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)

Ethanol/ether soluble apoproteins, comprising 17% of the total recovered surfactant-associated proteins, were isolated from rat lung surfactant and purified by silicic acid chromatography. The protein that eluted in 4:1 chloroform/methanol accounted for greater than 85% of protein in the ethanol/ether soluble fraction and was termed surfactant apoprotein Et (Apo Et). By sodium dodecyl sulfate polyacrylamide gel electrophoresis, this protein had an apparent molecular weight of approximately 10,500. Apo Et was evaluated for its effect on uptake of synthetic phospholipids in liposomal form by isolated granular pneumocytes (Type II alveolar epithelial cells) in primary culture. Liposomes were prepared to approximate the phospholipid composition of the alveolar surfactant, and uptake was measured by the accumulation of the radioactively labeled dipalmitoyl phosphatidyl choline fraction. The uptake of liposomal phosphatidylcholine by cells incubated for 2 h with Apo Et was increased by 61% over control. Most of the cell-associated phospholipid uptake was resistant to treatment with trypsin, suggesting an increased internalization of liposomal material in the presence of Apo Et. The effect of Apo Et on uptake was concentration and time dependent and was not associated with cell damage, phospholipase activity, or detergent properties of the protein. Apo Et had no significant effect on phosphatidylcholine uptake by granular pneumocytes maintained for 7 d in primary culture. Apo Et augmented the uptake of phospholipids by alveolar macrophages although total uptake by these cells was less than that observed with granular pneumocytes. Because Apo Et increases the rate of uptake of surfactant phospholipids by alveolar cells (granular pneumocytes and alveolar macrophages), this protein may represent a physiologically important regulator for clearance of lung surfactant phospholipids.
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PMID:An ethanol/ether soluble apoprotein from rat lung surfactant augments liposome uptake by isolated granular pneumocytes. 654 63

When soluble and particulate fractions of human platelet homogenate were chromatographed on DEAE-cellulose and hydroxylapatite columns, total phospholipase A2 (PLA2; phosphatide 2-acylhydrolase, EC 3.1.1.4) activity increased sharply. PLA2 activity detected in these partially purified preparations was approximately 12 times greater than that associated with the original homogenate. Chromatography of the particulate enzyme on a DEAE-cellulose column yielded one activity peak. Fractions eluted near the activity peak showed a trace of PLA2 activity but inhibited purified PLA2 stoichiometrically, suggesting the presence of an endogenous "inhibitor" in the homogenate. The inhibitor activity was heat-stable, trypsin insensitive, and extractable by chloroform/methanol, and thus appears to be associated with a lipid(s). PLA2 activity was Ca2+ dependent. The crude enzyme was variably stimulated by calmodulin, whereas the purified enzyme was not, suggesting that the effect of calmodulin on the crude enzyme is indirect. Our results suggest that human platelet PLA2 activity reported in the literature may have been underestimated, apparently due to the presence of an endogenous inhibitor.
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PMID:Marked increase of human platelet phospholipase A2 activity in vitro and demonstration of an endogenous inhibitor. 657 16

A nonganglioside factor(s) present in Sigma types II and III mixed bovine brain ganglioside preparations synergises with suboptimal amounts of serum to induce proliferation specifically in nondividing B 103 neuroblastoma cultures. The active substance is nondialysable and soluble in water as well as in chloroform-methanol mixtures of 1:1-4:1 (vol/vol). It is completely insoluble in ether and acetone at room temperature. Biological activity survives heating to 70 degrees C in the presence of 0.1 M HCl for 1 h as well as boiling at neutral pH. Loss of activity occurs on heating to 70 degrees C for 1 h with 1 M HCl or 1 M NaOH. The activity is insensitive to digestion with neuraminidase, trypsin, pronase, and phospholipases A2 and C. The factor cochromatographs with gangliosides on Dowex AG 50W and Sephadex G100 and is partially recovered with GM1 on DEAE-Sepharose, but may be isolated in a ganglioside-free fraction by sequential chromatography on Sephadex LH20 and silicic acid columns. The substance(s) has the properties of a water-soluble proteolipid protein, the amino acid composition being reported. It is not immunologically cross-reactive with antibodies to GM1 ganglioside or the major proteolipid protein of myelin.
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PMID:Characterization and partial purification of a ganglioside-associated mitogen. 661 41

