Isolation of a third bovine neurophysin.
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ABSTRACT:
SUBMITTER: Rauch R
PROVIDER: S-EPMC1185126 | biostudies-other | 1969 Nov
REPOSITORIES: biostudies-other
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Project description:Current evidence indicates that the ligand-facilitated dimerization of neurophysin is mediated in part by dimerization-induced changes at the hormone binding site of the unliganded state that increase ligand affinity. To elucidate other contributory factors, we investigated the potential role of neurophysin's short interdomain loop (residues 55-59), particularly the effects of loop residue mutation and of deleting amino-terminal residues 1-6, which interact with the loop and adjacent residues 53-54. The neurophysin studied was bovine neurophysin-I, necessitating determination of the crystal structures of des 1-6 bovine neurophysin-I in unliganded and liganded dimeric states, as well as the structure of its liganded Q58V mutant, in which peptide was bound with unexpectedly increased affinity. Increases in dimerization constant associated with selected loop residue mutations and with deletion of residues 1-6, together with structural data, provided evidence that dimerization of unliganded neurophysin-I is constrained by hydrogen bonding of the side chains of Gln58, Ser56, and Gln55 and by amino terminus interactions, loss or alteration of these hydrogen bonds, and probable loss of amino terminus interactions, contributing to the increased dimerization of the liganded state. An additional intersubunit hydrogen bond from residue 81, present only in the liganded state, was demonstrated as the largest single effect of ligand binding directly on the subunit interface. Comparison of bovine neurophysins I and II indicates broadly similar mechanisms for both, with the exception in neurophysin II of the absence of Gln55 side chain hydrogen bonds in the unliganded state and a more firmly established loss of amino terminus interactions in the liganded state. Evidence is presented that loop status modulates dimerization via long-range effects on neurophysin conformation involving neighboring Phe22 as a key intermediary.
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Project description:The interaction of oxytocin with bovine neurophysin II in 0.1 M-sodium phosphate, pH 5.8, was investigated by equilibrium-dialysis and sedimentation studies. Sigmoidality of the binding curve is attributed to isomerization, either hormone-induced or pre-existing, with preferential binding of oxytocin to one isomeric state. Results are consistent with a binding equation of the form r = (2P[S]+2PQ[S]2)/(1+2P[S]+PQ[S]2) and values of 0.7 X 10(5)M-1 and 1.3 X 10(5)M-1 for P and Q respectively. The significance of these two parameters in relation to current theories of allostery is also discussed.
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Project description:1. A procedure for the isolation of highly purified neurosecretory granules from the posterior lobe of the bovine pituitary gland is described. The preparation was free from contamination by the mitochondrial enzyme succinate dehydrogenase and the lysosomal enzyme cathepsin. 2. The biological activities of the neurosecretory granules were measured: the oxytocic activity was 11.61+/-1.30units and the pressor activity was 10.73+/-1.74units/mg. of protein. 3. A lysate of the isolated granules was shown to contain two proteins that appear to be identical with two of the constituents of neurophysin. 4. The constituents of neurophysin not present in neurosecretory granules could not be detected in any other subcellular fraction. It is suggested that the components of neurophysin not present in the neurosecretory granules arise as a result of the degradation of the two granular proteins.
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Project description:1. Bovine neurophysin-II contains 1mol of tyrosine residue/10000g of protein. This residue could be readily nitrated with tetranitromethane. On hydrolysis and amino acid analysis 1mol of 3-nitrotyrosine was found/10000g of protein. Starchgel electrophoresis at pH8.5 showed that nitration had converted the native protein into a single, more acidic species. The increase in acidity was consistent with the observed fall in pK of the tyrosine hydroxyl from 9.2 in native neurophysin to 7.3 in the nitrated protein. Further, the absence of any intermediate species, even under conditions of minimum substitution, confirmed that the molecular weight of the monomer is 10000. 2. O-Acetylation of the tyrosine residue was carried out with N-acetylimidazole, in conjunction with the reversible blocking of amino groups by citraconylation. The degree of O-acetylation, determined spectroscopically, was 0.9mol of O-acetyltyrosine/10000g of protein. 3. The hormone-binding ability of modified protein was tested by equilibrium dialysis and was found to be unchanged by either nitration or O-acetylation of the tyrosine residue. 4. Interaction of neurophysin-II and [8-arginine]-vasopressin gave rise to a characteristic difference spectrum with a peak at 286.8nm and shoulder at 279.6nm. Part of this hyperchromicity is thought to result from entry of the tyrosine residue at position 2 in the hormone into the hydrophobic environment of the binding site. With nitrated neurophysin-II a second peak appeared at 436nm, showing that the tyrosine of the protein is also perturbed. The very large red shift (84nm) in this region suggests that the 3-nitrotyrosyl residue not only enters a more hydrophobic environment on protein-hormone interaction, but is caused to ionize more fully by the approach of some positively charged group.
