Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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The metY gene coding for a minor form of the initiator tRNA is the first gene of a complex polycistronic operon also encoding the transcription termination factor NusA and the translation initiation factor IF2. The mixed tRNA-mRNA polycistronic transcript is cleaved by RNase III in a hairpin structure downstream from the tRNA. This cleavage separates the tRNA from the mRNA and initiates the rapid degradation of the 5' extremity of the downstream mRNA. Dissociation of the structural (tRNA) and informational (mRNA) RNAs from this operon is also achieved by independent transcription in vivo. The presence of two transcription terminators located downstream from metY produces a small tRNAMetf2 precursor transcript, whereas an internal promoter situated between metY and the first open reading frame directs the transcription of only the protein-coding part of the operon.
J Mol Biol 1989 Nov 20
PMID:Cleavage by RNase III in the transcripts of the met Y-nus-A-infB operon of Escherichia coli releases the tRNA and initiates the decay of the downstream mRNA. 248 Oct 42

Heterologous radioreceptor assays, commonly using ovine prolactin, may generate inconsistent results since prolactin (PRL) from one species may be recognized as growth hormone in another. Homologous radioreceptor assays (RRA) are most similar to the in vivo hormone-tissue receptor environment; however, lactogenic homologous RRAs have been reported only for mouse hepatic membranes. In this study, an assay system was developed to investigate homologous binding for porcine PRL in porcine uterine tissue. The pig does not produce a decidual PRL or a placental lactogen; yet, PRL affects uterine physiology during reproductively important events. Optimal binding conditions established for porcine PRL homologous RRA include 150 micrograms membrane, 45,000 cpm labeled porcine PRL and 500 microliters sodium phosphate buffer pH 7.6, incubated at 25 degrees C for 24 h. Binding of porcine PRL tracer is very low (less than 3%); however, when tissue is treated with the chaotropic agent, MgCl2 (4 M), binding increases from 3 to 28%. Dissociation kinetics show a rate of 3.79 X 10(-6)/s initially, and then 1.63 X 10(-6)/s. Competition for labeled PRL on binding sites with unlabeled porcine PRL results in 80% displacement with unlabeled porcine prolactin (NSB) of 7% at 1000 ng. Affinity constant generated from homologous inhibition assays is 0.326 X 10(8) M-1. Porcine growth hormone (GH), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) do not displace porcine PRL tracer. These data describe a lactogenic homologous RRA for porcine endometrial membranes similar to that previously reported for murine hepatic tissue. Homologous RRAs may allow elucidation of PRL receptor characteristics with more similarity to the in vivo hormone-receptor milieu.
Mol Cell Endocrinol 1989 Jul
PMID:Porcine endometrial prolactin receptors detected by homologous radioreceptor assay. 250 73

recA protein forms stable filaments on duplex DNA at low pH. When the pH is shifted above 6.8, recA protein remains stably bound to nicked circular DNA, but not to linear DNA. Dissociation of recA protein from linear duplex DNA proceeds to a non-zero endpoint. The kinetics and final extent of dissociation vary with several experimental parameters. The instability on linear DNA is most readily explained by a progressive unidirectional dissociation of recA protein from one end of the filament. Dissociation of recA protein from random points in the filament is eliminated as a possible mechanism by several observations: (1) the requirement for a free end; (2) the inverse and linear dependence of the rate of dissociation on DNA length (at constant DNA base-pair concentration); and (3) the kinetics of exposure of a restriction endonuclease site in the middle of the DNA. Evidence against another possible mechanism, ATP-mediated translocation of the filament along the DNA, is provided by a novel effect of the non-hydrolyzable ATP analog, ATP gamma S, which generally induces recA protein to bind any DNA tightly and completely inhibits ATP hydrolysis. We find that very low, sub-saturating levels of ATP gamma S completely stabilize the filament, while most of the ATP hydrolysis continues. If these levels of ATP gamma S are introduced after dissociation has commenced, further dissociation is blocked, but re-association does not occur. These observations are inconsistent with movement of recA protein along DNA that is tightly coupled to ATP hydrolysis. The recA nucleoprotein filament is polar and the protein binds the two strands asymmetrically, polymerizing mainly in the 5' to 3' direction on the initiating strand of a single-stranded DNA tailed duplex molecule. A model consistent with these results is presented.
J Mol Biol 1989 Feb 20
PMID:Dissociation pathway for recA nucleoprotein filaments formed on linear duplex DNA. 253 35

