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排序方式: 共有279条查询结果,搜索用时 296 毫秒
1.
J M Stadel  R Rebar  S T Crooke 《Biochemistry》1987,26(18):5861-5866
Preincubation of turkey erythrocytes with isoproterenol is associated with (1) 50-60% attenuation of agonist-stimulated adenylate cyclase activity, (2) altered mobility of the beta-adrenergic receptor on sodium dodecyl sulfate-polyacrylamide gels, and (3) increased phosphorylation of the beta-adrenergic receptor. Using a low-cross-linked polyacrylamide gel, the beta-adrenergic receptor protein from isoproterenol-desensitized cells, labeled with 32P or with the photoaffinity label 125I-(p-azidobenzyl)carazolol, can be resolved into a doublet (Mr congruent to 37,000 and Mr congruent to 41,000) as compared to a single Mr congruent to 37,000 beta-adrenergic receptor protein from control erythrocytes. The appearance of the doublet was dependent on the concentration of agonist used to desensitize the cells. Incubation of erythrocytes with dibutyryl-cAMP did not promote formation of the doublet but decreased agonist-stimulated adenylate cyclase activity 40-50%. Limited-digestion peptide maps of 32P-labeled beta-adrenergic receptors using papain revealed a unique phosphopeptide in the larger molecular weight band (Mr congruent to 41,000) of the doublet from the agonist-desensitized preparation that was absent in the peptide maps of the smaller band (Mr congruent to 37,000), as well as control or dibutyryl-cAMP-desensitized receptor. These data provide evidence that maximal agonist-induced desensitization of adenylate cyclase coupled beta-adrenergic receptors in turkey erythrocytes occurs by a two-step mechanism.  相似文献   
2.
Mammalian phosphoinositide-specific phospholipase C isoenzymes   总被引:4,自引:0,他引:4  
Procaryotic and eucaryotic cells have evolved multiple pathways for communication with their external environment. The inositol 1,4,5-trisphosphate/diacylglycerol second messenger system is an example of such a signal transduction pathway which is present in multicellular eucaryotic organisms. Binding of an agonist to a specific cell surface receptor promotes rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate. The pivotal enzyme for this second messenger system is phosphoinositide-specific phospholipase C which hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate the two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. Recently, much progress has been made in the purification, characterization and cDNA cloning of multiple PI-PLC isoenzymes. The results of the recent studies on phosphoinositide-specific phospholipase C are reviewed.  相似文献   
3.
We have reconstituted protein translocation across plasma membrane vesicles of Escherichia coli using purified proOmpA and trigger factor, a 63 kd soluble protein. Treatment of membrane vesicles with urea inactivates them for translocation unless a factor present in cytoplasmic extracts is added during the translocation reaction. Sedimentation analysis showed that the stimulatory activity is of distinctly higher mol. wt than trigger factor. Cytoplasmic extracts from a strain that greatly overproduces the SecA protein are highly enriched in the stimulatory activity for untreated membranes and restore translocation to urea-treated membranes, suggesting that this protein is the stimulatory factor. This assay was used to monitor the isolation of SecA protein from the overproducing strain. The purified protein is soluble, yet binds peripherally to membranes with high affinity and supports translocation. Using pure proOmpA, SecA protein, trigger factor and urea-treated membranes, the protein export process was resolved into binding and translocation steps. We find that proOmpA binds to membrane vesicles with or without SecA protein, but that translocation only occurs when SecA was bound prior to proOmpA.  相似文献   
4.
E Crooke  B Guthrie  S Lecker  R Lill  W Wickner 《Cell》1988,54(7):1003-1011
We have isolated large amounts of E. coli outer-membrane protein A precursor (proOmpA). Purified proOmpA is active in membrane assembly, and this assembly is saturable with respect to the precursor protein. A proOmpA-Sepharose matrix allows affinity isolation of trigger factor, a soluble, 63,000 dalton monomeric protein that stabilizes proOmpA in assembly competent form. Comparison of trigger factor's amino-terminal sequence with those in a computer data bank and with those encoded by sec genes, as well as groEL and heat shock gene dnaK, suggests that trigger factor is encoded by a previously undescribed gene. Trigger factor and proOmpA form a 1:1 complex that can be isolated by gel filtration. Purified canine signal recognition particle (SRP) can also stabilize proOmpA for membrane insertion. This postribosomal activity of SRP suggests a unifying theme in protein translocation mechanisms.  相似文献   
5.
Isoproterenol-induced desensitization of turkey erythrocyte adenylate cyclase is accompanied (1) by a decrease in the mobility of beta-adrenergic receptor proteins, specifically photoaffinity labeled with 125I-(p-azidobenzyl)carazolol (125I-PABC), on sodium dodecyl sulfate (SDS)-polyacrylamide gels and (2) by a 2-3-fold increase in phosphate incorporation into the beta receptor [Stadel, J.M., Nambi, P., Shorr, R. G. L., Sawyer, D. F., Caron, M. G., & Lefkowitz, R. J. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3173]. Analysis of 32P-labeled beta receptors partially purified by affinity chromatography and subsequently hydrolyzed in 6 N HCl revealed that the beta receptor from control erythrocytes contained only phosphoserine and that agonist-promoted phosphorylation of the receptor in desensitized cells occurred on serine residues. Comparison of limited-digest peptide maps of 125I-PABC-labeled beta receptors from control and desensitized erythrocytes reveals distinctly different sensitivities of the two beta receptors to cleavage by chymotrypsin and Staphylococcus aureus protease. The altered mobility of the 125I-PABC-labeled beta receptor from desensitized erythrocytes was eliminated when 5 M urea was included in the SDS-polyacrylamide gels. Limited-digest peptide mapping of 32P-labeled beta receptors from control and desensitized cells with the protease papain identified a unique phosphorylated peptide in desensitized preparations. Our results are consistent with the hypothesis that the altered mobility of beta-receptor proteins on SDS gels following desensitization is due to changes in conformation promoted by prolonged exposure to agonists.  相似文献   
6.
