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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Calcium-dependent transglutaminase (TGase) activity, determined by incorporation of [1,4-14C]diaminobutane dihydrochloride (putrescine) into casein, was demonstrated in a light membrane fraction prepared from bovine calf testicular homogenates. Purification of these membranes by sucrose density gradient centrifugation produced a follicle-stimulating hormone (FSH) receptor-enriched fraction containing TGase activity which cosolubilized with the FSH receptor and could be incorporated with detergent-solubilized receptor into liposomes. In the present study, we show that calcium increases specific binding of FSH to receptor in a concentration-related manner, and is associated with an increase (13.2-fold at 20 mM) in the affinity (Ka) of the receptor with no significant (P greater than 0.05) change in receptor concentration. Treatment of the light membrane fraction with monodansylcadaverine (MDC, 1 mM), a specific inhibitor of TGase, did not affect specific binding of FSH, but resulted in only a 3.9-fold increase in Ka at 20 mM calcium with no change in receptor concentration. Specific binding of FSH to receptor at 4 degrees C was also enhanced by calcium. Scatchard analysis of competitive binding inhibition data showed a Ka at 20 mM calcium similar to that observed with MDC. Dissociation of [125I]hFSH-receptor complexes formed at 30 degrees C in the presence of calcium was significantly less than dissociation of complexes formed at 30 degrees C in the absence of calcium. When [125I]hFSH-receptor complexes were formed at 30 degrees C in the presence of calcium and dissociated in calcium-deficient buffer, dissociation increased 3-fold. Similar results were obtained in the presence of MDC.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Endocrinol 1992 Sep
PMID:Stabilization of follicle-stimulating hormone-receptor complexes may involve calcium-dependent transglutaminase activation. 135 84

The Salmonella typhimurium basal body, a part of the flagellar rotary motor, consists of four rings (denoted M, S, P and L) and a coaxial rod. Using low-dose electron microscopy and image averaging methods on negatively stained and frozen-hydrated preparations, we examined whole basal body complexes and subcomplexes obtained by dissociation in acid. Dissociation occurs in steps, allowing us to obtain images of substructures lacking the M ring, lacking the M and S rings, and lacking the M and S rings and the proximal portion of the rod. We obtained images of the L and P ring subcomplex. The existence of a subcomplex missing only the M ring suggests either that the S and M rings derive from two different proteins, or that the M ring is a labile domain of a single protein, which makes up both rings. At the 25 to 30 A resolution of our averaged images, the L, P and S rings appear cylindrically symmetric. Images of the M ring show variability that may be due to differences in angular orientation of the grid, but equally could be due to structural variations. Three-dimensional reconstructions of these structures from the averaged images reveal the internal structure and spatial organization of these components.
J Mol Biol 1992 Jan 05
PMID:Substructure of the flagellar basal body of Salmonella typhimurium. 137 May 43

The state of genes microinjected into mouse embryos was followed from the one-cell to the blastocyst stage using the polymerase chain reaction (PCR). Microinjected DNA was detected in all one-, two-, and four-cell injected embryos and in 44% of morula and 26% of blastocysts. Head-to-tail ligation of microinjected genes, a common feature of stably integrated transgene arrays, was detected in all embryos after injection of microinjected genes and occurred irrespective of the structure at the ends of the injected genes. Sensitivity of microinjected DNA to a methylation-dependent restriction endonuclease Dpn I was lost in all embryos by the two-cell stage (24 hr), indicating a change in DNA methylation, independent of transgene integration. Dissociation of blastomeres prior to compaction revealed a mosaic distribution of the microinjected DNA within the embryo and supports the notion that injected genes form a limited number of arrays, which segregate independently until they integrate into the genome or are degraded.
Mol Reprod Dev 1992 Dec
PMID:Fate of microinjected genes in preimplantation mouse embryos. 147 74

A constitutive estrogen-binding protein (EBP) has been identified in the cytosol of Pseudomonas aeruginosa, a Gram-negative bacterium. All 14 strains tested contained the EBP. Estradiol binding was rapid and maximal binding occurred by 90 min at 0 degrees C. Dissociation of estradiol from the binding protein occurred at a rate of 4.6 fmol/min with a t1/2 of 42 min. EBP binding was destroyed by protease treatment and at high temperature. Sodium molybdate had no effect on binding. The Kd determined by Scatchard analysis was 3.9 nM and the Bmax was 323 fmol/mg protein. The EBP sedimented at 8.9 S on sucrose density gradients. The presence of 0.4 M KCl increased estradiol binding 6-fold but did not cause a shift in the sedimentation value. Gel filtration of the native protein gave an estimated molecular weight of 215,000 and a Stokes radius of 50.2 A. Steroid binding specificity, in order of decreasing affinity, was estradiol, estrone, dihydrotestosterone, estriol, testosterone, progesterone and promegestone. Other steroid hormones tested did not compete for estradiol binding. Identification of an EBP in a bacterium allows a comparative analysis of other steroid-binding proteins in unicellular microorganisms.
J Steroid Biochem Mol Biol 1992 Aug
PMID:Identification of an estrogen-binding protein in Pseudomonas aeruginosa. 150 10

