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)

To examine the relationship between pre-protein cleavage and nascent chain glycosylation placental mRNA was translated in a reconstituted ascites cell-free system containing microsomal membranes prepared from tunicamycin-treated or untreated ascites tumor cells. In the absence of membranes, first trimester RNA directed the synthesis of the pre-form of the alpha subunit of human chorionic gonadotropin, whereas, in the presence of normal membranes, first trimester RNA directed the synthesis of a glycosylated form of the alpha subunit. Cell-free lysates containing membranes derived from tunicamycin-treated cells synthesized an alpha subunit protein with little, if any, carbohydrate. This protein was apparently sequestered into membranes since it was resistant to the action of trypsin which was added after translation. The pre-peptide of the alpha subunit protein was removed by treated membranes as determined by amino acid sequence analyses. The non-glycosylated protein pre-placental lactogen was also cleaved to its mature form by tunicamycin membranes. These data strongly suggest that, in vitro, glycosylation is not obligatory for pre-protein cleavage and sequestration of these placental protein hormones.
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PMID:Processing of placental peptide hormones synthesized in lysates containing membranes derived from tunicamycin-treated ascites tumor cells. 56 37

Monolayer cultures of human midterm and term placentae have been established following trypsin dispersion of placental minces. Maintenance of endocrine function was monitored by the concentrations of specific hormones in the culture media. At either gestational age the cultures 1) secret estradiol-17beta(1) and estrone (in a ratio of about 1:20) and aromatize 3H- or 14C-dehydroepiandrosterone sulfate and 14C-androstenedione, estrogen production being markedly enhanced by addition of dehydroepiandrosterone (10(-6)7) to the culture medium; 2) metabolize 3H-pregnenolone to progesterone and 14C-cortisol to cortisone; and 3) produce increasing amounts of chorionic gonadotropin and decreasing amounts of placental lactogen during the first week in culture. It is proposed that the model is highly suited to the study of factors affecting hormonogenesis by the human placenta whether they be of maternal or of fetal origin.
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PMID:Short term culture of human midterm and term placenta: parameters of hormonogenesis. 60 61

A recombinant Escherichia coli strain was constructed for the overexpression of bovine placental lactogen (bPL), using a bPL structural gene containing 9 of the rare arginine codons AGA and AGG. When high level bPL synthesis was induced in this strain, cell growth was inhibited and bPL accumulated to less than 10% of total cell protein. In addition, about 2% of the recombinant bPL produced from this strain exhibited an altered trypsin digestion pattern. Amino acid residues 74 through 109 normally produce 2 tryptic peptides, but the altered form of bPL lacked these two peptides and instead had a new peptide which was missing arginine residue 86 and one of the two flanking leucine residues. The codon for arginine residue 86 was AGG and the codons for the flanking leucine residues 85 and 87 were TTG. When 5 of the 9 AGA and AGG codons in the bPL structural gene were changed to more preferred arginine codons, cell growth was not inhibited and bPL accumulated to about 30% of total cell protein. When bPL was purified from this modified strain, which included changing the arginine codon at position 86 from AGG to CGT, none of the altered form of bPL was produced. These observations are consistent with a model in which translational pausing occurs at the arginine residue 86 AGG codon because the corresponding arginyl-tRNA species is reduced by the high level of bPL synthesis, and a translational hop occurs from the leucine residue 85 TTG codon to the leucine residue 87 TTG codon. This observation represents the first report of an error in protein synthesis due to an in-frame translational hop within an open reading frame.
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PMID:Novel in-frame two codon translational hop during synthesis of bovine placental lactogen in a recombinant strain of Escherichia coli. 148 Apr 91

