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 aim of the present study is the isolation of subpopulations of steroid-producing cells in the ovary of the newly hatched chicken. Cells were obtained by fractional trypsin dissociation of the ovary and isopycnic separation in a continuous metrizamide gradient (0-30%). Testosterone and 17 beta-estradiol secretion was measured by radioimmunoassay in the incubation medium of the isolated cells. Six fractions of ovarian cells were studied. Fraction II (density 1.080) contained typical steroidogenic cells with a positive reaction for 3 beta-hydroxysteroid dehydrogenase, delta 5-isomerase. This fraction secreted testosterone (3.7 ng/10(6) cells/2 hr) but no 17 beta-estradiol secretion was detectable. The majority (85%) of cells obtained in fraction VI (density 1.150) were relatively undifferentiated and contained polyribosomes, mitochondria with lamellar cristae, and few rough endoplasmic reticulum; only 3-5% of the cells of this fraction were similar to those of fraction II. In fraction VI the highest level of 17 beta-estradiol secretion was found (2.9 ng/10(6) cells/2 hr) whereas testosterone was at a minimum level (0.06 ng/10(6) cells/2 hr). Results strongly suggest the existence of two cell subpopulations in the inmature chicken ovary: typical steroidogenic and poorly differentiated cells which secrete testosterone and 17 beta-estradiol, respectively.
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PMID:Identification of steroidogenic cell subpopulations in the ovary of the newly hatched chicken. 341 Feb 93

Peroxisomes isolated from rat liver were incubated with [3H]squalene and [3H]mevalonate and the subsequent incorporation of radioactivity into cholesterol studied. The isolated lipids became labeled after incubation with both precursors. In contrast to findings with microsomes, trypsin and detergent treatment of peroxisomes did not influence the rate of cholesterol synthesis. In addition, the luminal content of peroxisomes could alone mediate this synthetic process. Upon treatment of rats with various inducers of peroxisomes and of the endoplasmic reticulum, as well as upon feeding with cholesterol and cholestyramine, large differences in the pattern of in vitro incorporation of [3H]mevalonate into the cholesterol of peroxisomes and microsomes were observed. Injection of this precursor also resulted in high initial labeling of peroxisomal cholesterol in vivo. These experiments indicate that cholesterol synthesis may also occur in peroxisomes.
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PMID:In vitro labeling of peroxisomal cholesterol with radioactive precursors. 344 27

IL 1 activity, as assayed by the proliferation of responsive mouse thymocytes and a human astrocytoma cell line, was detected on the membrane of 1% paraformaldehyde-fixed activated human monocytes. Resting, unactivated monocytes did not display IL 1 activity. Maximum induction of membrane IL 1 was obtained from monocytes treated with polyclonal activators, such as LPS or Staphylococcus aureus, whereas adherence was a weak inducer of membrane IL 1. Isolated cell compartments as plasma membranes, crude lysosomes, and crude cytosol from activated human monocytes expressed significant IL 1 activity, whereas the endoplasmic reticulum showed no IL 1 activity. Exposure to trypsin of either fixed, activated human monocytes or cell compartments from unfixed monocytes, revealed biologically active IL 1 in the membrane, crude lysosome, and crude cytosol, but not in the endoplasmic reticulum. The IL 1 activity in the purified cytosol, prepared by extraction with digitonin, was considerably increased by the trypsin treatment, whereas the increase in IL 1 activity within crude lysosomes and plasma membranes was less. The cell compartments from nonactivated monocytes did not express active IL 1 and trypsin treatment revealed no active IL 1, suggesting the absence of a pool of the trypsin-sensitive form of IL 1. The data confirm the presence of membrane-bound IL 1 in activated human monocytes and indicate that an inactive precursor molecule can be found in the cytosol of such cells. Furthermore, the absence of IL 1 activity either in its active form or as the inactive precursor in the endoplasmic reticulum suggests that IL 1 is not a conventionally secreted protein. Because IL 1 was found in the cytosol as a precursor and in the lysosomal fractions in an active form, these data suggest that after the synthesis and processing of the cytosolic precursor, the 17-kda IL 1, is released via lysosomal vesicles.
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PMID:Subcellular localization of human monocyte interleukin 1: evidence for an inactive precursor molecule and a possible mechanism for IL 1 release. 349 84

