scholarly journals Nonequilibrium kinetics of a cyclic GMP-binding protein in dictyostelium discoideum

1982 ◽  
Vol 94 (2) ◽  
pp. 271-278 ◽  
Author(s):  
PJM Van Haastert ◽  
H Van Walsum ◽  
FJ Pasveer
Keyword(s):  
Dictyostelium Discoideum ◽  
Cyclic Gmp ◽  
Binding Proteins ◽  
Binding Protein ◽  
Kinetic Constants ◽  
Nonequilibrium Conditions ◽  
Kinetics Of ◽  
Stimulation Of

Chemoattractants added to cells of the cellular slime mold dictyostelium discoideum induce a transient elevation of cyclic GMP levels, with a maximum at 10 s and a recovery of basal levels at approximately 25 s after stimulation. We analyzed the kinetics of an intracellular cGMP binding protein in vitro and in vivo. The cyclic GMP binding protein in vitro at 0 degrees C can be described by its kinetic constants K(1)=2.5 x 10(6) M(- 1)s(-1), k(-1)=3.5 x 10(-3)s(-1), K(d)=1.4 x 10(-9) M, and 3,000 binding sites/cell. In computer simulation experiments the occupancy of the cGMP binding protein was calculated under nonequilibrium conditions by making use of the kinetic constants of the binding protein and of the shape of the cGMP accumulations. These experiments show that under nonequilibrium conditions by making use of the kinetic constants of the binding protein and the shape of the cGMP accumulations. These experiments show that under nonequilibrium conditions the affinity of the binding protein for cGMP is determined by the rate constant of association (k(1)) and not by the dissociation constant (k(d)). Experiments in vivo were performed by stimulation of aggregative cells with the chemoattractant cAMP, which results in a transient cGMP accumulation. At different times after stimulation with various cAMP concentrations, the cells were homogenized and immediately thereafter the number of binding proteins which were not occupied with native cGMP were determined. The results of these experiments in vivo are in good agreement with the results of the computer experiments. This may indicate that: (a) The cGMP binding protein in vivo at 22 degrees C can be described by its kinetic constants: K(1)=4x10(6)M(-1)s(-1) and K(-1)=6x10(-3)s(-1). (b) Binding the cGMP to its binding protein is transient with a maximum at about 20-30 s after chemotactic stimulation, followed by a decay to basal levels, with a half-life of approximately 2 min. (c) The cGMP to its binding proteins get half maximally occupied at a cGMP accumulation of δ[cGMP](10)=2x10(-8) M, which corresponds to an extracellular stimulation of aggregative cells by 10(-10) M cAMP. (d) Since the mean basal cGMP concentration is approximately 2x10(-7) M, the small increase of cGMP cannot be detected accurately. Therefore the absence of a measurable cGMP accumulation does not argue against a cGMP function. (e) There may exist two compartments of cGMP: one contains almost all the cGMP of unstimulated cells, and the other contains cGMP binding proteins and the cGMP which accumulates after chemotactic stimulation. (f) From the kinetics of binding, the cellular responses to the chemoattractant can be divided into two classes: responses which can be mediated by this binding protein (such as light scattering, proton extrusion, PDE induction, and chemotaxis) and responses which cannot be (solely) mediated by this binding protein such as rlay, refractoriness, phospholipids methylation, and protein methylation.

Possible role of cyclic GMP in stimulus-secretion coupling by salt gland of the duck

1979 ◽  
Vol 237 (5) ◽  
pp. C200-C204 ◽  
Author(s):  
D. J. Stewart ◽  
J. Sax ◽  
R. Funk ◽  
A. K. Sen
Keyword(s):  
Cyclic Amp ◽  
Guanylate Cyclase ◽  
Cyclic Gmp ◽  
Salt Gland ◽  
Domestic Ducks ◽  
Stimulus Secretion Coupling ◽  
Stimulation Of

Stimulation of salt galnd secretion in domestic ducks in vivo increased the cyclic GMP concentration of the tissue, but had no effect on cyclic AMP levels. Methacholine, which is known to stimulate sodium transport by the glands both in vivo and in vitro, stimulated ouabain-sensitive respiration in salt gland slices. Cyclic GMP stimulated ouabain-sensitive respiration to the same extent as methacholine. Guanylate cyclase stimulators, hydroxylamine and sodium azide, also stimulated ouabain-sensitive respiration. The stimulation of ouabain-sensitive respiration by methacholine was blocked either by atropine or by removal of calcium from the incubation medium. The stimulation of ouabain-sensitive respiration by cyclic GMP still occurred in the absence of calcium. The above observations seem to indicate that cyclic GMP acts as a tertiary link in the process of stimulus-secretion coupling in the tissue.

scholarly journals The 36-kilodalton embryonic-type cytoplasmic polyadenylation element-binding protein in Xenopus laevis is ElrA, a member of the ELAV family of RNA-binding proteins.