Binding of 1,4-dihydropyridine Ca2+ channel ligands was characterized as a function of membrane potential using saturation, competition, and kinetic measurements in cultured neonatal rat ventricular myocytes. The 1,4-dihydropyridine antagonist [3H]PN 200-110 bound to polarized cells (5.8 mM K+) with a KD value of 3.53 X 10(-9) M and a Bmax value of 50.1 fmol/mg of protein. In depolarized cells (50 mM K+), a KD value of 6.33 X 10(-11) M was found, reflecting a 55-fold increase in affinity; Bmax did not change upon depolarization. Dissociation rates (k-1) of [3H]PN 200-110 binding were faster in polarized cells (0.53 min-1) than in depolarized cells (0.018 min-1), but association rates (k1 of 2.17 X 10(8) and 2.27 X 10(8) min-1M-1 were not different in polarized and depolarized cells. The KD values calculated from the ratio of k-1/k1 accorded well with those determined from equilibrium binding assays. The enantiomers of Bay K 8644 and 202-791 and a series of nifedipine analogs inhibited specific binding of [3H]PN 200-110 in depolarized cells. In polarized cells, the affinities of the S-enantiomers (activators) were close to those in depolarized cells; however, the affinities of R-enantiomers (antagonists) were 50- to 65-fold lower. The effects of both (S)- and (R)-Bay K 8644 on [3H]PN 200-110 binding were mediated through increased apparent KD values, without changes in Bmax and nH. In depolarized cells, l-D600 and d-D600 partially inhibited [3H]PN 200-110 binding to a maximum of 71% and 56%, respectively; in polarized cells, l-D600 (d-D600 not measured) was ineffective on [3H]PN 200-110 binding. d-(cis)-Diltiazem, but not l-(cis)-diltiazem, partially inhibited (maximum 30%) specific binding of [3H]PN 200-110 in depolarized cells, but potentiated (maximum 79%) binding in polarized cells. The potentiating effect of d-(cis)-diltiazem was mediated through an increase in affinity without change in Bmax of [3H]PN 200-110 binding. (S)-Bay K 8644 potentiated 45Ca2+ uptake into the cells, with an EC50 value of 4.26 X 10(-10) M; concentrations higher than 10(-7) M were inhibitory, producing a biphasic concentration-response relationship. (R)-Bay K 8644 inhibited 80 mM K+-stimulated 45Ca2+ uptake with an IC50 value of 2.11 X 10(-9) M. These pharmacologic values correlate well with the binding affinities.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1989 Apr
PMID:Voltage-dependent binding of 1,4-dihydropyridine Ca2+ channel antagonists and activators in cultured neonatal rat ventricular myocytes. 253 61

Heat-labile specific binding sites for [3H]3-methylhistidine2-TRH [( 3H]Me-TRH) were demonstrated on chicken adenohypophysial membranes. These binding sites were of both high affinity (dissociation constant, Kd = 15.53 nM) and low capacity (maximum binding capacity, Bmax = 8.73 pmol/g tissue) and of low affinity (Kd = 104.5 nM) and high capacity (Bmax = 32.41 pmol/g). Binding of [3H]Me-TRH to the pituitary membranes was greater at 4 degrees C than at 39 degrees C and occurred with rate constants (k1) of 1.6 x 10(6) and 3.39 x 10(6) M-1 min-1 respectively. Dissociation of [3H]Me-TRH binding at 4 degrees C occurred with a rate constant (k-1) of 0.125 min-1. Binding sites for [3H]Me-TRH were found in the cephalic lobe of the pituitary gland (the location of most lactotrophs and thyrotrophs) and in the caudal lobe (the location of most somatotrophs). The number of binding sites was greater in the caudal lobe than in the cephalic lobe, although the affinity of [3H]Me-TRH binding did not differ. The binding of [3H]Me-TRH to caudal lobe membranes was displaced by Me-TRH, TRH, pGlu-His-Pro-Gly-NH2 and [Glu1]-TRH, with half-maximal effective doses of 33 nM, 70.7 nM, 1.23 microM and 22 microM respectively, but not by [Phe2]-TRH, TRH free acid or His-Pro-diketopiperazine. The number of caudal lobe binding sites for [3H]Me-TRH in old birds was less than that in young ones, and the number of binding sites was increased in birds deprived of food for 48 h. TRH-induced GH secretion in birds would thus appear to be mediated by specific receptors on caudal lobe somatotrophs, and these results suggest that physiological changes in GH secretion (during growth and periods of fasting) are causally related to the abundance of TRH-binding sites.
J Mol Endocrinol 1989 Jul
PMID:Thyrotrophin-releasing hormone (TRH)-induced growth hormone secretion in fowl: binding of TRH to pituitary membranes. 254 27