Surface epithelium and submucosal glands of the ferret trachea undergo extensive postnatal development. This study examined developmental changes in rates of release and types of high molecular weight glycoconjugates secreted by explanted ferret tracheas. Digestion with bovine testicular hyaluronidase separated the high molecular weight glycoconjugates into two types, hyaluronidase-resistant mucins and hyaluronidase-susceptible glycosaminoglycans. Release rates were measured under unstimulated conditions and in the presence of known secretagogues. The unstimulated rate of release of total 3H-glycoconjugates was 4-fold higher at birth than after complete maturation. The mucin content varied from 39 to 74% of total 3H-glycoconjugates; however, no age-related pattern was observed for mucin/glycosaminoglycan ratios. The rate of release of 3H-mucins was 6-fold higher at birth than in the adult but rapidly dropped to adult levels by 28 days of age. The secretory cells in the tracheal epithelium of newborn ferrets had more abundant rough endoplasmic reticulum than did mature goblet cells, suggesting increased synthesis of secretory product. Response to methacholine and trypsin, both known stimulators of mucin release, was not observed until 28 and 54 days of age, respectively. Incorporation of 35S-sulfate into mucins relative to that for 3H-glucosamine increased with age, consistent with increasingly acidic histochemical staining properties of secretory cells. These developmental differences in rates of release, modulation of release, and relative sulfation of mucins may represent changes in secretory and synthetic mechanisms of the secretory cells.
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PMID:Developmental changes in glycoconjugate secretion by ferret tracheas. 353 88

The v-sis oncogene and its cellular homolog c-sis encode chain B of platelet-derived growth factor. Cells transformed by v-sis produce a platelet-derived growth factor-related molecule which is able to stimulate the platelet-derived growth factor receptor in an autocrine fashion. Site-directed mutagenesis was used to construct several mutations which substitute charged residues for hydrophobic residues in the proposed signal sequence of the v-sis gene product. Two of these mutations resulted in the synthesis of altered v-sis gene products with an unexpected nuclear location and a loss of biological activity. We also report here the intracellular localization of the v-sis gene product to the endoplasmic reticulum-Golgi compartment, where signal sequence cleavage and N-linked glycosylation occur. The v-sis gene product contains no transmembrane regions, as it is completely protected within isolated microsomes from trypsin proteolysis. Site-directed mutagenesis was also used to alter a proposed proteolytic processing site in the v-sis gene product. This mutant v-sis gene, which encodes Asn-Ser in place of Lys-Arg at residues 110 to 111, was found to retain full biological activity.
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PMID:Biosynthesis of the v-sis gene product: signal sequence cleavage, glycosylation, and proteolytic processing. 353 1

The properties of the antiestrogen-binding protein have been examined in mouse tissues, a species in which nonsteroidal antiestrogens are virtually pure agonists. As in other species studied, this protein was distributed in all tissues - highest levels being in the liver. Subcellular fractionation of mouse liver showed that 82% of the antiestrogen-binding protein was associated with the rough endoplasmic reticulum where it was confined to the membranous component. The antiestrogen-binding protein was also present in smooth endoplasmic reticulum, nuclei and cytosol. Its concentration in intact nuclei was at least 10-times higher than levels previously reported in intact rat liver nuclei. Binding of [3H]tamoxifen to the murine antiestrogen-binding protein was of high affinity (Kd = 1 nM) and was inhibited by unsaturated fatty acids and 7-ketocholesterol. In general, cis-isomers of unsaturated fatty acids were more effective binding inhibitors than trans-isomers. The antiestrogen-binding protein solubilized from rough endoplasmic reticulum membranes by the zwitterionic detergent CHAPS, had a molecular mass of approx. 700 kDa and a sedimentation coefficient of about 19 S. [3H]Tamoxifen binding capacity of the solubilized protein was abolished by trypsin and nonspecific proteinases but not by clostripain or Staphylococcus aureus V8 proteinase, suggesting that lysine residue(s) may be involved in [3H]tamoxifen binding.
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PMID:Murine antiestrogen-binding protein: characterization, solubilization and modulation by lipids. 367 52

The objective of this study was to establish a method by which trophectodermal cells originating from individual preimplantation bovine embryos could be perpetuated in monolayer culture. A single, Day-11 bovine embryo collected nonsurgically from a mixed-breed beef cow was cultured in Ham's F10 medium supplemented with fetal bovine serum, sodium pyruvate, insulin, and epidermal growth factor. After 13 d in culture the embryo had adhered to the surface of the plastic culture vessel and a monolayer covering 0.3 cm2 had developed in the manner of a tissue explant. The monolayer was successfully dispersed using trypsin-EDTA and the cells were passaged. Expansion to a 25-cm2 flask was achieved by the 4th passage. By passaging cultures at a dilution ratio of 1:2, cells were maintained for 38 passages before growth slowed. Transfers beyond the 44th passage were unsuccessful. The cell line, designated BE-13, was successfully frozen and thawed at the 9th, 12th, 15th, and 20th passages. The cell line contains both mono- and binucleate cells with a prominent rough endoplasmic reticulum characteristic of ruminant trophoblast cells. Susceptibility to eight bovine viruses was demonstrated. Such cell lines may provide inexpensive systems for the study of trophoblast metabolism and for investigation of the role of the trophoblast in the pathogenesis of selected bovine abortifacient diseases. Because of their range of viral susceptibility, these cells might also be useful for diagnostic purposes.
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PMID:Monolayer culture of cells originating from a preimplantation bovine embryo. 368 Jan 2