1997 ◽  
Vol 17 (11) ◽  
pp. 6402-6409 ◽  
Author(s):  
L Wu ◽  
P J Good ◽  
J D Richter
Keyword(s):  
Xenopus Laevis ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Dominant Negative ◽  
Cytoplasmic Polyadenylation ◽  
Element Binding Protein

The translational activation of several maternal mRNAs in Xenopus laevis is dependent on cytoplasmic poly(A) elongation. Messages harboring the UUUUUAU-type cytoplasmic polyadenylation element (CPE) in their 3' untranslated regions (UTRs) undergo polyadenylation and translation during oocyte maturation. This CPE is bound by the protein CPEB, which is essential for polyadenylation. mRNAs that have the poly(U)12-27 embryonic-type CPE (eCPE) in their 3' UTRs undergo polyadenylation and translation during the early cleavage and blastula stages. A 36-kDa eCPE-binding protein in oocytes and embryos has been identified by UV cross-linking. We now report that this 36-kDa protein is ElrA, a member of the ELAV family of RNA-binding proteins. The proteins are identical in size, antibody directed against ElrA immunoprecipitates the 36-kDa protein, and the two proteins have the same RNA binding specificity in vitro. C12 and activin receptor mRNAs, both of which contain eCPEs, are detected in immunoprecipitated ElrA-mRNP complexes from eggs and embryos. In addition, this in vivo interaction requires the eCPE. Although a number of experiments failed to define a role for ElrA in cytoplasmic polyadenylation, the expression of a dominant negative ElrA protein in embryos results in an exogastrulation phenotype. The possible functions of ElrA in gastrulation are discussed.

Function of Tubulin Binding Proteins in Vivo

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 69-80 ◽  
Author(s):  
James A Fleming ◽  
Leticia R Vega ◽  
Frank Solomon
Keyword(s):  
Binding Proteins ◽  
Binding Protein ◽  
Yeast Cells ◽  
Gene Products ◽  
Salvage Pathway ◽  
Tubulin Binding ◽  
Mutant Cells ◽  
Β Tubulin

Abstract Overexpression of the β-tubulin binding protein Rbl2p/cofactor A is lethal in yeast cells expressing a mutant α-tubulin, tub1-724, that produces unstable heterodimer. Here we use RBL2 overexpression to identify mutations in other genes that affect formation or stability of heterodimer. This approach identifies four genes—CIN1, CIN2, CIN4, and PAC2—as affecting heterodimer formation in vivo. The vertebrate homologues of two of these gene products—Cin1p/cofactor D and Pac2p/cofactor E—can catalyze exchange of tubulin polypeptides into preexisting heterodimer in vitro. Previous work suggests that both Cin2p or Cin4p act in concert with Cin1p in yeast, but no role for vertebrate homologues of either has been reported in the in vitro reaction. Results presented here demonstrate that these proteins can promote heterodimer formation in vivo. RBL2 overexpression in cin1 and pac2 mutant cells causes microtubule disassembly and enhanced formation of Rbl2p-β-tubulin complex, as it does in the α-tubulin mutant that produces weakened heterodimer. Significantly, excess Cin1p/cofactor D suppresses the conditional phenotypes of that mutant α-tubulin. Although none of the four genes is essential for viability under normal conditions, they become essential under conditions where the levels of dissociated tubulin polypeptides increase. Therefore, these proteins may provide a salvage pathway for dissociated tubulin heterodimers and so rescue cells from the deleterious effects of free β-tubulin.

scholarly journals Transient kinetics of a cGMP-dependent cGMP-specific phosphodiesterase from Dictyostelium discoideum.