The ejaculated porcine spermatozoa were fractionated into the cytosol, membrane, midpiece plus tail (flagella) and head fractions, and their adenylate cyclase activities were measured. About 65% of the total activity was located in the flagella fraction. For all the fractions, Mn2+-dependent adenylate cyclase activity was about 20 times higher than Mg2+-dependent activity, and guanine nucleotides, fluoride, and other reagents tested did not activate adenylate cyclase. The results suggest that the GTP-dependent regulatory subunit is absent in porcine spermatozoa. The porcine seminal plasma was found to stimulate the adenylate cyclase activity in spermatozoa. The stimulating factor in porcine seminal plasma was partially purified by gel filtration and the molecular weight of the factor appeared to be between 200 and 300. The partially purified factor is heat stable and is not inactivated by treatment with Pronase, trypsin, phospholipase A2 or D but is inactivated by acid hydrolysis. It is easily soluble in water, partially soluble in methanol, and insoluble in ethanol, ethyl ether, chloroform, or acetone. The activation of sperm adenylate cyclase by the factor occurred without a time lag. The activating effect was dose-dependent, saturated at high dose, and ascribed to the increase of the maximal velocity (Vmax). The effect of the factor appears to be limited to adenylate cyclase in spermatozoa; the factor activated adenylate cyclase both in porcine and bovine spermatozoa but failed to activate those in other porcine tissues. The factor was shown to activate the enzyme not only in the ejaculated spermatozoa but also in the epididymal sperm. The factor was also found to elevate the cAMP level in the intact porcine spermatozoa. The factor enhanced the motility of corpus and cauda epididymal spermatozoa. These findings indicate the possibility that this factor initiates the spermatozoan motility upon ejaculation through directly activating adenylate cyclase.
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PMID:Activation of spermatozoan adenylate cyclase by a low molecular weight factor in porcine seminal plasma. 663 Feb 19

Somatic neurohybrid SB21B1 cells grown in serum exhibit limited capacity to bind 125I-labeled tetanus toxin and cannot synthesize gangliosides higher than GM2. By 6 h after supplementing the culture medium with pure or mixtures of brain gangliosides, binding of 125I-labeled tetanus toxin to cells increases approximately 8-fold compared to that of nonsupplemented cells. The uptake of added gangliosides is a saturable process and is facilitated by serum removal (2.1-fold) or substitution of growth factors for serum (3.8-fold). Enhancement of tetanus toxin binding to cells depends on the ganglioside species and concentration; GT1b (25 micrograms/ml) is, respectively, two and three times as effective as GD1b and GM1 in increasing toxin binding. Reconstitution of ganglioside-mediated tetanus toxin binding activity is a reversible phenomenon; removal of medium gangliosides causes a 3-fold drop in toxin binding by 24 h, after which an apparent plateau for at least 3 days above the basal level is established. As in cerebral cultures, binding of toxin to ganglioside-supplemented neurohybrid cells exhibits salt and sialidase sensitivity and is enhanced 2.6-fold at 37 degrees C compared to 0-4 degrees C. The resultant temperature-dependent toxin-cell association is sialidase insensitive. Fixation of cells by formaldehyde or treatment of ganglioside-supplemented cells with trypsin has no substantial effect on ganglioside-mediated binding of the toxin. Methanol/chloroform treatment of cells causes a 91.4% loss of binding activity.
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PMID:Gangliosides mediate association of tetanus toxin with neural cells in culture. 671 26

We have used photoaffinity labeling to investigate the distribution and function of daunomycin binding sites in Sarcoma 180 cells. When native daunomycin is irradiated at 366 or 488 nm in the presence of cells, the drug is irreversibly incorporated into cellular molecules. The cellular acceptor for the photoincorporation cannot be extracted by chloroform-methanol nor can it be degraded by DNase. However, the drug acceptor is susceptible to trypsin digestion. These results show that the photoincorporation site is composed of protein but not of lipid or DNA. Furthermore, the fact that photoincorporation proceeds equally well at 0 degrees (where drug does not accumulate inside the cells) as compared to 37 degrees (where free drug concentrates in the cells) suggests that the labeling reaction occurs principally at the cell surface. The photolabeling process is not highly specific since it is not saturable at high drug concentrations and cannot be competed for by unlabeled daunomycin. When 2 X 10(5) daunomycin molecules are incorporated per Sarcoma 180 cell, the cells can still accumulate free drug. This result suggests that the photolabeling reaction does not occur at the drug transport locus. Photoincorporation of daunomycin also does not affect the viability of Sarcoma 180 cells, as judged by a cloning assay. Thus, there is probably no surface receptor for the drug which mediates cytotoxicity when occupied. This result is as expected from previous work predicting that the mechanism of daunomycin involves disruption of some generalized membrane property like fluidity. However, in a series of Sarcoma 180 sublines selected for increasing resistance to daunomycin, the photoincorporation increases in direct proportion to drug sensitivity. Consequently, daunomycin appears to be capable of photoaffinity labeling a cell surface protein which, although not directly involved in the mechanism of cytotoxicity is implicated in the expression of drug resistance.
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PMID:Photoaffinity labeling of the Sarcoma 180 cell surface by daunomycin. 671 90