Phosphatidylcholine is synthesized on the cytoplasmic surface of the endoplasmic reticulum and transported to the lumenal monolayer by a protein transporter, a phosphatidylcholine "flippase" (Bishop, W. R., and Bell, R. M. (1985) Cell 42, 51-60). Since the endoplasmic reticulum contains enzymes involved in phosphatidylcholine turnover that have different locations within the organelle, transport systems may exist for phosphatidylcholine metabolites. To test the hypothesis that rat liver microsomes contain a lysophosphatidylcholine transporter, sn-1-monobutyroylphosphatidylcholine was employed. Since this homolog is highly water-soluble, transport of lysophosphatidylcholine could be measured using standard transport methods. sn-1-Monobutyroylphosphatidylcholine entered the lumenal compartment of microsomal vesicles. Transport was saturable and dependent on time and on amount of microsomes and required an intact permeability barrier. sn-1-Monobutyroylphosphatidylcholine transport was inhibited by treatment of microsomes with trypsin, N-ethylmaleimide, and trinitrobenzene-sulfonic acid. These findings suggest that sn-1-monobutyroylphosphatidylcholine transport is protein-mediated. sn-1-Monobutyroylphosphatidylcholine transported into microsomes was degraded to glycerophosphorylcholine. Glycerophosphorylcholine was also transported across the microsomal membrane. Glycerophosphorylcholine transport was also saturable and dependent on time, amount of microsomes, and an intact permeability barrier but was not inhibited by treatment with trypsin or the two protein modification agents. Thus, separate and distinct transport systems exist for phosphatidylcholine metabolites. Molecular events of phosphatidylcholine turnover in the endoplasmic reticulum are discussed.
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PMID:Assembly of the endoplasmic reticulum phospholipid bilayer. Transporters for phosphatidylcholine and metabolites. 368 Feb 61

Solubilization by sodium deoxycholate and trypsin of some metabolic enzymes of unrelated compounds associated with endoplasmic reticulum membranes was carried out. The effects of urea, butanol and detergents on the retinol content in the membranes were studied. It was shown that retinol deficiency causes changes in the interactions of NADH-arylesterase with microsomal membrane components that are manifested in the decrease of the activating effect of butanol and low detergent concentrations on the NADH-reductase activity as well as in the increase in the damaging effect of urea and high detergent concentrations on the enzyme activity. Under conditions of retinol deficiency, the degree of solubilization of NADH-reductase, hydroxylase and arylesterase in the presence of sodium deoxycholate is enhanced. After treatment of liver microsomes of retinol-deficient animals with trypsin or with a trypsin-sodium cholate mixture, the content of these enzymes in the supernatant becomes much greater than that in liver microsomes of vitamin A-deficient rats. It is assumed that retinol deficiency causes of weakening of hydrophobic interactions within the membrane as well as partial translocation of the enzymes from the hydrophobic to the hydrophilic layer.
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PMID:[Effect of retinol deficiency on the activity and solubility of various enzymes of the endoplasmic reticulum membranes in the rat liver]. 369 12

Previous attempts in several laboratories, including ours, to purify oligosaccharyl-transferase have met with limited success because of the lability of the membrane-associated enzyme after solubilization with detergents. In an effort to identify the enzyme in face of this lability, we recently developed a photoaffinity reagent to label the active site [J. K. Welply, P. Shenbagamurthi, F. Naider, H. R. Park, and W. J. Lennarz (1985) J. Biol. Chem. 260, 6459-6465]. In this report, the preparations of a more sensitive selective labeling probe, 125I-labeled N alpha-3-(4-hydroxyphenylpropionyl)-Asn-Lys-(N epsilon-p-azidobenzoyl)-Thr-NH2, is described. Using this new probe, we have confirmed, independently of catalytic activity, that hen oviduct oligosaccharyltransferase is tightly associated with the endoplasmic reticulum membrane. The 125I-labeled oligosaccharyltransferase was released from the membrane by detergent and strong alkali treatments but not by sonication, high salt, or hypotonic shock. However, all procedures that released the enzyme from the membrane resulted in a dramatic loss of enzyme activity. Treatment of sealed microsomal membrane vesicles with phospholipase A resulted in nearly complete enzyme inactivation; in contrast, phospholipase C or D had moderate or little effect, respectively. Taken together, these results suggest that the hydrophobic environment of the membrane is required for oligosaccharyltransferase activity. Trypsin treatment of intact vesicles diminished enzyme activity by nearly 70%, but it had no effect on the binding affinity of the enzyme for the 125I-labeled photoaffinity probe. This result suggests that the polypeptide acceptor portion of oligosaccharyltransferase is lumenally disposed, and that a trypsin-sensitive, cytoplasmically oriented domain or another subunit binds the carbohydrate donor, dolichol-PP-oligosaccharide.
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PMID:Studies on properties of membrane-associated oligosaccharyltransferase using an active site-directed photoaffinity probe. 370 33


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