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1.
Human erythrocyte clathrin and clathrin-uncoating protein   总被引:2,自引:0,他引:2  
Clathrin, a Mr = 72,000 clathrin-associated protein, and myosin were purified in milligram quantities from the same erythrocyte hemolysate fraction. Erythrocyte clathrin closely resembled brain clathrin in several respects: (a) both are triskelions as visualized by electron microscopy with arms 40 nm in length with globular ends and a flexible hinge region in the middle of each arm, and these triskelions assemble into polyhedral "cages" at appropriate pH and ionic strength; (b) both molecules contain heavy chains of Mr = 170,000 that are indistinguishable by two-dimensional maps of 125I-labeled peptides; and (c) both molecules contain light chains of Mr approximately 40,000 in a 1:1 molar ratio with the heavy chain. Erythrocyte clathrin is not identical to brain clathrin since antibody raised against the erythrocyte protein reacts better with erythrocyte clathrin than with brain clathrin and since brain clathrin contains two light chains resolved on sodium dodecyl sulfate gels while the light chain of erythrocyte clathrin migrates as a single band. The erythrocyte Mr = 72,000 clathrin-associated protein is closely related to a protein in brain that mediates ATP-dependent disassembly of clathrin from coated vesicles and binds tightly to clathrin triskelions (Schlossman, D. M., Schmid, S. L., Braell, W. A., and Rothman, J. E. (1984) J. Cell Biol. 99, 723-733). The erythrocyte and brain proteins have identical Mr on sodium dodecyl sulfate gels and identical maps of 125I-labeled peptides, share antigenic sites, and bind tightly to ATP immobilized on agarose. Clathrin and the uncoating protein are not restricted to reticulocytes since equivalent amounts of these proteins are present in whole erythrocyte populations and reticulocyte-depleted erythrocytes. Clathrin is present at 6,000 triskelions/cells, while the uncoating protein is in substantial excess at 250,000 copies/cell.  相似文献   

2.
There is an increased synthesis of proteins in the molecular weight region of 100,000 72,000-74,000 and 37,000 two hours after treatment of HeLa cells for 10 min at 45 degrees C. In vitro translation, using a rabbit reticulocyte cell-free protein synthesising system, of HeLa cell cytoplasmic RNA shows that the prominent 72,000-74,000 Mr heat shock protein band comprises seven polypeptide species (namely alpha d beta gamma delta epsilon zeta) and these polypeptides are directly encoded by both polyadenylated and nonpolyadenylated mRNA.  相似文献   

3.
During the lytic infection of monkey and mouse cells with simian virus 40 and polyoma virus, respectively, the preferentially increased synthesis of two host proteins of 92,000 and 72,000 Mr was observed by 15 to 20 h after infection besides the general stimulation of most cellular proteins. The incubation of uninfected monkey and mouse cell cultures for 30 to 60 min at 43.5 degrees C induced the enhanced synthesis of at least three proteins of 92,000, 72,000 and 70,000 Mr, the last one being the major heat shock protein of mammalian cells. Two-dimensional gel electrophoresis and partial proteolytic digestion confirmed that the same 92,000- and 72,000-Mr proteins are stimulated by virus infection and thermal treatment. In simian virus 40-infected CV-1 cells, we also observed the weak stimulation of a 70,000-Mr protein comigrating in gel electrophoresis with the major heat shock protein. The 92,000-, 72,000- and 70,000-Mr proteins of monkey cells are structurally very similar to the corresponding proteins of mouse cells. In immunoprecipitations, no specific association of these proteins to simian virus 40 T antigens was noticed.  相似文献   

4.
Uncoating ATPase is a member of the 70 kilodalton family of stress proteins   总被引:84,自引:0,他引:84  
The synthetic peptide, VGIDLGTTYSC, derived from the heat shock-induced genes human hsp70, Drosophila hsp70, S. cerevisiae YG100, and E. coli dnaK, elicited antibodies that recognized two constitutive proteins in bovine extracts. One of these proteins, 71 kd, has previously been identified as uncoating ATPase, an enzyme that releases clathrin from coated vesicles. This immunological data complemented the result that uncoating ATPase was indistinguishable from the constitutive mammalian 71 kd stress protein by either partial proteolytic mapping or two-dimensional gel analysis. In addition, affinity-purified uncoating ATPase antibodies recognize proteins in yeast identified as the gene products of the heat shock or heat shock cognate genes YG100 and YG102. The results show that uncoating ATPase is a member of the 70 kd heat shock protein family.  相似文献   