Conditionally lethal Escherichia coli mutants in secY (prlA) show defective export of proteins to the periplasm and outer membrane. It has been proposed that this gene and other sec genes must act on pro-OmpA at an early stage of protein synthesis in order to allow later translocation to occur. We have described a temperature-sensitive mutation in which the secYts function is impaired at the nonpermissive temperature (Ito, K. (1984) Mol. Gen. Genet. 197, 204-208). A plasmid bearing the wild-type secY gene under the control of the lactose operon (Shiba, K., Ito, K., Yura, T., and Cerretti, D. P. (1984) EMBO J. 3, 631-635) has been introduced into this mutant strain. We now report that the in vivo chase of pulse-labeled full length pro-OmpA to mature OmpA is accelerated by inducing the synthesis of the wild-type secY protein at the end of the period of pulse labeling. We have also assayed the requirements for secY function for in vitro protein translocation. Membranes derived from secY ts cells which were incubated at 42 degrees C were inactive in vitro in the post-translational uptake and processing of pro-OmpA. Thus, the secY protein can act post-translationally, enhancing the translocation of completed pro-OmpA polypeptide chains across the plasma membrane.  相似文献   
7.
Yeast metallothionein function in metal ion detoxification   总被引:10,自引:0,他引:10  
A genetic approach was taken to test the function of yeast metallothionein in metal ion detoxification. A yeast strain was constructed in which the metallothionein locus was deleted (cup1 delta). The cup1 delta strain was complemented with normal or mutant metallothionein genes under normal or constitutive regulatory control on high copy episomal plasmids. Metal resistance of the cup1 delta strain with and without the metallothionein-expressing vectors was analyzed. The normally regulated metallothionein gene conferred resistance only to copper (1000-fold); constitutively expressed metallothionein conferred resistance to both copper (500-fold) and cadmium (1000-fold), but not to mercury, zinc, silver, cobalt, nickel, gold, platinum, lanthanum, uranium, or tin. Two mutant versions of the metallothionein gene were constructed and tested for their ability to confer metal resistance in the cup1 delta background. The first had a deletion of a highly conserved amino acid sequence (Lys-Lys-Ser-Cys-Cys-Ser). The second was a hybrid gene consisting of the sequences coding for the first 20 amino acids of the yeast protein fused to the monkey metallothionein gene. Expression of these genes under the CUP1 promoter provided significant protection from copper, but none of the other metals tested. These results demonstrate that there is significant flexibility in the structural requirements for metallothionein to function in copper detoxification and that yeast metallothionein is also capable of detoxifying cadmium under conditions of constitutive expression.  相似文献   
8.
9.
J Garner  E Crooke 《The EMBO journal》1996,15(9):2313-2321
The capacity of DnaA protein to initiate DNA synthesis at the chromosomal origin is influenced profoundly by the tightly bound nucleotides ATP and ADP. Acidic phospholipids can catalyze the conversion of inactive ADP-DnaK protein into the active ATP form. Proteolytic fragments of the nucleotide form of DnaA protein were examined to determine regions of the protein critical for functional interaction with membranes. A 35 kDa chymotryptic and 29 kDa tryptic fragment retained the tightly bound nucleotide. The fragments, whose amino-termini are within three residues of each other, but differ at their carboxyl ends, showed strikingly different behavior when treated with acidic phospholipids. The larger chymotryptic fragment released the bound nucleotide in the presence of acidic, but not neutral phospholipids. In contrast, the smaller tryptic fragment was inert to both forms of phospholipids. Acidic membranes, but not those composed of neutral phospholipids, protect from tryptic digestion a small portion of the segment that constitutes the difference between the 29 and 35 kDa fragments. The resulting 30 kDa tryptic fragment, which possesses this protected region, interacts functionally with acidic membranes to release the bound effector nucleotide. Inasmuch as the anionic ganglioside GM1, a compound structurally dissimilar to acidic glycerophospholipids, efficiently releases the nucleotide from DnaA protein, an acidic surface associated with a hydrophobic environment is the characteristic of the membrane that appears crucial for regulatory interaction with DnaA protein.  相似文献   
10.
The degradation of many proteins requires their prior attachment to ubiquitin. Proteolytic substrates are characteristically multiubiquitinated through the formation of ubiquitin-ubiquitin linkages. Lys-48 of ubiquitin can serve as a linkage site in the formation of such chains and is required for the degradation of some substrates of this pathway in vitro. We have characterized the recessive and dominant effects of a Lys-48-to-Arg mutant of ubiquitin (UbK48R) in Saccharomyces cerevisiae. Although UbK48R is expected to terminate the growth of Lys-48 multiubiquitin chains and thus to exert a dominant negative effect on protein turnover, overproduction of UbK48R in wild-type cells results in only a weak inhibition of protein turnover, apparently because the mutant ubiquitin can be removed from multiubiquitin chains. Surprisingly, expression of UbK48R complements several phenotypes of polyubiquitin gene (UB14) deletion mutants. However, UbK48R cannot serve as a sole source of ubiquitin in S. cerevisiae, as evidenced by its inability to rescue the growth of ubi1 ubi2 ubi3 ubi4 quadruple mutants. When provided solely with UbK48R, cells undergo cell cycle arrest with a terminal phenotype characterized by replicated DNA, mitotic spindles, and two-lobed nuclei. Under these conditions, degradation of amino acid analog-containing proteins is severely inhibited. Thus, multiubiquitin chains containing Lys-48 linkages play a critical role in protein degradation in vivo.  相似文献   
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