共查询到20条相似文献,搜索用时 31 毫秒
1.
Gabriel G. Limas Matilde Salinas Ignacio Moneo Stefan Fischer Brigitte Wittmann-Liebold Enrique Méndez 《Planta》1990,181(1):1-9
Ten new proteins from rice (Oryza saliva L. cv. Bahia) including four protein-synthesis inhibitors and two immunoglobulin E (IgE)-binding proteins have been isolated and characterized. These proteins as well as one previously known component, -globulin, were purified from a 0.5 M NaCl extract of rice endosperm by a new, apparently non-denaturing, isolation procedure developed for rice proteins. The method is based on extractions of this complex protein mixture with a diluted volatile salt solution and an aqueous solution of ethanol. This preliminary step results in an improvement in the separation of these proteins, thus facilitating their subsequent purification by reversed-phased high-performance liquid chromatography. These new proteins have similar relative molecular masses (Mrs) from 11000 to 17000. The purity of the proteins was analyzed by micro two-dimensional gel electrophoresis. Four of these components were found to be in-vitro protein-synthesis inhibitors in a cell-free system from rat brain. The NH2-terminal amino-acid sequences of these four inhibitors were determined from 12 to 26 cycles after direct blotting of the separated proteins from electrophoresis gels. Three of these proteins with Mrs between 16000 and 17000 showed a high degree of homology ranging from 57% to 75% but seem to be unrelated to the fourth inhibitor. In addition, the -globulin and one of the new low-molecular-weight proteins of Mr 12500 seemed to show allergenic properties since they bound IgE antibodies from the sera of hypersensitive patients. Boths proteins have blocked NH2-terminal amino acids.Abbreviations HMW
high molecular weight
- IgE
immunoglobulin E
- LMW
low molecular weight
- Mr
relative molecular mass
- PAGE
polyacrylamide gel electrophoresis
- RP-HPLC
reversed-phase high-performance liquid chromatography
- SDS
sodium dodecyl sulphate
We thank F. Soriano and F. Colillia for technical assistance, and Shirley McGrath for secretarial work. We also appreciate the cheerful assistance of the members of Instituto Nacional de Semillas, specially Mr. L. Solaices, who provided samples of rice. This work was supported by a grant from Comision Asesora de Investigación Ciéntifica y Técnica. 相似文献
2.
Background
Pollens are important triggers for allergic asthma and seasonal rhinitis, and proteases released by major allergenic pollens can injure airway epithelial cells in vitro. Disruption of mucosal epithelial integrity by proteases released by inhaled pollens could promote allergic sensitisation.Methods
Pollen diffusates from Kentucky blue grass (Poa pratensis), rye grass (Lolium perenne) and Bermuda grass (Cynodon dactylon) were assessed for peptidase activity using a fluorogenic substrate, as well as by gelatin zymography. Following one- or two-dimensional gel electrophoresis, Coomassie-stained individual bands/spots were excised, subjected to tryptic digestion and analysed by mass spectrometry, either MALDI reflectron TOF or microcapillary liquid chromatography MS-MS. Database searches were used to identify allergens and other plant proteins in pollen diffusates.Results
All pollen diffusates tested exhibited peptidase activity. Gelatin zymography revealed high Mr proteolytic activity at ~ 95,000 in all diffusates and additional proteolytic bands in rye and Bermuda grass diffusates, which appeared to be serine proteases on the basis of inhibition studies. A proteolytic band at Mr ~ 35,000 in Bermuda grass diffusate, which corresponded to an intense band detected by Western blotting using a monoclonal antibody to the timothy grass (Phleum pratense) group 1 allergen Phl p 1, was identified by mass spectrometric analysis as the group 1 allergen Cyn d 1. Two-dimensional analysis similarly demonstrated proteolytic activity corresponding to protein spots identified as Cyn d 1.Conclusion
One- and two-dimensional electrophoretic separation, combined with analysis by mass spectrometry, is useful for rapid determination of the identities of pollen proteins. A component of the proteolytic activity in Bermuda grass diffusate is likely to be related to the allergen Cyn d 1. 相似文献3.