5.
Interferon-treated mouse and human cells show enhanced levels of a protein kinase activity which is manifested by the phosphorylation of endogenous Mr = 67,000 and 72,000 proteins, respectively. Such kinase activity can be assayed after its partial purification on poly(I) X poly(C)-Sepharose. Under these experimental conditions, the apparent km of the kinase for ATP is 1.0 X 10(-6) M and 2.5 X 10(-6) M in enzyme fractions from mouse L-929 and human HeLa cells, respectively. The Mr = 67,000 and 72,000 proteins are phosphorylated by their serine and threonine residues, the ratio of which is modified in preparations from interferon-treated cells. Both of these phosphoproteins are composed of several subspecies with similar isoelectric points (pIs) in the range of 7.2 to 8.2. This heterogeneity is due to the number of phosphate groups per molecule of protein. Accordingly, the pIs of highly phosphorylated proteins are at a less basic pH (7.2 to 7.5). Furthermore, highly phosphorylated proteins show an increase in their apparent molecular weights compared to partially phosphorylated ones. This corresponds to an increase of Mr = 1,500. Partial proteolysis of the 32P-labeled Mr = 67,000 and 72,000 proteins by Staphylococcus aureus V8 protease, alpha-chymotrypsin and thrombin, indicated that these phosphoproteins differ in their polypeptide structure. Phosphorylation of the Mr = 67,000 and 72,000 proteins in enzyme fractions from control L-929 and HeLa cells is enhanced by mixing with extracts from interferon-treated heterologous cells. Proteins, Mr = 67,000 and 72,000, therefore, may serve as suitable substrates for an exogenous kinase, thus indicating that the substrate in enzyme fractions from control cells is less phosphorylated because of a low level of kinase activity.  相似文献   

6.
The intracellular location of the major induced mammalian heat shock (or stress) protein (Mr = 72,000) has been determined by both biochemical and immunological methods. This protein, shown here to be comprised of at least three structurally related isoforms, is produced at high levels within 30 min to 1 h following heat treatment of cells. Biochemical fractionation of cells grown under heat shock showed that following its synthesis a portion of the 72,000-Da protein (and its isoforms) becomes associated with the nucleus while some remains in the cytoplasm. Indirect immunofluorescence studies using antiserum directed against the major isoforms of the 72,000-Da protein were carried out in normal and heat-shocked cells as well as in cells grown under stress by exposure to either an amino acid analogue or to sodium arsenite. Diffuse cytoplasmic and nuclear staining was observed in cells grown at 37 degrees C. In cells grown under heat shock conditions, both the cytoplasmic staining and the nuclear staining were found to increase with the nuclear staining consisting of both granular and patch-like structures, the latter being coincident with phase-dense nucleoli. In the case of cells exposed to amino acid analogues or to sodium arsenite, only cytoplasmic and to a lesser extent nuclear staining was observed, i.e. no localized nucleolar fluorescence was observed. Following return of heat shock-treated cells to normal growth temperatures, both the synthesis of the 72,000-Dalton stress protein and its nucleolar staining were found to diminish.  相似文献   