The biochemical mechanism of action of prolactin is unknown. This hormone enters the blood stream and binds to receptors predominantly in the monomeric form. A structural analysis of mammalian and piscine prolactin based on the present-day concepts of proteolytic processing of the hormone molecules in target tissues has been carried out. The experimental data suggest that prolactin molecules are protected from exopeptidase influence by their terminal cyclic peptides. The highly conservative proline-2 residues increase the resistance of the mammalian hormone N-terminal fragment to the effects of many aminopeptidases. Structurally the C-terminal cyclic peptides of prolactin, growth hormone and placental lactogen were shown to be homologous to peptides inhibiting trypsin-like proteinases. A structural analysis of the N-terminal domain of mammalian prolactin revealed the important role of Pro-2 and Pro-4 residues at positions adjacent with and inside the disulfide moiety. It is assumed that these proline residues and the cyclic structure are necessary for the manifestation of the inhibiting effect of the mammalian prolactin N-terminal dodecapeptide on proline-specific proteinases. It is assumed that proteolytic degradation of prolactin molecules in target tissues may induce the secretion of functionally active peptides.
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PMID:[Analysis of the structure of prolactin terminal fragments as potential substrates of serine and proline-specific proteinases]. 174 7

Discrepancies exist in the reported purity and biological activity of ovine placental lactogen (oPL), and little structural characterization has been reported. Ovine PL was purified from fetal cotyledonary tissue (day 100 of gestation) by monitoring activity with a recombinant bovine GH (bGH) liver radioreceptor assay. Two hundred grams of tissue yielded 4.2 mg of oPL, with an approximately 1000-fold purification of oPL specific activity following initial tissue extraction. The oPL was radioiodinated and used in an ovine fetal liver (day 100 of gestation) radioreceptor assay to examine competitive displacement of oPL, ovine GH (oGH) and ovine prolactin (oPRL). The potency of oPL (ED50 = 0.18 nmol/l; ED50 is the quantity of ligand necessary to displace 50% of specifically bound 125I-labelled oPL) was greater than that of oGH (ED50 = 4.1 nmol/l) and oPRL (ED50 = 1.1 mumol/l) in competing for 125I-labelled oPL-binding sites. Attempts to sequence the NH2 terminus of oPL by vapour-phase sequencing indicated that the NH2 terminus was blocked. Purified oPL was subjected to trypsin and CnBr digestion, and two CnBr and six tryptic peptides were sequenced. The peptide sequences were compared with other PLs, oPRL and bGH for sequence similarity, and found to be most similar to bovine PL (bPL; 68% overall identity) and oPRL (47% overall identity). Complementary DNA (cDNA) clones were isolated for oPL by screening a lambda ZAP cDNA library with a cDNA coding for bPL. Three cDNAs were nucleotide sequenced, and their combined sequence included 41 nucleotides of 5'-untranslated region, the complete coding region of pre-oPL (708 nucleotides) and a portion of the 3' untranslated region (158 nucleotides).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purification and structural characterization of ovine placental lactogen. 238 Jun 52

As to endometrial cancer and endometrial hyperplasia, this study represents the localizations of the placental proteins and the tumor-associated antigens by both immunohistochemical light microscopy (ILM) and immunoelectron microscopy (IEM). The reagents examined include human placental lactogen (HPL), placental alkaline phosphatase (PAP), pregnancy-associated plasma protein A (PAPP-A), pregnancy-specific beta 1-glycoprotein (SP1), alpha 1-anti-trypsin (alpha-AT), and tissue polypeptide antigen (TPA). The tumor cells stained for HPL are localized and the SP1-positive cancer cells are almost entirely restricted to an infiltrating front. alpha-AT is shown in both tumor cells with marked cellular atypism and ones in deeply infiltrating foci. The immunoreactive frequency for the above six reagents in endometrial cancer is higher than that in endometrial hyperplasia. In endometrial cancer, the high frequency of an immunoreaction is confirmed in TPA (p less than 0.05). The combined stainings, i.e. either for TPA and SP1 or for TPA and PAPP-A, are efficient immunohistochemical screening methods to use in diagnosing endometrial cancer in the biopsied specimens (p less than 0.05).
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PMID:Immunohistochemical localization of placental proteins and tumor-associated antigens in endometrial cancer and endometrial hyperplasia. 244 80