Promastigotes and amastigotes of Leishmania tropica were surface-radioiodinated using the lactoperoxidase technique. Detergent lysates of the labeled organisms were analyzed by two-dimensional gel electrophoresis. Analysis of radioiodinated promastigote membrane proteins revealed six major and some minor acidic polypeptides. Analysis of the amastigote membrane proteins revealed six major proteins, mostly acidic, and some poorly resolved basic proteins. Four of the major membrane proteins appeared to be common to the two parasitic forms (Mr 67,000, Mr 50,000, Mr 68,000, and Mr 80,000). These polypeptides were recognized by antipromastigote antibodies as well as antibodies from CBA/H mice that had recovered from infection. Peptide mapping confirmed their homology in the two parasite forms. One polypeptide appeared to be specific for the promastigote (Mr 50,000) and two polypeptides appeared to be specific for the amastigote form of the parasite (Mr 94,000 and Mr 43,000). 相似文献
4.
Jenny van Odijk Sigrid Sjölander Peter Brostedt Magnus P. Borres Hillevi Englund 《Clinical and molecular allergy : CMA》2017,15(1):18
Background
IgE sensitization to storage proteins from nuts and seed is often related to severe allergic symptoms. There is a risk of immunological IgE cross-reactivity between storage proteins from different species. The potential clinical implication of such cross-reactivity is that allergens other than the known sensitizer can cause allergic symptoms. Previous studies have suggested that kiwi seed storage proteins may constitute hidden food allergens causing cross-reactive IgE-binding with peanut and other tree nut homologs, thereby mediating a potential risk of causing allergy symptoms among peanut ant tree nut allergic individuals. The objective of this study was to investigate the degree of sensitization towards kiwi fruit seed storage proteins in a cohort of peanut allergic individuals.Methods
A cohort of 59 adolescents and adults with peanut allergy was studied, and self reported allergies to a number of additional foods were collected. Quantitative IgE measurements to seed storage proteins from kiwi and peanut were performed.Results
In the cohort, 23 out of the 59 individuals were reporting kiwi fruit allergy (39%). The frequency of IgE sensitization to kiwi fruit and to any kiwi seed storage protein was higher among peanut allergic individuals also reporting kiwi fruit allergy (P = 0.0001 and P = 0.01). A positive relationship was found between IgE levels to 11S globulin (r = 0.65) and 7S globulin (r = 0.48) allergens from kiwi and peanut, but IgE levels to 2S albumin homologs did not correlate. Patients reporting kiwi fruit allergy also reported allergy to hazelnut (P = 0.015), soy (P < 0.0001), pea (P = 0.0002) and almond (P = 0.016) to a higher extent than peanut allergic individuals without kiwi allergy.Conclusions
Thirty-nine percent of the peanut allergic patients in this cohort also reported kiwi fruit allergy, they displayed a higher degree of sensitization to kiwi storage proteins from both kiwi and peanut, and they also reported a higher extent of allergy to other nuts and legumes. On the molecular level, there was a correlation between IgE levels to 11S and 7S storage proteins from kiwi and peanut. Taken together, reported symptoms and serological findings to kiwi in this cohort of patients with concurrent allergy to peanut and kiwi fruit, could be explained by a combination of cross-reactivity between the 11S and 7S globulins and co-sensitization to the 2S albumin Act d 13.5.
Rabbit kidney brush-border membrane vesicles were exposed to bacterial protease which cleaves off a large number of externally oriented proteins. Na+-dependent d-glucose transport is left intact in the protease-treated vesicles. The protease-treated membrane was solubilized with deoxycholate and the deoxycholate-extracted proteins were further resolved by passage through Con A-Sepharose columns. Sodium-dependent d-glucose activity was found to reside in a fraction containing a single protein band of Mr ? 165000 which is apparently a dimer of Mr ? 85 000. When reconstituted and tested for transport, this protein showed Na+-dependent, stereo-specific and phlorizin-inhibitable glucose transport. Transport activity is completely recovered and is 20-fold increased in specific activity. A similar isolate was obtained from rabbit small intestinal brush-border membranes and kidneys from several other species of animals. 相似文献
6.
《Comparative biochemistry and physiology. B, Comparative biochemistry》1993,104(3):551-556
- 1.1. Co-isolating proteins (Mr 170,000–220,000) from sodium channel preparations made from the electric organ of the electric eel (Electrophorus electricus) were detected on Western blots using monoclonal a antibodies.
- 2.2. Similar protein patterns were seen on immunoblots containing immunoprecipitated protein from eel muscle and brain tissues but not heart.
- 3.3. These co-isolating proteins could be separated from the mature TTX-sensitive channel protein (Mr 280,000) using a lentil lectin-Sepharose column.
