Participation of a trisaccharide-lipid in glycosylation of oviduct membrane glycoproteins.
Preincubation of a hen oviduct membrane preparation with UDP-Nactyl[14C]glucosamine and bacitracin, followed by incubation with GDP-mannose, leads to formation of a chloroform/methanol (2/1)-extractable glycolipid. Treatment of the lipid with mild acid results in the release of a trisaccharide shown to have the structure beta-mannosyl-N-acetylglucosamineyl-N-acetylglucosamine. Incubation of purified trisaccharide-lipid with oviduct membranes in the presence of sodium deoxycholate, Mn2+, and GDP-mannose leads to formation of a labeled glycoprotein with an apparent molecular weight of 25,000... (+info)
Endoribonuclease IV. A poly(A)-specific ribonuclease from chick oviduct. 1. Purification of the enzyme.
A new endoribonuclease, termed endoribonuclease IV, has been described. This enzyme has been isolated from chick oviducts and purified 15 000-fold in a 25% yield nearly to homogeneity. The nuclease, which specifically degrades poly(A), forms oligonucleotides of an average chain length of 10. These (A)-10 fragments are terminated by 3'-hydroxyl and 5'-phosphate groups. The enzyme has a pH optimum at 8.7, requires Mn2+ or Mg2+ as a cofactor, and has a molecular weight of about 45 000. (+info)
Endoribonuclease IV. 2. Further investigation on the specificity.
The poly(A)-specific endoribonuclease IV produces oligo(A) fragments of a chain length of 10 AMP nucleotides. One enzyme molecule performs 1700 cleavages per min; the cleavages occur randomly. The endoribonuclease IV is a nuclear enzyme which is present in the oviduct of quails in a concentration of 40 000 enzyme molecules per cell. Poly(A) segments on mRNA are selectively hydrolyzed by endoribonuclease IV; the poly(A)-free part of the RNA is not affected. After incubation with the enzyme, a residual oligo(A) stretch of 5 AMP nucleotides remains on poly(A)-rich RNA. The endoribonuclease IV does not disintegrate the polyribosomal complex after incubation in vitro and the enzyme has also no influence on the translational capacity of a cell-free protein-synthesizing system. (+info)
Quantitative estimates of cytoplasmic and nuclear oestrogen receptors in chick oviduct. Effect of oestrogen on receptor concentration and subcellular distribution.
(3H)Oestradiol exchange techniques were developed for the determination of specific oestrogen receptor site concentrations in the cytoplasm and nuclei of chick oviduct cells. Non-labelled, receptor-bound oestrogens were exchanged with (3H)oestradiol during a 24-h incubation at 20 degrees C, 2 h at 30 degrees C or 45 min at 3 degrees C. Both "soluble" and "insoluble" nuclear receptors were stable for at least 6 h at 30 degrees C and 3 degrees C but a proportion (approx. 30%) of cytoplasmic sites from withdrawn chickens were inactivated after 2 h at 20 degrees C. The magnum of 4-week-old immature chickens (weight = 15 mg) contained 0.20 pmol of oestrogen receptor which corresponds to 4275 receptor sites/cell, when it is assumed that all magnum cells have equal concentrations of receptor. In primarily stimulated chickens of similar age which had received 10x1 mg of oestradiol benzoate/day, the magnum weighed approximately 800 mg and contained 8.65 pmol of oestrogen receptor (4610 sites/cell). Withdrawal from primary oestrogenic stimulation for 3-6 weeks resulted in a 110 mg magnum which contained 1.20 pmol of receptor (2225 sites/cell). Oviducts from immature and withdrawn chickens had the majority (73-77%) of their oestrogen receptors sites in the receptor sites in the cytoplasmic fraction, while in primary stimulated chicken oviducts the majority (82%) of receptor sites were located in the nucleus. A single secondary injection of oestradiol, to oestrogen-withdrawn chickens, resulted in apparent translocation of cytoplasmic receptors to the nucleus during the first hour after injection. The magnitude of the decline in cytoplasmic receptor, and the concurrent increase in nuclear receptor concentration, was dose-dependent between 2 and 100 mug oestradiol/kg body weight. Larger doses of oestradiol up to 1 mg/kg did not increase the concentration of nuclear receptor above the maximum level seen at 100 mug oestradiol/kg. The initial rapid accumulation of nuclear receptor sites was followed by a period of progressive decline. The initial rapid accumulation of nuclear receptor sites was followed by a period of progressive decline. By 15 h after an injection of 100 mug oestradiol/kg, the concentration of nuclear sites had reached pre-injection levels. During the same time period, the depleted cytoplasmic receptor levels were replenished such that they reached control values by 12 h and were about 150% of the pre-injection level at 24 h. (+info)
Identification of the novel player deltaEF1 in estrogen transcriptional cascades.