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Project description:Bovine foamy virus (BFV) is endemic in many countries, but has not been reported in Japan. A syncytium-forming virus was isolated from peripheral blood leukocytes of clinically healthy cattle on a farm in Kanagawa prefecture during a periodic epidemiological survey of viral diseases. The isolate was propagated in primary fetal bovine muscle cells and subsequently passaged in Madin-Darby bovine kidney cells. Since the isolate appeared to be distinct from the viruses with syncytium-forming ability previously isolated in Japan, we attempted to identify it using genomic analyses and electron microscopy. A phylogenetic analysis revealed that the isolate belongs to the bovine foamy virus cluster and is highly similar to a BFV strain isolated in China. A sero-epidemiological survey was performed using agar gel immunodiffusion test with the isolated virus as the antigen, and five of the 57 cattle tested were found to be seropositive.
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Project description:1. The native hormone-binding proteins, neurophysin-I and -II, have been isolated from acetone-desiccated bovine pituitary posterior lobes. 2. Neurophysin-I and -II are present in approximately equal quantities in the tissue and are localized in the neurosecretory granules. 3. The apparent molecular weight, determined by equilibrium sedimentation of neurophysin-I, was 19000 and that of neurophysin-II was 21000; their sedimentation coefficients, S(20,w), were 1.66 and 2.02s respectively. 4. Neurophysin-I and -II are similar in amino acid composition. Neurophysin-II was distinguished from neurophysin-I by the absence of histidine. 5. The proteins form complexes with oxytocin as well as with vasopressin. Complexes of both proteins with [8-arginine]-vasopressin have been crystallized. 6. Bioassay of the pressor and oxytocic activities of the crystals shows that neurophysin-I binds three molecules of either vasopressin or oxytocin whereas neurophysin-II binds only two molecules of each hormone per molecule of protein. Complexes containing two molecules of oxytocin and one molecule of [8-arginine]-vasopressin per molecule of protein are formed by neurophysin-I and -II; both proteins appear to possess three polypeptide-binding sites/molecule.
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Project description:G-protein-coupled receptors transmit stimuli (light, taste, hormone, neurotransmitter, etc.) to the intracellular signaling systems, and rhodopsin (Rh) is the most-studied G-protein-coupled receptor. Rh possesses an 11-cis retinal as the chromophore, and 11-cis to all-trans photoisomerization leads to the protein structural changes in the cytoplasmic loops to activate G-protein. Microbial rhodopsins are similar heptahelical membrane proteins that function as bacterial sensors, light-driven ion-pumps, or light-gated channels. Microbial rhodopsins possess an all-trans retinal, and all-trans to 13-cis photoisomerization triggers protein structural changes for each function. Despite these similarities, there is no sequence homology between visual and microbial rhodopsins, and microbial rhodopsins do not activate G-proteins. However, it was reported that bacteriorhodopsin (BR) chimeras containing the third cytoplasmic loop of bovine Rh are able to activate G-protein, suggesting a common mechanism of protein structural changes. Here we design chimeric proteins for Natronomonas pharaonis sensory rhodopsin II (SRII, also called pharaonis phoborhodopsin), which has a two-orders-of-magnitude slower photocycle than BR. Light-dependent transducin activation was observed for most of the nine SRII chimeras containing the third cytoplasmic loop of bovine Rh (from Y223, G224, Q225 to T251, R252, and M253), but the activation level was 30,000-140,000 times lower than that of bovine Rh. The BR chimera, BR/Rh223-253, activates a G-protein transducin, whereas the activation level was 37,000 times lower than that of bovine Rh. We interpret the low activation by the chimeric proteins as reasonable, because bovine Rh must have been optimized for activating a G-protein transducin during its evolution. On the other hand, similar activation level of the SRII and BR chimeras suggests that the lifetime of the M intermediates is not the simple determinant of activation, because SRII chimeras have two-orders-of-magnitude's slower photocycle than the BR chimera. Activation mechanism of visual and microbial rhodopsins is discussed on the basis of these results.
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