- 4.4. The 180 kDa proteins do not appear to be channel-related and can be detected as contaminants in electroplax sodium channel preparations using the monoclonal antibodies described here.
7.
Bernardi ML Giangrieco I Camardella L Ferrara R Palazzo P Panico MR Crescenzo R Carratore V Zennaro D Liso M Santoro M Zuzzi S Tamburrini M Ciardiello MA Mari A 《PloS one》2011,6(11):e27856
Background
Food allergy is increasingly common worldwide. Tools for allergy diagnosis measuring IgE improved much since allergenic molecules and microarrays started to be used. IgE response toward allergens belonging to the same group of molecules has not been comprehensively explored using such approach yet.Objective
Using the model of lipid transfer proteins (LTPs) from plants as allergens, including two new structures, we sought to define how heterogeneous is the behavior of homologous proteins.Methods
Two new allergenic LTPs, Act d 10 and Act c 10, have been identified in green (Actinidia deliciosa) and gold (Actinidia chinensis) kiwifruit (KF), respectively, using clinically characterized allergic patients, and their biochemical features comparatively evaluated by means of amino acid sequence alignments. Along with other five LTPs from peach, mulberry, hazelnut, peanut, mugwort, KF LTPs, preliminary tested positive for IgE, have been immobilized on a microarray, used for IgE testing 1,003 allergic subjects. Comparative analysis has been carried out.Results
Alignment of Act d 10 primary structure with the other allergenic LTPs shows amino acid identities to be in a narrow range between 40 and 55%, with a number of substitutions making the sequences quite different from each other. Although peach LTP dominates the IgE immune response in terms of prevalence, epitope recognition driven by sequence heterogeneity has been recorded to be distributed in a wide range of behaviors. KF LTPs IgE positive results were obtained in a patient subset IgE positive for the peach LTP. Anyhow, the negative results on homologous molecules allowed us to reintroduce KF in patients'' diet.Conclusion
The biochemical nature of allergenic molecule belonging to a group of homologous ones should not be taken as proof of immunological recognition as well. The availability of panels of homologous molecules to be tested using microarrays is valuable to address the therapeutic intervention. 相似文献8.
Nouri HR Sankian M Vahedi F Afsharzadeh D Rouzbeh L Moghadam M Varasteh A 《Molecular biology reports》2012,39(3):3169-3178
Chenopodium album pollen is one of the main sources of pollen allergy in desert and semi-desert areas and contains three identified allergens,
so the aim of this study is comparison of the diagnostic potential of C. album recombinant allergens in an allergenic cocktail and C. album pollen extract. Diagnostic potential of the allergenic cocktail was investigated in 32 individuals using skin prick test
and obtained results were compared with the acquired results from C. album pollen extract. Specific IgE reactivity against the pollen extract and allergenic cocktail was determined by ELISA and western
blotting tests. Inhibition assays were performed for the allergenic cocktail characterization. The exact sensitization profile
of all patients was identified which showed that 72, 81 and 46% of allergic patients had IgE reactivity to rChe a 1, rChe
a 2 and rChe a 3, respectively. Almost all of C. album allergic patients (30/32) had specific IgE against the allergenic cocktail. In addition, there was a high correlation between
IgE levels against the allergenic cocktail and IgE levels against the pollen extract. The allergenic cocktail was able to
completely inhibit IgE binding to natural Che a 1, Che a 2 and Che a 3 in C. album extract. In addition, positive skin test reactions were seen in allergic patients that tested by the allergenic cocktail.
The reliable results obtained from this study confirmed that the allergenic cocktail with high diagnostic potential could
be replaced with natural C. album allergen extracts in skin prick test and serologic tests. 相似文献
9.
《生物化学与生物物理学报:生物膜》1985,813(2):338-342
Deprivation of two strains of Paracoccus denitrificans of iron elicited substantial siderophore production, and the associated cell envelope protein changes were examined. In strain DSM 413 four new outer membrane proteins in the Mr range 84000 to 76000 were induced, and an Mr 23000 protein disappeared, upon iron deprivation. In strain DSM 65 three new outer membrane proteins in the Mr range 81000 to 76000 were induced. 相似文献
10.
Cap binding protein (CBP)-related polypeptides were identified in different cytoplasmic RNP particles of embryonic chick muscles using monoclonal antibody to purified CBP. A single immunoreactive peptide (Mr 78000) was present in preparations of both free mRNP particles and a novel 10 S translation inhibitory RNP particle. In contrast, proteins isolated from these particles showed two new low-Mr immunoreactive peptides (Mr 43000 and Mr 29000). No CBP related protein could be detected in polysomal mRNP, although an immunoreactive Mr 43000 CBP-related protein was present in polysomes. The relevance of the association of different CBP-related polypeptides with cytoplasmic RNP particles and polysomes are discussed. 相似文献
11.
