Effect of pertussis toxin on the heart muscarinic-cholinergic receptors and their function

Administration of pertussis toxin to rats induced a significant increase in heart rate that was evident as soon as 24 hours after the administration of the toxin and that persisted for at least 15 days. Electrical stimulation of the vagus decreased dramatically the heart rate of control animals but was unable to do it so in rats treated with pertussis toxin. In cardiac membranes muscarinic agonists decreased adenylate cyclase activity (approximately equal to 20-25%); no effect was observed in membranes obtained from toxin-treated animals. Agonist displacement of antagonist binding [( 3H] Quinuclidinyl benzilate) indicated that treatment with pertussis toxin decreased the proportion of receptors in the high affinity state for agonists. All these data suggest that blockade of the parasympathetic tone plays a key role in the induction of tachycardia by pertussis toxin.     

Disulfides of the lutropin receptor

Affinity cross-linking of the lutropin receptor with 125I-human choriogonadotropin (hCG) on porcine granulosa cells produced four distinct homone-receptor complexes under reducing conditions. They contain 18-, 24-, 28-, and 34-kDa components (Ji, I., Bock, J. H., and Ji, T. H. (1985) J. Biol. Chem. 260, 12815-12821). Photoaffinity labeling and cross-linking produced 136-, 102-, and 74-kDa hCG-receptor complexes under reducing conditions and the 136-kDa complex under nonreducing conditions. In addition, the unreduced 102-kDa complex was seen in photoaffinity labeling but not in cross-linking. When the unreduced 136-kDa complex was reduced, the 102- and 74-kDa complexes were generated, indicating release of the 34- and the 28-kDa components in two steps. When the unreduced 102-kDa complex was reduced, the 74-kDa complex was produced, indicating the release of a 28-kDa component. The 74-kDa complex could not be reduced but was cleaved by alkaline treatment to produce the hCG alpha beta dimer. The results indicate that the 24-kDa component is released from the 74-kDa complex, since the apparent mass of the hCG alpha beta dimer on gels is 50 kDa. The 24-kDa component appears to be the initial site for photoaffinity labeling or cross-linking and to be disulfide linked to the 28-kDa component which is in turn disulfide linked to the 34-kDa component. These intercomponent disulfides exist in some receptors but not all. Formation of the disulfide-linked 136-kDa band required the presence of a sulfhydryl-blocking agent, N-ethylmaleimide. In particular, the 34-kDa component was vulnerable to reduction. There was no significant evidence of disulfides between the hormone and any of the receptor components.     

二氧化硫熏气对小麦叶片中1-氨基环丙烷-1-羧酸、1-(丙二酰氨基)环丙烷-1-羧酸含量的影响及与乙烯产生的关系

在SO_2熏气9h过程中,小麦叶片中乙烯先上升,约6h达高峰,后下降;ACC含量则随熏气时间的延长而上升。停止熏气,乙烯继续下降,ACC含量也明显降低。MACC含量从熏气3h后不断上升,脱离接触后仍继续增加。6-BA预处理对SO_2引起的乙烯和ACC上升有促进作用,但对MACC含量无明显影响。SO_2熏气提高了乙烯形成酶活性。6-BA预处理对SO_2伤害有保护作用。对逆境乙烯的产生与调节作用进行了讨论。     

Kinetic studies of the unfolding-refolding of horse muscle phosphoglycerate kinase induced by guanidine hydrochloride

The kinetics of the unfolding and refolding of horse muscle phosphoglycerate kinase were studied with three different signals: fluorescence emission intensity at 336 nm (excitation at 292 nm), ellipticity at 220 nm, and enzyme activity. The results corroborate the conclusion on the existence of intermediates in the folding pathway obtained from equilibrium studies. Kinetic studies showed at least two phases of refolding, as revealed by fluorescence as well as by circular dichroism measurements. During the fast phase, an intermediate was formed with a fluorescence intensity higher than that of the native protein, but devoid of enzyme activity. The fluorescence emission spectrum of this intermediate was determined. Only the slow phase was detected for the unfolding process; it was not attributable to proline isomerization. Several models were assumed, and simulated kinetics derived from these models were compared with the experimental results. A plausible one accounting for most of the data is proposed.     

Composition of cross-linked 125I-follitropin-receptor complexes

Both of the alpha and beta subunits of intact human follitropin (FSH) were radioiodinated with 125I-sodium iodide and chloramine-T and could be resolved on sodium dodecyl sulfate-polyacrylamide gels. Radioiodinated FSH was affinity-cross-linked with a cleavable (nondisulfide) homobifunctional reagent to its membrane receptor on the porcine granulosa cell surface as well as to a Triton X-100-solubilized form of the receptor. Cross-linked samples revealed three additional bands of slower electrophoretic mobility, corresponding to 65, 83, and 117 kDa, in addition to the hormone bands. The hormone alpha beta dimer band corresponded to 43 kDa. Formation of the three bands requires the 125I-hormone to bind specifically to the receptor with subsequent cross-linking. Binding was prevented by an excess of the native hormone but not by other hormones. A monofunctional analog of the cross-linking reagent failed to produce the three bands. Reagent concentration-dependent cross-linking revealed that their formation was sequential; smaller complexes formed first and then larger ones. When gels of cross-linked complexes were treated to cleave covalent cross-links and then electrophoresed in a second dimension, 18-, 22-, and 34-kDa components were released, in addition to the alpha and beta subunits of the hormone.     

Production of human c-myc protein in insect cells infected with a baculovirus expression vector.

A cDNA fragment coding for human c-myc was inserted into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. Insect cells infected with the recombinant virus produced significant amounts of c-myc protein, which constituted the major phosphoprotein component in these cells. By immunoprecipitation and immunoblot analysis, two proteins of 61 and 64 kilodaltons were detected with c-myc-specific antisera. The insect-derived proteins were compared with recombinant human c-myc-encoded proteins synthesized in Escherichia coli and Saccharomyces cerevisiae cells. The c-myc gene product was found predominantly in the nucleus by subcellular fractionation of infected insect cells.     

Unfolding-refolding transition of a hinge bending enzyme: horse muscle phosphoglycerate kinase induced by guanidine hydrochloride

The unfolding-refolding transition of horse muscle phosphoglycerate kinase induced by guanidine hydrochloride was studied under equilibrium conditions using four different signals: fluorescence intensity at 336 nm, UV difference absorbance at 286 and 292 nm, ellipticity at 220 nm, and enzyme activity. From the following arguments, we found that the process deviates from a two-state model and intermediates are significantly populated even at equilibrium: (1) the noncoincidence of the transition curves and (2) the asymmetry of the transition curve obtained from CD measurements. From these different data and the thermodynamic analysis, it was suggested that the two domains of the horse muscle phosphoglycerate kinase refold independently of one another with different equilibrium constants, the most favorable constant referring to the folding of the C-terminal domain which contains all tryptophans.     

接触二氧化硫后小麦叶片中逆境乙烯的生物合成

AVG和AOA强烈抑制二氧化硫处理小麦叶片中乙烯产生和ACC合成,对MACC的形成也有一定的抑制作用。CoGl_2明显抑制乙烯产生,而ACC大量积累,MACC含量则未因ACC增加而相应增加。DNP和CCCP也抑制乙烯产生,但前者引起ACC大量积累,后者引起ACC含量下降。CHI对乙烯产生和ACC形成均显示强烈的抑制作用,同时也明显抑制MACC形成。这表明小麦叶片接触SO_2引起的逆境乙烯也是循蛋氨酸→SAM→ACC→乙烯途径。