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Peroxisomal membrane proteins (PMPs) from the Swiss-Webster mouse are analyzed and compared to those of rats and humans using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. A purification procedure for fresh mouse, rat, or human biopsy liver which enriches peroxisomal/mitochondrial marker enzyme ratios over 100-fold is characterized. When analyzed by SDS-PAGE, membranes of purified liver peroxisomes are shown to contain the same complement of 145-, 70-, 55-, 36-, and 22-kDa PMPs in rats, mice, and humans. A rabbit polyclonal antibody raised against mouse peroxisomal membranes demonstrates immunoreactivity to 145- and 70-kDa proteins in fresh liver homogenates from all three species and in control or Zellweger syndrome fibroblasts from humans. Human autopsy or placental tissues which were refrigerated before analysis exhibited 105-, 55-, and 36-kDa peptides which may be derived from the 145- and 70-kDa peptides. Such conversions, if related to degradation, may explain difficulties in purifying peroxisomes from human autopsy specimens. Variable amounts of the 55-kDa peptide also occurred in mouse adrenal and lung, and the conversion of higher to lower molecular weight PMPs could not be demonstrated by in vitro incubation of mouse liver. Further definition of the structure and variability of mammalian PMPs should be helpful in understanding polyenzymopathies such as Zellweger syndrome. 相似文献
14.
Characterization of the Fc gamma receptor on human platelets 总被引:4,自引:0,他引:4
IgG-containing immune complexes may play a role in the immune destruction of human platelets by interacting with an Fc gamma receptor on the platelet surface. We studied the platelet Fc gamma receptor and characterized its interaction with IgG ligand and anti-Fc gamma receptor monoclonal antibodies. Oligomers of IgG, but not monomeric IgG, bound to platelets and the number of binding sites was significantly increased at low ionic strength. Ligand-binding studies indicated that normal human platelets express a single Fc gamma receptor (Fc gamma RII) with 8559 +/- 852 sites per cell, Kd = 12.5 +/- 1.7 X 10(-8) M using trimeric IgG. Results of studies with bivalent and Fab monoclonal anti-Fc gamma RII were consistent with each Fc gamma receptor expressing two epitopes recognized by the antibody. The number of Fc gamma binding sites and affinity of binding were unchanged by the presence of 2.0 mM Mg2+ or 10 micrograms/ml cytochalasin B. Platelet stimulation with thrombin or ADP in the presence of fibrinogen also did not alter the number of Fc gamma binding sites or the affinity of binding. However, platelets preincubated with 5 microM dexamethasone expressed a decreased number of Fc gamma binding sites as well as decreased IgG-dependent platelet aggregation. Platelets from patients with Glanzmann's thrombasthenia and from patients with the Bernard Soulier syndrome expressed a normal number and affinity of Fc gamma binding sites. The data suggest that platelet Fc gamma RII binding of trimeric IgG occurs independent of actin filament interaction, Mg2+, ADP, or thrombin and does not require GPIIb/IIIa or GPIIb/IIIa-fibrinogen interaction. Furthermore, this receptor appears to be normally expressed on GPIb-deficient platelets and susceptible to modulation by glucocorticoids. Finally, the Fc gamma-binding protein was isolated from whole platelets as a 220-kDa protein which upon reduction dissociates into 50,000 Mr subunits. 相似文献
15.
Aorta smooth myosin contains two types of light chain, LC20 and LC17, which fold together with the N-terminal region of each heavy chain to form the globular head region of myosin. We demonstrate an altered conformation of LC20 after its separation from heavy chain by high concentrations of urea, on the basis of the following evidende: 1) A polyclonal antibody against LC20 was not able to recognize this conformationally altered form; 2) Myosin reconstituted from heavy chains and urea-dissociated light chains exhibited extremely low ATPase activity. Circular dichroism unfolding profiles showed that light chains dissociated from heavy chains by SDS appeared to be more stable than those generated by urea dissociation. 相似文献
16.
The cAMP receptor on the surface of aggregation competent Dictyostelium discoideum cells specifically binds [3H]cAMP in an oscillatory manner with a periodicity of 2 min. The oscillatory cAMP-binding component is developmentallly regulated and has the nucleotide specificity expected for recognition of chemotactic signals. The concentration dependence of the peak amplitudes of cAMP binding exhibit an apparent threshold at 10(-8) M cAMP. The threshold concentration for cAMP binding that we measure is consistent with the concentration dependence of signal relay (cAMP secretion) and the chemotactic response. The kinetic data of binding and dissociation are very rapid, consistent with the time course of oscillations in receptor capacity (affinity). Specific binding oscillations are destroyed by heat or chymotrypsin but are insensitive to trypsin or glycosidase. A plasma membrane localization of receptor is supported by enrichment of cAMP binding in a plasma membrane preparation from differentiated cells. Receptor oscillations with a 2-min period are preserved in the membrane preparations, and the peak amplitudes are increased about 10-fold consistent with the enrichment of other plasma membrane markers. The alternating change in the receptor's binding capacity for cAMP may be the basis of the relay refractory period as well as the primary oscillator involved in the generation of postreceptor events such as stimulation of adenylate cyclase, cAMP secretion, and cellular movement, all of which have been previously shown to oscillate. 相似文献
17.