Herpes Simplex Virus DNA Cleavage and Packaging: Association of Multiple Forms of UL15-Encoded Proteins with B Capsids Requires at Least the UL6, UL17, and UL28 Genes
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The UL15 gene of herpes simplex virus (HSV) is one of several genes required for the packaging of viral DNA into intranuclear B capsids to produce C capsids that become enveloped at the inner nuclear membrane. A rabbit antiserum directed against UL15-encoded protein recognized three proteins with apparent Mrs of 79,000, 80,000, and 83,000 in highly purified B capsids. The 83,000-Mr protein was detected in type C capsids and comigrated with the product of a UL15 cDNA transcribed and translated in vitro. The 83,000- and 80,000-Mr proteins were readily detected in purified virions. Inasmuch as (i) none of these proteins were detectable in capsids purified from cells infected with HSV-1(ΔUL15), a virus lacking an intact UL15 gene, and (ii) corresponding proteins in capsids purified from cells infected with a recombinant virus [HSV-1(R7244), containing a 20-codon tag at the 3′ end of UL15] were decreased in electrophoretic mobility relative to the wild-type proteins, we conclude that the proteins with apparent Mrs of 83,000, 80,000, and 79,000 are products of UL15 with identical C termini. The 79,000-, 80,000-, and 83,000-Mr proteins remained associated with B capsids in the presence of 0.5 M guanidine HCl and remained detectable in capsids treated with 2.0 M guanidine HCl and lacking proteins associated with the capsid core. These data, therefore, indicate that UL15-encoded proteins are integral components of B capsids. Only the 83,000-Mr protein was detected in B capsids purified from cells infected with viruses lacking the UL6, UL17, or UL28 genes, which are required for DNA cleavage and packaging, suggesting that capsid association of the 80,000- and 79,000-Mr proteins requires intact cleavage and packaging machinery. These data, therefore, indicate that capsid association of the 80,000- and 79,000-Mr UL15-encoded proteins reflects a previously unrecognized step in the DNA cleavage and packaging reaction. 相似文献
12.
《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1998,706(1):63-71
Cod fish is one of the foods most frequently involved in allergy. Only the cod allergen Gad c I, a 12.3 kDa parvalbumin, has been purified and characterized. Recently, we have detected allergen bands which have not previously been described, in particular a 41 kDa protein, by Western-blot. In the present work, this protein has been purified from a crude cod extract by ammonium sulfate fractionation, hydroxyapatite chromatography and preparative electrophoresis; a single band with an Mr of 41×−3 was found in silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition and the isoelectric point of the protein were determined. The purified protein (p41) was shown to bind specifically to reaginic IgE from sera of cod-allergic individuals and to a monoclonal anti-parvalbumin which recognizes specifically the first calcium binding site of parvalbumins. p41 may therefore contain a calcium binding site corresponding to an IgE-epitope similar to that of Gad c I. 相似文献
13.
The mammalian neurofilament triplet proteins (210, 160 and 68 × 103Mr proteins) are resolved by anion exchange chromatography in the presence of urea. Upon dialysis against physiological buffers at 37 °C only the 68 × 103Mr protein shows self-assembly into morphologically normal intermediate-sized filaments. Addition of 210 × 103Mr protein to 68 × 103Mr protein leads to shorter filaments, which upon embedding reveal a rough surface and whisker-like protrusions that are not present on the smooth surface of filaments assembled from 68 × 103Mr protein alone. Certain emerging principles of neurofilament structure are discussed, emphasizing a possible relation between neurofilaments and other intermediate-sized filaments. 相似文献
14.