When human placental microsomes were heated in boiling water or exposed to trypsin, 30 to 40% of the 5-ene,3-ketosteroid isomerase activity was stable. Aqueous suspensions of chloroform:methanol extracts of microsomes also catalyzed isomerization of 5-pregnene-3,20-dione, activity being associated with the polar lipid fraction. The trypsin- and heat-stable activities, as well as that of resuspended microsomal lipids, showed a dependence on buffer composition and concentration. Little activity was detected in water at pH 7.0. Relative activities in various buffers were Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) greater than Pipes (1,4-piperazinediethanesulfonic acid) greater than potassium phosphate greater than Mes(4-morpholineethanesulfonic acid). The data suggest that the occurrence of membrane lipid-dependent nonenzymatic catalysis could contribute to the isotope exchange with solvent observed in previous studies of the mechanism of isomerization catalyzed by placental microsomes. The ability of the membrane lipid phase to catalyze steroid isomerization under certain conditions and the fact that this activity is subject to modifications by exogenous agents may have more general implications for an understanding of possible effects of xenobiotics on steroid hormone formation and action in vivo.
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PMID:Characterization of a nonenzymatic component in the isomerization of 5-pregnene-3,20-dione catalyzed by human placental microsomes in vitro. 687 Feb 67

The cytoplasmic membranes and a cytoplasmic fraction of Staphylococcus aureus L-forms increased the incorporation of [3H]thymidine by human lymphocytes in the presence of fetal bovine serum. Both fractions stimulated cord blood lymphocytes as well as adult peripheral lymphocytes, suggesting the possibility that the observed effect was not due to an antigen-specific reaction, but to an immunologically nonspecific action. The membrane mitogen(s) was resistant to trypsin, although it was partially solubilized by trypsin treatment. The mitogen(s) could not be extracted with a chloroform-methanol mixture (2:1, v/v), although the chloroform-methanol soluble fraction was strongly mitogenic to murine splenocytes. Human serum which was added to the assay system in place of fetal bovine serum definitely suppressed the mitogenic effect of both cytoplasmic membranes and the cytoplasmic fraction, especially the latter.
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PMID:Mitogenic effect of cytoplasmic membranes and a cytoplasmic fraction of Staphylococcus aureus L-forms on human peripheral blood lymphocytes. 697 57

The first step in the entry of blood lymphocytes into lymph node is the binding of these cells to venules lined by high endothelium (HEV). This recognition event has been studied using a model system in which rat thoracic duct lymphocytes (TDL) adhere selectively to HEV when overlaid onto lymph node sections. We now report that this binding is increased by a lymph fraction designated adherence enhancing factor (AEF). Lymph depleted of cells and chylomicra was fractionated with (NH4)2SO4. Material that precipitated between 60 and 80% saturation was dissolved in buffer and extracted with chloroform/methanol; the aqueous phase was used as the source of AEF. The factor was nondialyzable, trypsin sensitive, and heat stable (70 degrees C, 30 min). Lymph node sections pretreated with AEF showed a 2- to 3-fold increase in the number of TDL bound to HEV and a 5 to 10-fold increase in the number of HEV with more than 5 adherent lymphocytes. The effect of AEF was specific: 1) No increase in lymphocyte binding to non-HEV areas of the tissue section occurred. 2) The binding of both TDL and spleen lymphoid cells to HEV was enhanced, but thymocyte attachment was not promoted. We conclude that AEF has biologic properties that might enable it to play a physiologic role in lymphocyte recirculation.
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PMID:Lymphocyte recognition of lymph node high endothelium. III. Enhancement by a component of thoracic duct lymph. 702 79

Chick embryos, chick embryo fibroblasts, and Rous sarcoma virus-transformed chick embryo fibroblasts contain a factor that preferentially blocks the accumulation of DNA-directed RNA polymerase II transcripts. The factor was detected by inhibition of transcription in a cell-free assay system utilizing partially purified RNA polymerase II from calf thymus, soluble factors from HeLa cells, and a purified DNA template. At low concentrations, it specifically prevents the accumulation of RNA polymerase II transcripts; at higher concentrations, it blocks the accumulation of other transcripts. The factor has been partially purified by sequential chromatography on BioRex 70, DNA-cellulose, Bio-Gel P-6, and HPX-87 from extracts of chicken embryos. The activity was resistant to treatment with trypsin, pronase, or micrococcal nuclease. A partial characterization of the molecule indicates that (i) it has an apparent molecular mass of about 200-300 daltons, (ii) it is stable at pH 2 and pH 12 and to heating at 100 degrees C, (iii) it is not extractable by ether or chloroform:methanol, (2:1, v/v), and (iv) it is labile to heating at 800 degrees C. These data suggest that it is a small, hydrolphilic compound probably organic in nature. The factor is active in a transcription assay utilizing either the Rous sarcoma virus Long Terminal Repeat promoter or the chick alpha 2 (Type I) collagen-promoter as DNA templates. The accumulation of promoter-specific transcripts is blocked in a cell-free assay utilizing either Rous sarcoma virus-chick embryo fibroblast extracts or HeLa S-100 factors and calf thymus RNA polymerase II. In the absence of S-100, the factor does not appreciably affect the accumulation of randomly initiated transcripts produced by calf thymus RNA polymerase II on a DNA template; this result indicates the factor interacts directly or indirectly with some component(s) of HeLa S-100 to prevent the accumulation of RNA.
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PMID:Chicken embryo extracts contain a factor that preferentially blocks the accumulation of RNA polymerase II transcripts in a cell-free system. 713 Jan 91


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