We studied the functional significance of the binding of angiotensin-II (AII) to human placentas. Human trophoblastic cell suspensions were prepared by trypsin digestion of minced tissue. Cell incubations with increasing doses of [125I](SAR1)AII, ranging from 0.01-2.5 nmol/L, were carried out for 20 min at 37 C. The results indicated the presence of specific low capacity [4300 +/- 1300 (+/- SE) sites/cell], high affinity (Kd = 0.38 +/- 0.06 nmol/L) binding sites for [125I](Sar1)AII. This binding was specific for AII analogs. When placental cells were preloaded with 40 microCi/mL [3H]myoinositol for 2 h at 37 C, AII stimulation resulted in a dose-dependent increase in inositol phosphate (InsP) production [EC50 = 1.4 +/- 0.4 (+/- SE) nmol/L], as measured by ion exchange chromatography. (Sar1)AII also stimulated InsP production, with an EC50 of 0.3 +/- 0.2 nmol/L. AII-stimulated production of InsP was completely blocked by the antagonist (Sar1,Ala8)AII. AII also stimulated human placental lactogen release from trophoblastic cells in a dose-dependent fashion. The EC50 was 18 +/- 9 pmol/L, and the stimulation was blocked by (Sar1,Ala8)AII, as found for AII-stimulated InsP production. These results suggest that stimulation of human placental lactogen release by AII may be mediated by activation of phospholipase-C, which, in turn, produces phosphoinositide breakdown. The results, therefore, provide evidence of a physiological role for the renin-angiotensin system within the human placenta.
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PMID:Angiotensin II stimulates both inositol phosphate production and human placental lactogen release from human trophoblastic cells. 254 60

High density lipoprotein (HDL3) binds with high affinity to many types of cells, but controversy exists concerning the nature and biological significance of the binding. We have recently demonstrated that HDL and apoproteins (apo)-AI, -AII, and -CI stimulate a specific and dose-dependent increase in placental lactogen (hPL) release from human trophoblast cells. To examine the possible relationship between HDL3 binding and stimulation of hPL release, we have characterized the binding of [125I]HDL3 to an enriched fraction of hPL-producing trophoblast cells. Binding studies were performed on trophoblast cells isolated by isopycnic centrifugation of collagenase/hyaluronidase-dispersed placental tissue and apo-E free-HDL3 (density, 1.125-1.215 g/ml). Scatchard analysis of binding studies performed at 37 C for 2 h revealed two classes of binding sites: 1) high affinity binding sites with a Kd of 9.7 +/- 2.2 micrograms/ml (1.3 x 10(-7) M) and 9.8 +/- 3.2 x 10(5) binding sites/trophoblast cell, and 2) low affinity binding sites with a Kd of 172.8 +/- 64.8 micrograms/ml (2.3 x 10(-6) M) and an estimated 3.2 x 10(6) sites/cell. As has been found in hepatocytes and other cells, the number of HDL3-binding sites per trophoblast cell (but not the binding affinity) decreased at lower incubation temperatures. In addition, HDL3 binding to trophoblasts cells did not require calcium and was not affected by prior treatment of the cells with pronase or trypsin. HDL3-binding sites on trophoblast cells, however, were not specific for HDL3. Low density lipoprotein (density, 1.063-1.055 g/ml), which does not stimulate hPL release, was nearly as potent on a molar basis as HDL3 in binding to the high and low affinity binding sites on trophoblast cells. Furthermore, nitrated HDL3, which does not compete for high affinity binding to trophoblast cells, stimulated hPL release. Although the characteristics of HDL3 binding to trophoblast cells are similar to those of other cells, these results strongly suggest that the binding of HDL3 to high affinity binding sites is not essential for HDL-mediated hPL release.
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PMID:High density lipoprotein3 binding and biological action: high affinity binding is not necessary for stimulation of placental lactogen release from trophoblast cells. 258 47