Kinetic parameters and equilibrium association constants (K) are reported for a panel of anti-bovine serum albumin (BSA) monoclonal antibodies (MAb) immobilized onto agarose particles. For 12 covalently immobilized MAb of moderate affinity (K = 0.25 x 10(8)-1.2 x 10(8) M-1) measured dissociation time constants varied two orders of magnitude, from 2.1 to 410 min. Directly measured association rate parameters agree with values calculated from measured equilibrium and dissociation rate parameters. Dissociation time constants and equilibrium association constants were also determined for eight MAb immobilized biospecifically (via their Fc regions). A significantly lower K was observed with those MAb which were covalently immobilized as opposed to biospecifically immobilized. These decreases in K appear to reflect decreased association rates rather than increased dissociation rates. The data suggest that, for the MAb described herein, dissociation rates do not correlate with equilibrium association constants.
Mol Immunol 1989 Feb
PMID:Dissociation kinetics of antigen-antibody interactions: studies on a panel of anti-albumin monoclonal antibodies. 264 12

We have studied the interactions of the Sp1 and IID transcription factors with a simple RNA polymerase II promoter. The adenovirus E1B core promoter consists essentially of a GC box and a TATA box, binding sites for the Sp1 and IID transcription factors, respectively. The E1B promoter is accurately transcribed in vitro using a mammalian transcription system. Sp1 activates E1B transcription in vitro in reactions using IID factor isolated from either human or yeast cells. In DNase I footprinting studies, Sp1 bound rapidly to its recognition sequence even at 0 degrees C (t1/2 less than 1 min). In contrast, yeast IID bound more slowly (t1/2 approximately 6 min at 25 degrees C) and required thermal energy for stable binding to the TATA box sequence. Dissociation rates were measured by the addition of specific oligonucleotide competitors to preformed DNA-protein complexes. Sp1 dissociates rapidly (t1/2 less than 1 min) at 25 degrees C, while yeast IID dissociates with an estimated t1/2 of 1 h at 25 degrees C. Sp1 and yeast IID bound to the E1B promoter simultaneously but independently. The rates of binding and dissociation of these factors were not significantly affected by the presence of the other factor. Bound Sp1 factor did not alter or enhance the yeast IID footprint. Oligonucleotide challenge of in vitro transcription reactions indicated that Sp1 also did not enhance the binding of the human IID factor to the E1B promoter. Thus the Sp1 factor activates transcription of the E1B gene by a mechanism that does not enhance the DNA-binding activity of the IID factor. Sp1 factor activates E1B transcription by 5- to 10-fold in vitro. Under these in vitro transcription conditions, transcripts due to reinitiation from an individual promoter complex contribute only a small portion of the total yield of E1B transcripts. Thus Sp1 cannot activate transcription by increasing the rate of initiation events per complex. Instead it appears that Sp1 acts by increasing the number of productive transcription complexes formed in vitro.
Mol Cell Biol 1989 Aug
PMID:Sp1 activates transcription without enhancing DNA-binding activity of the TATA box factor. 267 69