The core structures of microvilli from absorptive cells of the intestinal epithelium are primarily composed of calmodulin (Mr 16,000), actin (Mr 43,000), villin (Mr 95,000) and a protein of Mr 110,000. We have isolated this protein and raised antibodies against it. The antibodies interact specifically with villin and Mr 110,000 polypeptides present in isolated microvilli or brush borders. However, after absorption on an immobilized villin preparation, these antibodies still immunoprecipitate the Mr 110,000 protein but not villin. Thus, these two proteins appear to share some antigenic determinants but also contain other determinants specific for each protein. Immunolocalization studies have been performed using specific antibodies against the Mr 110,000 protein. Immunofluorescent studies on thin frozen sections of intestinal cells show that this protein is located in the brush border and at the basolateral faces of these polarized cells. Immunoferritin studies on rat brush borders demembranated with the detergent Triton X-100 show the association of the Mr 110,000 protein with core filaments of microvilli, as well as with some filaments localized in the terminal web network.Using sealed, right-side-out vesicles prepared from pig intestinal mucosa in the presence of Ca2+ and Mg2+, a polypeptide of Mr 140,000 was found to be a major component of the Triton X-100 insoluble pellet. This protein is a minor component of an equivalent pellet obtained from isolated microvilli prepared in the presence of EDTA. The significance of this Mr 140,000 polypeptide associated with the core residue of intestinal microvilli is discussed. 相似文献
15.
Cytosolic and nuclear forms of the glucocorticoid receptor were characterized using immunochemical techniques. Antibodies were raised in rabbits to an Mr 58,000 fragment of the transformed (DNA-binding) glucocorticoid receptor purified from rat liver cytosol by DNA-cellulose chromatography and polyacrylamide gel electrophoresis. Antibodies reacted with the transformed receptor form in a radioimmunoassay for glucocorticoid receptor. Western blot analysis of antibody reactivity revealed a single Mr 185,000 receptor form in rat liver cytosol but a smaller Mr 85,000 form in nucleosol, indicating the Mr 85,000 form is the transformed receptor. Furthermore, western blot analysis indicates that the Mr 185,000 receptor undergoes proteolysis during receptor purification and in vitro transformation processes by generating immunochemically similar proteins of smaller molecular weights. An identical Mr 185,000 glucocorticoid receptor was detected in cytosols of four rat tissues; liver, brain, adrenal medulla, and thymus. The glucocorticoid receptor was localized to the cytoplasm and nucleus of rat adrenal medulla cells by immunohistochemistry, demonstrating the existence in vivo of the transformed receptor and translocation of the receptor from cytoplasm to nucleus. 相似文献
16.
George D. Yocum Karl H. Joplin David L. Denlinger 《Archives of insect biochemistry and physiology》1991,18(4):239-249
Diapausing pharate first instars of the gypsy moth, Lymantria dispar, respond to high temperature (37–41°C) by suppressing normal protein synthesis and synthesizing a set of seven heat shock proteins with Mrs of 90,000, 75,000, 73,000, 60,000, 42,000, 29,000, and 22,000 as determined by SDS-PAGE. During recovery at 25°C from heat shock, synthesis of the heat shock proteins gradually decreases over a period of 6 h, while normal protein synthesis is restored. A subset of these same heat shock proteins is also expressed during recovery at 4°C or 25°C from brief exposures to low temperature (-10 to 20°C), and its expression is more intense with increased severity of cold exposure. During recovery at 4°C after 24 h at ?20°C, both 90,000 and 75,000 Mr heat shock proteins are expressed for more than 96 h. While normal protein synthesis is suppressed during heat shock and recovery from heat shock, normal protein synthesis coincides with synthesis of the heat shock proteins during recovery from low temperatures, thus implying that expression of the heat shock proteins is not invariably linked to suppression of normal protein synthesis. Western transfer, using a monoclonal antibody that recognizes the inducible form of the human 70,000 Mr heat shock protein, demonstrates that immunologically related proteins in the gypsy moth are expressed at 4°C and during recovery from cold and heat shock. 相似文献
17.
