from human cells. Similarity of their AMP-binding domains. 221 88

We observed that culture medium conditioned with fetal rat long bones stimulated cyclic AMP production by canine renal cortical membranes. This cyclase-stimulating activity (CSA) was retained by an ultrafiltration membrane with a molecular weight cutoff of 5000; three biologically active peaks with an approximate molecular weight of 18,000-25,000, 9000-12,000, and 4000-6000 were separated by high-performance liquid chromatography. The biologic activity was destroyed by trypsin digestion. The stimulation of adenylate cyclase by the medium and by the three peaks was inhibited by [N-leu8,18,Tyr34]parathyroid hormone-(3-34)-amide and by [Tyr34]parathyroid hormone-(7-34)amide. Preincubation of the bone culture medium and of the three peaks with an antibody raised against human parathyroid hormone-(1-34) did not decrease the biologic activity more than incubation with nonimmune serum. However, the biologic activity of the three active peaks was significantly suppressed after preincubation with an antiserum directed against the N-terminal region of the parathyroid hormone-related peptide of malignancy. The release of CSA into the bone culture medium was enhanced by parathyroid hormone induction and by 1,25-dihydroxycholecalciferol. It was decreased by calcitonin. We conclude that fetal murine bones in culture release peptides that stimulate the adenylate cyclase of renal cortical membranes. These peptides are antigenically similar to the parathyroid hormone-related peptide of malignancy. Their release from bones is modulated by hormones that control bone resorption.
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PMID:Release of parathyroid hormonelike peptides by fetal rat long bones in culture. 239 1

Activation of mast cells by bridging of IgE-receptors or concanavalin A (Con A) results in a rapid initial rise and fall in cyclic AMP (cAMP) levels followed by a second rise in cAMP levels and histamine release (Sullivan, T. et al. (1976) J. Immunol. 117, 713-716; Lewis, R.A. et al. (1979) J. Immunol. 123, 1663-1668; Ishizaka, T. et al. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6812-6816). trans-4-Guanidinomethylcyclohexanecarboxylic acid 4-tert-butylphenyl ester (GMCHA-OPhBut), a strong trypsin inhibitor and an anti-allergic agent (Muramatu, M. et al. (1982) Hoppe-Seyler's Z. Physiol. Chem. 363, 203-211; Takei, M. et al. Agents Actions, in press), strongly and dose-dependently inhibited the initial and second rises in cAMP levels, and release of histamine from rat mast cells by Con A, anti-IgE and antigen. Addition of GMCHA-OPhBut after the initial rise in cAMP inhibited the second rise in cAMP and histamine release. These results suggested a possible participation of a trypsin-like proteinase, probably pH 7 tryptase present in rat mast cells, in the activation of adenylate cyclase by the above secretagogues, and the initial rise in cAMP was not directly related to the latter events. The second rise in cAMP is induced by prostaglandin D2 (PGD2), a metabolic product of arachidonic acid. PGD2 elevated the cAMP levels in mast cells whereas no histamine was secreted. GMCHA-OPhBut did not inhibit the increase in cAMP by PGD2. Therefore, the strong inhibitory effect of GMCHA-OPhBut on the second rise in cAMP might depend on the inhibition of an earlier process than the activation of adenylate cyclase by PGD2.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Role of cyclic AMP during histamine release. Histamine release is not directly related to increase in cyclic AMP levels in rat mast cells activated by concanavalin A, anti-IgE, antigen, prostaglandin D2 and isoproterenol. 246 44

Serine protease inhibitors with a specificity for trypsin inhibit interferon-gamma (INF-gamma)-induced HLA-DR expression on a hybrid human epidermal cell line (H12), dermal fibroblasts, and primary keratinocytes. Protease inhibitors with a specificity for chymotrypsin or papain fail to inhibit IFN-gamma. The inhibitory effect of the trypsin inhibitors is similar to that of glucocorticoids in that it is a transient event, fading with length of exposure to IFN-gamma, and is reversed by the addition of dibutyryl cyclic AMP (dbcAMP) and phospholipase C(PLC) from Clostridium perfringens. In H12 cells, dbcAMP and PLC enhance the IFN-gamma induction of HLA-DR, but do not induce in the absence of INF-gamma. Evidence suggests that the protease inhibitors, as well as dbcAMP and PLC, may modulate HLA-DR expression at a post-translational site as well as during IFN-gamma signal transduction. These results suggest that trypsin-like protease activity may be required for cellular HLA-DR antigen expression following exposure to IFN-gamma.
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PMID:Trypsin inhibitors inhibit induction by interferon-gamma of HLA-DR antigen expression on human skin cells. 247 85

The functional significance of the molecular interaction of Ca2(+)-ATPase in the sarcoplasmic reticulum (SR) membrane was examined using intermolecular cross-linking of Ca2(+)-ATPase with N,N'-(1,4-phenylene)bismaleimide (PBM). When SR vesicles were allowed to react with 1 mM PBM at pH 7 and 23 degrees C for various intervals and subjected to SDS-PAGE, the amount of the major band of monomeric ATPase decreased with a half life of about 20 min. Higher orders of oligomers were concurrently formed without accumulation of any particular species of oligomer. When SR vesicles were allowed to react with 1 mM PBM in the presence of 1 mM adenyl-5'-imidodiphosphate (AMP-PNP), the rate of oligomerization was markedly reduced and the amount of dimeric Ca2(+)-ATPase increased with time. After 1 h, more than 40% of the Ca2(+)-ATPase had accumulated in the dimeric form. When 1 mol of fluorescein isothiocyanate (FITC) was bound per mol of ATPase, the effects of AMP-PNP on the cross-linking with PBM were completely abolished. When SR vesicles were treated with PBM in the presence of 0.1 mM vanadate in Ca2+ free medium, the oligomerization of the Ca2(+)-ATPase by PBM was strongly inhibited. The vanadate effect on the cross-link formation was completely removed by the presence of Ca2+ and AMP-PNP in the reaction medium. When SR vesicles were pretreated with PBM in the presence of AMP-PNP and digested with trypsin for a short time, the dimeric ATPase was degraded to a peptide with an apparent molecular mass of about 170 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of adenyl-5'-imidodiphosphate and vanadate Ion on the intermolecular cross-linking of Ca2(+)-ATPase in the sarcoplasmic reticulum membrane with N,N'-(1,4-phenylene)bismaleimide. 253 24


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