Highly purified functional cytotrophoblasts have been prepared from human term placentae by adding a Percoll gradient centrifugation step to a standard trypsin-DNase dispersion method. The isolated mononuclear trophoblasts averaged 10 microns in diameter, with occasional cells measuring up to 20-30 microns. Viability was greater than 90%. Transmission electron microscopy revealed that the cells had fine structural features typical of trophoblasts. In contrast to syncytial trophoblasts of intact term placentae, these cells did not stain for hCG, human placental lactogen, pregnancy-specific beta 1-glycoprotein or low mol wt cytokeratins by immunoperoxidase methods. Endothelial cells, fibroblasts, or macrophages did not contaminate the purified cytotrophoblasts, as evidenced by the lack of immunoperoxidase staining with antibodies against vimentin or alpha 1-antichymotrypsin. The cells produced progesterone (1 ng/10(6) cells . 4 h), and progesterone synthesis was stimulated up to 8-fold in the presence of 25-hydroxycholesterol (20 micrograms/ml). They also produced estrogens (1360 pg/10(6) cells . 4 h) when supplied with androstenedione (1 ng/ml) as a precursor. When placed in culture, the cytotrophoblasts consistently formed aggregates, which subsequently transformed into syncytia within 24-48 h after plating. Time lapse cinematography revealed that this process occurred by cell fusion. The presumptive syncytial groups were proven to be true syncytia by microinjection of fluorescently labeled alpha-actinin, which diffused completely throughout the syncytial cytoplasm within 30 min. Immunoperoxidase staining of cultured trophoblasts between 3.5 and 72 h after plating revealed a progressive increase in cytoplasmic pregnancy-specific beta 1-glycoprotein, hCG, and human placental lactogen concomitant with increasing numbers of aggregates and syncytia. At all time points examined, occasional single cells positive for these markers were identified. RIA of the spent culture media for hCG revealed a significant increase in secreted hCG, paralleling the increase in hCG-positive cells and syncytia identified by immunoperoxidase methods. We conclude that human cytotrophoblasts differentiate in culture and fuse to form functional syncytiotrophoblasts.
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PMID:Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae. 351 58

The factors that regulate the release of human placental lactogen (hPL) are poorly understood. To determine whether hPL is regulated by a factor(s) in pregnancy serum, placental explants were exposed for up to 9 h to a pool of serum samples from 50 women in the third trimester. In static explant cultures, the addition of the serum (0.6-10.8 mg protein/ml) caused a dose-dependent and reversible increase in hPL release during a 6-h period. The maximum release by the explants exposed to pregnancy serum was 200-250% greater than that of control explants, and the half-maximal dose was 2-3 mg/ml. Perifusion of placental explants with 15% pregnancy serum (final concentration, 10.5 mg protein/ml) also caused a significant release in hPL within 15 min, which reached a maximum of 200-225% above control levels. Two other pools of pregnancy serum samples as well as individual samples from four pregnant women also stimulated hPL release. Although pregnancy serum significantly stimulated hPL release, there was no increase in either the release of hCG or trichloroacetic acid-precipitable 35S-labeled proteins. Serum from nonpregnant women and men, as well as bovine serum, also stimulated hPL release, but their potencies were only 20-25% that of pregnancy serum. Chicken and porcine serum (10.8 mg/ml each) caused only small (less than 10%) increases in hPL release, and purified human albumin and ovalbumin had no effect. Dialysis or ultrafiltration of pregnancy serum using membranes with mol wt exclusions of 10K daltons caused no loss of activity. Delipidation of pregnancy serum with acetone-ethanol or acid-charcoal also caused no loss of activity, but treatment with trypsin caused greater than 95% loss of activity. Purification of the stimulatory activity by successive chromatographies on Sephadex G-150, Cibacron blue, and Sephadex G-75 resulted in an approximately 800-fold increase in specific activity. Approximately 90% of the total activity eluted from Sephadex G-75 with an apparent mol wt of 31,000, the remainder eluted in the void volume. Although partially purified pregnancy serum stimulated hPL release, the active fractions did not affect the release of rat LH, FSH, or GH from rat pituitary cells or the release of PRL from human decidual explants. Incubation of placental explants in calcium-deficient medium blocked the stimulatory effect of the partially purified pregnancy serum by greater than 90%. These studies indicate that human serum contains a protein(s) that causes a specific, rapid, dose-dependent, and reversible increase in hPL release.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Characterization and partial purification of a serum protein which stimulates the release of human placental lactogen in vitro. 372 25


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