Binding of 125I-calcitonin gene-related peptide (125I-CGRP) to rat cerebellum membranes and the sensitivity to guanine nucleotides of binding were investigated. Cerebellum binding sites labeled by 125I-CGRP appear to be highly specific, inasmuch as CGRP inhibited binding with an IC50 of 100 pM but other peptides were inactive or much less active in displacing 125I-CGRP from these sites. 125I-CGRP binding sites in cerebellum membranes were saturable and of high affinity. Scatchard analysis of the saturation binding data revealed a homogeneous population of binding sites, with a KD of 224 +/- 28 pM and Bmax of 131 +/- 15 fmol/mg of protein. In the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) (100 microM), a single population of binding sites, with a KD of 464 +/- 77 pM and Bmax of 100 +/- 14 fmol/mg of protein, was observed. The kinetics of association of 125I-CGRP with cerebellum membranes were monophasic at all ligand concentrations tested. However, the observed association rate constant (kobs) was not dependent on [125I-CGRP] in a linear fashion in either the absence or the presence of GTP gamma S (100 microM). The kinetics of dissociation of 125I-CGRP from cerebellum membranes were multiexponential, with fast and slow dissociating components having rate constants of 0.34 +/- 0.01 and 0.025 +/- 0.001 min-1, respectively. The fast dissociating component represented 60 +/- 2% of the total specific binding sites. Dissociation of 125I-CGRP from cerebellum sites was much faster in the presence of GTP gamma S (100 microM) but still exhibited dissociation from two affinity components. The rate constants for these components of dissociation were 0.67 +/- 0.03 and 0.077 +/- 0.007 min-1, with the faster dissociating component representing 66 +/- 1% of the total specific binding sites. These findings provide the first evidence that CGRP receptors exist in multiple affinity states and that cerebellum CGRP receptors are regulated by guanine nucleotides. Our results also suggest the existence of two affinity states of the CGRP-receptor-guanine nucleotide-binding protein ternary complex.
Mol Pharmacol 1991 Jun
PMID:Multiple affinity forms of the calcitonin gene-related peptide receptor in rat cerebellum. 164 51

The null wx-ml allele contains a 409-bp Dissociation 1 (Ds1) element in exon 9 of the maize waxy (Wx) gene. In the absence of the autonomous Activator (Ac) element, the Ds1 element cannot transpose, and this allele encodes several Wx transcripts that arise following alternative splicing of Ds1 sequences from Wx pre-mRNA. Splicing involves the utilization of three 5' splice sites and three 3' splice sites. All but one of these splice sites are in Ds1 sequences near the ends of the element. The presence of 5' and 3' splice sites near the Ds1 termini and the element's small size and AT richness are features that distinguish Ds1 elements from all other known Ds elements. It is suggested that these features may enhance the ability of Ds1 to function as a mobile intron.
Mol Cell Biol 1991 Dec
PMID:The maize transposable Ds1 element is alternatively spliced from exon sequences. 165 27

The pharmacological, radioligand binding, and electrophysiological properties of FPL 64176, a new nondihydropyridine Ca2+ channel activator, were studied in rat tail artery, cardiac membranes, and A7r5 smooth muscle cells. FPL 64176 induced a contractile response, with an EC50 value of 2.11 x 10(-7) M. The maximum tension response to FPL 64176 was approximately 2-fold higher than that to (S)-Bay K 8644. FPL 64176 showed no significant inhibitory activity at concentrations up to 10(-5) M. The Ca2+ channel antagonists nifedipine, verapamil and diltiazem noncompetitively antagonized and completely relaxed the responses induced by FPL 64176. IC50 values of these three drugs were 5.22 x 10(-9), 1.31 x 10(-7), and 1.95 x 10(-7) M, respectively, for relaxing submaximum contractile responses to FPL 64176 (5 x 10(-7) M). The washout time for FPL 64176 was about 40 min, which was much longer than that for (S)-Bay K 8644 (within 1 min). FPL 64176 weakly inhibited (+)-[3H]PN 200-110, [3H]D888, and [3H]TA-3090 binding in rat cardiac membranes, with IC50 values of 1.04 x 10(-5) M and 7.03 x 10(-6) M for inhibition of (+)-[3H]PN 200-110 and [3H]TA-3090 binding, respectively, and with 23% inhibition of [3H]D888 binding at a FPL 64176 concentration of 1 x 10(-5) M. Dissociation kinetics of the three radioligands were allosterically accelerated by FPL 64176. Electrophysiological studies on the A7r5 smooth muscle cell line directly confirmed a large (approximately 14-fold) stimulatory effect on L-type Ca2+ current amplitude. The results suggest that FPL 64176 is a new type of Ca2+ channel activator with higher efficacy and a mechanism and site of action that are distinct from those for (S)-Bay K 8644.
Mol Pharmacol 1991 Nov
PMID:Pharmacological, radioligand binding, and electrophysiological characteristics of FPL 64176, a novel nondihydropyridine Ca2+ channel activator, in cardiac and vascular preparations. 171 69