Infestation of sheep with the louse Bovicola ovis is common worldwide and leads to an allergic dermatitis referred to as ‘scatter cockle’. IgE from an infested lamb was used in immunoaffinity chromatography to purify allergens from crude preparations of whole B. ovis and its faeces. SDS-PAGE of the affinity-purified eluates from both preparations showed a dominant band with Mr of 28.5 kDa. Spleen cells from a mouse immunised with B. ovis faecal antigens were used to produce hybridomas which were screened by ELISA to identify those producing monoclonal antibodies (mAb) to the allergens purified by IgE immunoaffinity chromatography. Western blotting demonstrated that all of the mAbs examined recognised the 28.5 kDa allergen. The allergen, purified using immunoaffinity columns constructed with one of the specific mAbs, was shown to cause immediate and late-phase responses on intradermal skin testing in B. ovis-infested but not in naïve lambs. Levels of serum IgE specific for the purified allergen were significantly higher in infested than in naïve lambs (P ? 0.0025). N-terminal and internal amino acid (aa) sequences obtained from the purified 28.5 kDa allergen were used to design primers to amplify a partial cDNA probe from B. ovis cDNA by PCR. The amplified probe was radiolabeled and used to screen a B. ovis cDNA library. The complete nucleotide sequence of the allergen was determined from the sequences of the positive clones from the library. The full-length cDNA encodes a 255 aa protein including a secretory leader sequence of 26 aas and a mature protein of 229 aas. The encoded protein showed strong homology to several hypothetical proteins of unknown function from diverse species and weak homology with lipid-binding proteins of Xenopus tropicalis and Galleria mellonella. This is the first allergen to be identified from a louse and it has been designated Bov o 1 in accordance with the criteria of the World Health Organization/International Union of Immunological Societies Allergen Nomenclature Subcommittee. 相似文献
18.
Steven G. Pueppke 《Archives of biochemistry and biophysics》1981,212(1):254-261
Peanut lectin (or an immunologically indistinguishable material) is present in seeds of 4556 genotypes of the peanut, Arachis hypogaea, and in 65 genotypes of related species of Arachis. Seeds of one line of A. villosa and three lines of unclassified Arachis spp. are devoid of the lectin. Peanut lectin from 116 A. hypogaea genotypes is resolved by isoelectric focusing into three related isolectin profiles, which are designated the V, S, and V2 types. Each is composed of from six to eight separate isolectins. Peanut lectin from A. monticola, A. pusilla, and one genotype of Arachis spp. is of the V type; isolectin profiles from other wild Arachis genotypes are variable, but comprise several distinct groups. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate resolves peanut lectin preparations from 37 genotypes into the lectin subunit of Mr 30,000 and a second polypeptide of Mr 60,000. Lectin preparations from five genotypes lack the Mr 60,000 polypeptide band and have a subunit that migrates slightly faster (and therefore probably is of lower molecular weight) than the subunits of all other tested lines. Peanut lectin preparations from 62 lines have specific hemagglutinating activities ranging from 1024 to 4196 with desialyzed human Type O erythrocytes. The lectin from one genotype exhibits substantially less hemagglutinating activity and is hemolytic. 相似文献
19.
Paul Williams Henrik Chart Elwyn Griffiths Pauline Stevenson 《FEMS microbiology letters》1987,44(3):407-412
Seventeen isolates of Klebsiella aerogenes, K. pneumoniae, K. oxytocum and K. edwardsii were examined for their ability to express iron-regulated outer membrane proteins (IROMPs) and high affinity iron-chelating agents (siderophores). In response to iron deprivation, all strains induced at least 4 IROMPs in the approximate Mr range 70 000–85 000 and the phenolate siderophore enterobactin. Six strains also produced the hydroxamate siderophore aerobactin. The Klebsiella enterobactin receptor was identified as an 81 000 Mr iron-repressible outer membrane (OM) protein which appears to be highly conserved and shows considerable antigenic homology with that of Escherichia coli. 相似文献
20.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1987,927(3):392-401
This study examined the changes in protein phosphorylation in response to cholinergic (muscarinic) stimulation of salivary secretion in the rat submandibular gland. Carbachol stimulation was associated with phosphorylation in a number of protein bands as detected by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and autoradiography. The molecular masses (Mr) of two proteins, in which the amount of phosphorylation more than doubled in response to carbachol, were 22 000 and 96 000. The Mr 96 000 protein precipitated at 120 000 × g while most of the Mr 22 000 protein remained in the supernatant at this speed. The effect of carbachol on the phosphorylation of the Mr 22 000 and 96 000 proteins was blocked by atropine, indicating that the cholinergic receptor involved is muscarinic. The time course of phosphorylation of the Mr 22 000 protein consisted of a rapid incrase in phosphorylation within the first min of carbachol stimulation. This increased phosphorylation persisted for less than 1 min. The increased phosphoryaltion of the Mr 96 000 protein also occurred within the first min but it persisted for at least 10 min. However, removal of the muscarinic agonist, carbachol, resulted in the rapid dephosphorylation of this protein. When the plasma membranes were purified, the Mr 96 000 protein was phosphorylated by ATP in the presence of Na+ and Mg2+. It was dephosphorylated by K+. This proves that the Mr 96 000 dalton protein is the α-subunit of the (Na+ + K+)-ATPase. 相似文献