1984 ◽  
Vol 98 (2) ◽  
pp. 709-716 ◽  
Author(s):  
P J Van Haastert ◽  
M M Van Lookeren Campagne
Keyword(s):  
Steady State ◽  
Dictyostelium Discoideum ◽  
Binding Sites ◽  
Catalytic Site ◽  
Transient Kinetics ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Rate Of Activation ◽  
Stimulation Of

Chemotactic stimulation of Dictyostelium discoideum cells induces a fast transient increase of cGMP levels which reach a peak at 10 s. Prestimulation levels are recovered in approximately 30 s, which is achieved mainly by the action of a guanosine 3',5'-monophosphate cGMP-specific phosphodiesterase. This enzyme is activated about fourfold by low cGMP concentrations. The phosphodiesterase has two distinct cGMP-binding sites: a catalytic site and an activator site. cAMP does not bind to either site; inosine 3',5'-monophosphate (cIMP) binds only to the catalytic site, whereas 8-bromoguanosine 3',5'-monophosphate (c-b8-GMP) preferentially binds to the activator site. For detailed kinetical measurements we have used [3H]cIMP as the substrate and c-b8-GMP as the activator. c-b8-GMP activated the hydrolysis of [3H]cIMP by reducing the Km, whereas the Vmax was not altered. The hydrolysis of [3H]cIMP was measured at 5-s intervals by using a new method for the separation of 5'-nucleotides from cyclic nucleotides. The hydrolysis of [3H]cIMP by nonactivated enzyme or by preactivated enzyme was linear with time, which indicates that a steady state is reached at the catalytic site within 5 s after addition of the substrate. In contrast, the hydrolysis of [3H]cIMP immediately after activation by 0.1 microM c-b8-GMP was not linear with time, but increased in a quasi-exponential manner with a time constant of 21 s. This suggests that a steady state at the activator site is only reached in 30-45 s after addition of the activator. The on-rate of activation (k1) was 3 X 10(5) M-1s-1 for c-b8-GMP and 1.4 X 10(5) M-1s-1 for cGMP. The off-rate of activation (k-1) was 0.03 s-1 for both c-b8-GMP and cGMP. The significance of these kinetic constants for the chemoattractant-mediated cGMP response in vivo is discussed.

EFFECT OF LUTEINIZING HORMONE ON THE CONCENTRATION OF CYCLIC GMP IN RABBIT OVARIES

1978 ◽  
Vol 79 (2) ◽  
pp. 251-252
Author(s):  
V. V. PATWARDHAN ◽  
A. LANTHIER
Keyword(s):  
Luteinizing Hormone ◽  
Cyclic Amp ◽  
Cyclic Gmp ◽  
Ovarian Tissue ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Direction Opposite ◽  
Stimulation Of

Laboratoire d'Endocrinologie, Hôpital Notre-Dame et Département de Medicine, Université de Montréal, Montréal, Canada (Received 28 June 1978) Cyclic AMP has been implicated as an intermediate in some of the actions of luteinizing hormone (LH) on ovarian tissues, such as stimulation of steroidogenesis (LeMaire & Marsh, 1975). Both in vitro (Marsh, Butcher, Savard & Sutherland, 1966) and in vivo (Armstrong, Dorrington & Robinson, 1976), stimulation with LH results in a rapid increase in the amount of cyclic AMP in ovarian tissues, which precedes the LH-induced increase in steroidogenesis. Recently, studies on rat ovaries (Grinwich, Ham, Hichens & Behrman, 1976; Ratner, 1976; Ratner & Sanborn, 1976) have indicated that the ovarian tissue content of cyclic GMP may also be regulated by LH, but in a direction opposite to that of cyclic AMP. In the rabbit, Goff & Major (1975) have shown that administration of human chorionic gonadotrophin (HCG) causes a biphasic increase

scholarly journals Heterogeneity and properties of hepatic dexamethasone-binding proteins

1979 ◽  
Vol 180 (1) ◽  
pp. 187-193 ◽  
Author(s):  
S L H Liu ◽  
T E Webb
Keyword(s):  
Rat Liver ◽  
Binding Proteins ◽  
Binding Protein ◽  
Novikoff Hepatoma ◽  
Liver Cytosol ◽  
Activated Complex ◽  
Simple Dissociation ◽  
Rat Liver Cytosol