In the absence of hormone, human progesterone receptors (PR) are recovered in the cytosolic fraction of cell lysates as a multimeric complex containing the steroid-binding polypeptide, heat shock protein-90 (hsp90), and heat shock protein-70 (hsp70). Activated forms of human PR that acquire the ability to bind to DNA are dissociated from hsp90, but retain association with hsp70. The present study has examined whether associated hsp70 has a function in receptor-DNA binding. When activated PR was bound to specific target DNA in a gel shift assay, no hsp70 was detectable in the PR-DNA complex, as evidenced by the failure of several antibodies to hsp70 to affect the mobility or the amount of complexes. To determine whether hsp70 might indirectly influence DNA-binding activity, we have examined the effect of hsp70 dissociation on PR-DNA-binding activity. Dissociation was achieved either by treatment of immunoaffinity-purified immobilized PR complexes with ATP or by the binding of PR complexes to ATP-agarose, followed by elution with high salt. Under both conditions, dissociation from hsp70 neither enhanced nor impaired the ability of PR to bind to specific DNA. These results suggest that hsp70 is not involved in PR binding to DNA, either directly by participating in DNA binding or indirectly by modulating PR-DNA-binding activity. This implies that hsp70 functions at an earlier stage in the receptor activation pathway. Consistent with the known involvement of hsp70 in stabilizing unfolded states of other target proteins, we propose that hsp70 may assist in nuclear transport of PR or in assembly-disassembly of the 8-10S multimeric complex.
Mol Endocrinol 1991 Dec
PMID:Evidence that heat shock protein-70 associated with progesterone receptors is not involved in receptor-DNA binding. 179 44

Studying biochemical events in human spermatogenesis requires separated populations of spermatogenic cells. Dissociation of these cells was performed by a Trypsin-DNAse method adapted from the technique used for rodents. Cell separation was performed by centrifugal elutriation. Seven populations were collected, one further purified by Percoll gradient centrifugation, giving nine different cell populations. The efficiency of the cell separation was evaluated by phase contrast microscopy, flow cytometric DNA analysis, and electron microscopy. Five populations were enriched in spermatids: two in round spermatids (87% and 73%), another in round (52%) and elongating (44%) spermatids, another constituted by 80% elongating spermatids, and the last by 90% elongated spermatids. Two of the four remaining populations were enriched in primary spermatocytes (74% and 54%); another population was the upper part of the Percoll gradient and constituted cytoplasmic lobes and residual bodies (89%); the last population was made up of various cells, with no specific enrichment. Electron microscopic observations revealed good preservation of the separated cells; only the flagella from elongated spermatids were lost. Furthermore, an unusual pattern of nucleoplasm distribution during stages 2-4 of spermatid differentiation was observed and its signification is discussed with regard to the shape of the human spermatozoon.
Mol Reprod Dev 1991 Nov
PMID:Preparation of spermatogenic cell populations at specific stages of differentiation in the human. 179 6

To characterize the immunoreactive glucocorticoid receptor (GR) protein present in "receptorless" (r-) mutants isolated from the glucocorticoid-sensitive (dexs) human leukemic cell line CEM-C7, binding of [3H]dexamethasone was determined in extracts prepared from the sensitive cell line 6TG1.1 and the r- mutant ICR27TK.3 after gentle freeze-thaw lysis and low-speed centrifugation. Under these conditions there was significant high-affinity binding activity in r- extracts assayed at 4 degrees C but not at 23 degrees C. Loss of binding at 23 degrees C was not a function of GR proteolysis or denaturation of the steroid-binding site and could be prevented by the addition of sodium molybdate. Dissociation of ligand from either activated or unactivated receptors in r- extracts was significantly more rapid than from receptors in extracts prepared from normal cells, suggesting that the defect in receptors in r- cells is the result of mutation in the ligand-binding site. While the rate of dissociation from unactivated receptors in r- extracts was linear, dissociation from receptors in extracts of 6TG1.1 cells was biphasic. Analysis of these dissociation curves, as well as dissociation from receptors in the B-cell line IM-9, indicated that the mutant gene present in r- cells is also present in the dexs parental cell line. This conclusion is consistent with our previous hypothesis (J.M. Harmon et al., Mol. Endocrinol., 3:734-743, 1989) that glucocorticoid-sensitive CCRF-CEM cells express both a normal (GR+) and a mutant (GR*) allele.
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PMID:Biochemical evidence that glucocorticoid-sensitive cell lines derived from the human leukemic cell line CCRF-CEM express a normal and a mutant glucocorticoid receptor gene. 191 46


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