Mapping of the adenosine 5'-triphosphate binding site of type II calmodulin-dependent protein kinase
The specificity of the ATP-binding site of the type II calmodulin-dependent protein kinase was probed with 25 analogues of ATP modified at various positions of the molecule. The analogues were compared by their ability to compete with ATP in the protein kinase reaction. The result of this comparison indicates that the enzyme is most sensitive to modifications at, or replacement of, the purine moiety. Changes at the triphosphate chain are much better tolerated, although the enzyme exhibited a selective sensitivity to changes in the conformation of this group. The smallest contribution to the specificity of ATP binding appears to be made by the ribose ring. The Ki values obtained for a subset of these analogues were compared to those previously reported for phosphorylase b kinase and the cyclic nucleotide dependent protein kinases [Flockhart, D. A., Freist, W., Hoppe, J., Lincoln, T. M., & Corbin, J. D. (1984) Eur. J. Biochem. 140, 289-295]. A striking similarity in the responses of these protein kinases to modifications of the ATP molecule suggests that the type II calmodulin-dependent protein kinase is related to these enzymes. Support for this conclusion was provided, recently, through comparisons of the deduced primary structures of the alpha and beta subunits of the type II calmodulin-dependent protein kinase with the protein sequences of the catalytic subunits of phosphorylase b kinase and cAMP-dependent protein kinase [Hanley, R. M., Means, A. R., Ono, T., Kemp, B. E., Burgin, K. E., Waxham, N., & Kelly, P. T. (1987) Science (Washington, D.C.) 237, 293-297; Bennett, M. K., & Kennedy, M. B. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1794-1798], which indicated areas of extensive homology. 相似文献
18.
Autophosphorylation plays an essential role in proteolytic activation of the type II calmodulin-dependent protein kinase (CaM kinase II). Limited proteolysis of CaM kinase II by trypsin, alpha-chymotrypsin, and Ca2+-stimulated neutral protease (calpain) yielded a catalytically active kinase fragment only when the holoenzyme was autophosphorylated prior to proteolysis. Slightly larger, inactive fragments were obtained from nonphosphorylated CaM kinase II, regardless of whether Ca2+/calmodulin or Mg2+/ATP were present or absent. The active fragment exhibited Ca2+/calmodulin-dependent kinase activity with kinetic parameters identical with those of the activated holoenzyme. The key autophosphorylation site of CaM kinase II was absent from the active fragment which indicates that proteolysis can effectively uncouple the activation state and Ca2+/calmodulin independence of the kinase from the action of phosphoprotein phosphatases. Because autophosphorylation exerts such a tight control over this irreversible process, proteolytic activation of CaM kinase II by intracellular proteases offers an attractive mechanism for prolonging the effects of Ca2+ at the synapse. 相似文献
19.
The semidian rhythm in flowering response of Pharbitis nil in relation to dark period time measurement and to a circadian rhythm 总被引:1,自引:0,他引:1
When seedlings of Pharbitis nil Choisy, cv. Violet, are exposed to a single inductive dark period at 27°C, brief interruptions with red light (R) can be promotive after 2–3 h of darkness but increasingly inhibitory to flowering up to the 8–9th h of darkness. This rhythmic response to R interruptions can be advanced in phase by > 1 h when the preceding light period is interrupted with far-red (FR) 2 h before darkness (FR -2 h) or with FR – 15 h, whereas FR –8 h or FR–22 h retard the rhythm. These shifts in the R interruption rhythm are paralleled by equal shifts in the length of the dark period required for flowering. Brief FR interruptions of darkness displayed a similar rhythm which was also advanced by FR –2 h and retarded by FR –8 h. We conclude therefore that the semidian rhythm in the light, which we have previously described, continues through at least the first 12 h of darkness, is manifested in the R interruption rhythm, and determines the critical night length. A circadian rhythm with a marked effect on flowering was also identified, but several lines of evidence suggest that the circadian and semidian rhythms have independent additive effects on flowering and do not appear to show phase interaction. 相似文献
20.
Methyl bromide (MeBr) has broad microbicidal activity, but its use as a disinfectant for food is limited by the resulting bromide residues. Increasing the MeBr concentration, exposure temperature, or exposure period of a treatment tended to increase both the microbicidal efficacy of MeBr and the bromide residues. Its sporicidal activity was less at high than at low relative humidity within the range of 20 to 99%. Both the efficacy and the resulting residues of a MeBr treatment varied inversely with the load of product in a fumigation chamber due to sorption of the fumigant. Fumigation tests with almond kernels inoculated with Escherichia coli or Salmonella typhimurium indicated that MeBr can be used to disinfect whole nut kernels without resulting in excessive bromide residues, although the MeBr level necessary is higher than that normally used for insect control. 相似文献