Dissociation and reassociation of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinases I and II were studied in intact AtT20 cells. Cells were stimulated with 50 microM forskolin to raise intracellular cAMP levels and induce complete dissociation of R and C subunits. After the removal of forskolin from the incubation medium cAMP levels rapidly declined to basal levels. Reassociation of R and C subunits was monitored by immunoprecipitation of cAMP-dependent protein kinase activity using anti-R immunoglobulins. The time course for reassociation of R and C subunits paralleled the loss of cellular cAMP. Total cAMP-dependent protein kinase activity and the ratio of protein kinase I to protein kinase II seen 30 min after the removal of forskolin was the same as in control cells. Similar results were seen using crude AtT20 cell extracts treated with exogenous cAMP and Mg2+. Our data showed that after removal of a stimulus from AtT20 cells inactivation of both cAMP-dependent protein kinase isoenzymes occurred by the rapid reassociation of R and C subunits to form holoenzyme. Our studies also showed that half of the type I regulatory subunit (RI) present in control cells contained bound cAMP. This represented approximately 30% of the cellular cAMP in nonstimulated cells. The cAMP bound to RI was resistant to hydrolysis by cyclic nucleotide phosphodiesterase but was dissociated from RI in the presence of excess purified bovine heart C. The RI subunits devoid of C may function to sequester cAMP and, thereby, prevent the activation of cAMP-dependent protein kinase activity in nonstimulated AtT20 cells.
Mol Endocrinol 1988 May
PMID:In situ reassociation of the regulatory and catalytic subunits of 3',5'-cyclic adenosine monophosphate-dependent protein kinase isoenzymes in AtT20 cells. 284 55

The influence of pH, temperature, ethylene glycol, urea, chaotropic anions and excess unlabelled secreted mouse prolactin (smPRL) on the dissociation kinetics of 125I-iodosmPRL from mouse hepatic receptors was investigated. The destabilization of smPRL-receptor complexes by chaotropic anions followed the typical trend of the Hofmeister series: I- greater than Br- greater than Cl- greater than F-. Increasing the temperature of the dissociation reaction from 8 degrees C to 23 degrees C and 30 degrees C caused partial dissociation of 125I-iodosmPRL-receptor complexes. Dissociation of 125I-iodosmPRL from mouse hepatic receptors was pH dependent, with the slowest rate of dissociation occurring at pH 8 and the fastest rate of dissociation occurring at pH 5 and 6. Both ethylene glycol and urea accelerated the rate of dissociation of 125I-iodosmPRL from mouse hepatic receptors in a concentration-dependent manner. Dissociation of 125I-iodosmPRL from mouse hepatic receptors was 6-fold faster in the presence of excess unlabelled smPRL than in its absence. The results of these investigations suggest that both protonation/de-protonation reactions and hydrophobic interactions play important roles in stabilizing the smPRL-receptor complex. In addition, they suggest that cooperative interactions may be involved in the binding of smPRL to mouse hepatic receptors.
Mol Cell Endocrinol 1986 Feb
PMID:Studies on dissociation of mouse prolactin from mouse hepatic receptors. 300 86

Dissociation of charged residues on the surface of immunoglobulins was analysed by an Mn2+ probe ESR method that has been developed in our previous work. Several kinds of IgG proteins and their Fab and Fc fragments were used for the experiments. The pH dependence of the intensity of ESR signals was analysed. It was shown that the number of Asp, Glu and His residues on the surface of Fc is about twice as many as that of Fab. The accessible surface area of amino acid residues calculated using X-ray crystallographic data is quite consistent with the present ESR experiments. This indicates that the number of the Asp, Glu and His residues on the surface of IgG molecules in solution is similar to that in the crystal. The Mn2+-probe ESR method was also applied to other classes of immunoglobulins, i.e. IgA and IgM. It was demonstrated that the IgA protein, which is known to lack the ability to bind Clq, has on the surface of it a smaller number of Asp, Glu and His residues as compared to IgG and IgM proteins. On the basis of these results obtained by the Mn2+-probe ESR method, we suggest that the Clq molecule, which is a basic protein, interacts favorably with the Fc portion whose surface is more negatively charged with Asp and Glu residues, compared to the Fab portion. Fine adjustment of fitting of the head of the Clq molecule into the CH2 domain of the Fc portion presumably follows for optimum binding. It was also demonstrated that Ser and Thr residues are much more abundant on the surface of Fab than in the case of Fc. We suggest that the Ser and Thr residues on the surface of Fab play an important role for binding of C4b upon activation of the complement system.
Mol Immunol 1986 Mar
PMID:Mn2+-probe ESR method for the analyses of the dissociation of charged residues on the surface of immunoglobulins. 301 23


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