Evidence from experiments in vivo and in vitro is presented for the presence of three species of dexamethasone-binding proteins in rat liver, which are identified by chromatography on Sepharose 6B or by isoelectric focusing. Although two of these species (DI and DII) possess properties characteristic of a true receptor, the third binding protein (i.e. DIII), which migrates most slowly on Sepharose 6B, but has stability properties similar to protein DII, exhibits a 3-fold lower affinity for dexamethasone and the activated complex neither binds to DNA-cellulose nor translocates to the nucleus. Only the predominant liver receptor (DI), which is eluted first from Sepharose 6B, is present in Novikoff-hepatoma cytosol, suggesting that the major and minor species are not interconverted through simple dissociation during their isolation. The binding activities of all three species in the liver cytosol increase approx. 2-fold in vivo after adrenalectomy and show a transient 2-fold fall in vivo after the administration of cortisol. These changes in vivo in protein DIII shows a marked lag compared with those in proteins DI and DII, which change in parallel. It is therefore proposed that rat liver cytosol contains two dexamethasone receptors and a dexamethasone-binding protein that may be derived from these receptors.

Lactoferrin-binding proteins in Bifidobacterium bifidum

2002 ◽  
Vol 80 (1) ◽  
pp. 91-94 ◽  
Author(s):  
Woan-Sub Kim ◽  
Tetsuya Tanaka ◽  
Haruto Kumura ◽  
Kei-ichi Shimazaki
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Binding Proteins ◽  
Binding Protein ◽  
Bifidobacterium Bifidum ◽  
Molecular Weights ◽  
In Vivo Experiments ◽  
Bactericidal Effects

Lactoferrin is an iron-binding glycoprotein and its bacteriostatic and bactericidal effects on gram-positive and gram-negative bacteria are well known. On the other hand, it is known that certain kinds of lactic acid bacteria are resistant to its antibacterial effects. Moreover, it is reported that lactoferrin promotes the growth of bifidobacteria in in vitro and in vivo experiments. In our experiments, lactoferrin-binding protein was found both in the membrane and cytosolic fractions of Bifidobacterium bifidum Bb-11. The bifidobacteria were grown in anaerobic conditions with lactobacilli MRS broth containing cysteine, harvested by centrifugation, and processed by sonication. The lactoferrin-binding proteins on the PVDF-membrane transferred after SDS-PAGE were detected by far-Western (western-Western) method using biotinylated lactoferrin and streptavidin-labelled horse radish peroxidase. The molecular weights of the lactoferrin binding protein detected in the membrane fraction were estimated to be 69 kDa and those in cytosolic fractions were 20, 35, 50, and 66 kDa.Key words: lactoferrin, Bifidobacterium, lactic acid bacteria, binding protein.

Characterization of mercury-binding proteins in rat blood plasma

2018 ◽  
Vol 54 (54) ◽  
pp. 7439-7442 ◽  
Author(s):  
Yiling Li ◽  
Bin He ◽  
Qiying Nong ◽  
Guangbo Qu ◽  
Lihong Liu ◽  
...  
Keyword(s):  
Blood Plasma ◽  
Binding Proteins ◽  
Binding Protein ◽  
Protein Profiles ◽  
Rat Blood

Mercury-binding protein profiles in plasma in vitro and in vivo, which were unclear previously, were systematically investigated.

Effect of pH and inhibitors on beta-amyloid fibril assembly

1996 ◽  
Vol 54 ◽  
pp. 752-753
Author(s):  
Beverly E. Maleeff ◽  
Timothy K. Hart ◽  
Stephen J. Wood ◽  
Ronald Wetzel
Keyword(s):  
Amyloid Fibril ◽  
Peptide Fragment ◽  
Hydrophobic Peptides ◽  
Amyloid Fibril Formation ◽  
Effects Of Ph ◽  
Severity Of The Disease ◽  
Kinetics Of ◽  
The Brain

Alzheimer's disease is characterized post-mortem in part by abnormal extracellular neuritic plaques found in brain tissue. There appears to be a correlation between the severity of Alzheimer's dementia in vivo and the number of plaques found in particular areas of the brain. These plaques are known to be the deposition sites of fibrils of the protein β-amyloid. It is thought that if the assembly of these plaques could be inhibited, the severity of the disease would be decreased. The peptide fragment Aβ, a precursor of the p-amyloid protein, has a 40 amino acid sequence, and has been shown to be toxic to neuronal cells in culture after an aging process of several days. This toxicity corresponds to the kinetics of in vitro amyloid fibril formation. In this study, we report the biochemical and ultrastructural effects of pH and the inhibitory agent hexadecyl-N-methylpiperidinium (HMP) bromide, one of a class of ionic micellar detergents known to be capable of solubilizing hydrophobic peptides, on the in vitro assembly of the peptide fragment Aβ.

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