Although many genes are regulated by estrogen, very few have been shown to directly bind the estrogen receptor complex. Therefore, transcriptional cascades probably occur in which the estrogen receptor directly binds to a target gene that encodes another transcription factor that subsequently regulates additional genes. Through the use of a differential display assay, a transcription factor has been identified that may be involved in estrogen transcriptional cascades. This report demonstrates that transcription factor deltaEF1 is induced eightfold by estrogen in the chick oviduct. Furthermore, the regulation by estrogen occurs at the transcriptional level and is likely to be a direct effect of the estrogen receptor complex, as it does not require concomitant protein synthesis. A putative binding site was identified in the 5'-flanking region of the chick ovalbumin gene identifying it as a possible target gene for regulation by deltaEF1. Characterization of this binding site revealed that deltaEF1 binds to and regulates the chick ovalbumin gene. Thus, a novel regulatory cascade that is triggered by estrogen has been defined. (+info)
The Caenorhabditis elegans mel-11 myosin phosphatase regulatory subunit affects tissue contraction in the somatic gonad and the embryonic epidermis and genetically interacts with the Rac signaling pathway.
Caenorhabditis elegans embryonic elongation is driven by cell shape changes that cause a contraction of the epidermal cell layer enclosing the embryo. We have previously shown that this process requires a Rho-associated kinase (LET-502) and is opposed by the activity of a myosin phosphatase regulatory subunit (MEL-11). We now extend our characterization and show that mel-11 activity is required both in the epidermis during embryonic elongation and in the spermatheca of the adult somatic gonad. let-502 and mel-11 reporter gene constructs show reciprocal expression patterns in the embryonic epidermis and the spermatheca, and mutations of the two genes have opposite effects in these two tissues. These results are consistent with let-502 and mel-11 mediating tissue contraction and relaxation, respectively. We also find that mel-11 embryonic inviability is genetically enhanced by mutations in a Rac signaling pathway, suggesting that Rac potentiates or acts in parallel with the activity of the myosin phosphatase complex. Since Rho has been implicated in promoting cellular contraction, our results support a mechanism by which epithelial morphogenesis is regulated by the counteracting activities of Rho and Rac. (+info)
Inhibition of an ecto-ATP-diphosphohydrolase by azide.
Cell surface ATPases (ecto-ATPases or E-ATPases) hydrolyze extracellular ATP and other nucleotides. Regulation of extracellular nucleotide concentration is one of their major proposed functions. Based on enzymatic characterization, the E-ATPases have been divided into two subfamilies, ecto-ATPases and ecto-ATP-diphosphohydrolases (ecto-ATPDases). In the presence of either Mg2+ or Ca2+, ecto-ATPDases, including proteins closely related to CD39, hydrolyze nucleoside diphosphates in addition to nucleoside triphosphates and are inhibited by millimolar concentrations of azide, whereas ecto-ATPases appear to lack these two properties. This report presents the first systematic kinetic study of a purified ecto-ATPDase, the chicken oviduct ecto-ATPDase (Strobel, R.S., Nagy, A.K., Knowles, A.F., Buegel, J. & Rosenberg, M.O. (1996) J. Biol. Chem. 271, 16323-16331), with respect to ATP and ADP, and azide inhibition. Km values for ATP obtained at pH 6.4 and 7.4 are 10-30 times lower than for ADP and the catalytic efficiency is greater with ATP as the substrate. The enzyme also exhibits complicated behavior toward azide. Variable inhibition by azide is observed depending on nucleotide substrate, divalent ion, and pH. Nearly complete inhibition by 5 mm azide is obtained when MgADP is the substrate and when assays are conducted at pH 6-6.4. Azide inhibition diminishes when ATP is the substrate, Ca2+ as the activating ion, and at higher pH. The greater efficacy of azide in inhibiting ADP hydrolysis compared to ATP hydrolysis may be related to the different modes of inhibition with the two nucleotide substrates. While azide decreases both Vmax and Km for ADP, it does not alter the Km for ATP. These results suggest that the apparent affinity of azide for the E.ADP complex is significantly greater than that for the free enzyme or E.ATP. The response of the enzyme to three other inhibitors, fluoride, vanadate, and pyrophosphate, is also dependent on substrate and pH. Taken together, these results are indicative of a discrimination between ADP and ATP by the enzyme. A mechanism of azide inhibition is proposed. (+info)
Analyses of oviductal pars recta-induced fertilizability of coelomic eggs in Xenopus laevis.
The acquisition of fertilizability in coelomic eggs of Xenopus laevis has been shown to be correlated with the physical, biochemical, and ultrastructural alterations of the egg envelope [coelomic envelope (CE)] induced during the passage of eggs through the pars recta portion of the oviduct. However, no direct evidence that the pars recta renders eggs fertilizable has yet been presented. In this study, we show that coelomic eggs are highly fertilizable when they are incubated with continuous shaking for 4 h at 15 degrees C in pars recta extract (PRE) derived from females prestimulated by pregnant mare serum gonadotropin. The PRE from pituitary-stimulated Bufo japonicus was as potent as homologous PRE in rendering Xenopus eggs fertilizable. Incubation of coelomic eggs in PRE for 30 min induced a dramatic increase in the rates of sperm binding to the envelope to a level equivalent to that exhibited by the envelope from uterine eggs (VEs). The CE-to-VE ultrastructural conversion and a 43k-to-41k hydrolysis of the envelope glycoprotein component started 5 min after, and were completed by 15 min after, the start of incubation in PRE and were accompanied by an exposure of a new N-terminal sequence typical to gp41. Thus, the biochemical and ultrastructural conversions and the sperm-binding activity of the envelope induced by PREs, although being prerequisite, were not sufficient to render coelomic eggs fully accessible to fertilizing sperm. (+info)