7.
Brain ankyrin. Purification of a 72,000 Mr spectrin-binding domain   总被引:19,自引:0,他引:19  
Polypeptides of Mr = 190,000-220,000 that cross-react with erythrocyte ankyrin were detected in immunoblots of membranes from pig lens, pig brain, and rat liver. The cross-reacting polypeptides from brain were cleaved by chymotrypsin to fragments of Mr = 95,000 and 72,000 which are the same size as fragments obtained with erythrocyte ankyrin. The brain 72,000 Mr fragment associated with erythrocyte spectrin, and the binding occurred at the same site as that of erythrocyte ankyrin 72,000 Mr fragment since (a) brain 72,000 Mr fragment was adsorbed to erythrocyte spectrin-agarose and (b) 125I-labeled erythrocyte spectrin bound to brain 72,000 Mr fragment following transfer of the fragment from a sodium dodecyl sulfate gel to nitrocellulose paper, and this binding was displaced by erythrocyte ankyrin 72,000 Mr fragment. Brain 72,000 Mr fragment was purified about 400-fold by selective extraction and by continuous chromatography on columns attached in series containing DEAE-cellulose followed by erythrocyte spectrin coupled to agarose, and finally hydroxylapatite. The brain 72,000 Mr fragment was not derived from contaminating erythrocytes since peptide maps of pig brain and pig erythrocyte 72,000 Mr fragments were distinct. The amount of brain 72,000 Mr fragment was estimated as 0.28% of membrane protein or 39 pmol/mg based on radioimmunoassay with 125I-labeled brain fragment and antibody against erythrocyte ankyrin. Brain spectrin tetramer was present in about the same number of copies (30 pmol/mg of membrane protein) based on densitometry of Coomassie blue-stained sodium dodecyl sulfate gels. The binding site on brain spectrin for both brain and erythrocyte ankyrin 72,000 Mr fragments was localized by electron microscopy to the midregion of spectrin tetramers about 90 nM from the near end and 110 nM from the far end. These studies demonstrate the presence in brain membranes of a protein closely related to erythrocyte ankyrin, and are consistent with a function of the brain ankyrin as a membrane attachment site for brain spectrin.  相似文献   

8.
Brain ankyrin was purified from pig brain membranes in milligram quantities by a procedure involving affinity chromatography on erythrocyte spectrinagarose. Brain ankyrin included two polypeptides of Mr = 210,000 and 220,000 that were nearly identical by peptide mapping and were monomers in solution. Brain ankyrin and erythrocyte ankyrin are closely related proteins with the following properties in common: 1) shared antigenic sites, 2) high-affinity binding to the spectrin beta subunit at the midregion of spectrin tetramers, 3) a binding site for the cytoplasmic domain of the erythrocyte anion channel, 4) a binding site for tubulin, 5) a similar domain structure with a protease-resistant domain of Mr = 72,000 that contains the spectrin-binding activity and domains of Mr = 95,000 (brain ankyrin) or 90,000 (erythrocyte ankyrin) that contain binding sites for both tubulin and the anion channel. Brain ankyrin is present at about 100 pmol/mg of membrane protein in demyelinated membranes based on radioimmunoassay with antibody raised against brain ankyrin and affinity purified on brain ankyrin-agarose. Brain spectrin tetramers are present at 30 pmol/mg of membrane protein. Brain ankyrin thus is present in sufficient amounts to attach spectrin to membranes. Brain ankyrin also may attach microtubules to membranes independently of spectrin and has the potential to interconnect microtubules and spectrin-associated actin filaments.  相似文献   

9.
At elevated temperatures, germinating conidiospores of Neurospora crassa discontinue synthesis of most proteins and initiate synthesis of three dominant heat shock proteins of 98,000, 83,000, and 67,000 Mr and one minor heat shock protein of 30,000 Mr. Postemergent spores produce, in addition to these, a fourth major heat shock protein of 38,000 Mr and a minor heat shock protein of 34,000 Mr. The three heat shock proteins of lower molecular weight are associated with mitochondria. This exclusive synthesis of heat shock proteins is transient, and after 60 min of exposure to high temperatures, restoration of the normal pattern of protein synthesis is initiated. Despite the transiency of the heat shock response, spores incubated continuously at 45 degrees C germinate very slowly and do not grow beyond the formation of a germ tube. The temperature optimum for heat shock protein synthesis is 45 degrees C, but spores incubated at other temperatures from 40 through 47 degrees C synthesize heat shock proteins at lower rates. Survival was high for germinating spores exposed to temperatures up to 47 degrees C, but viability declined markedly at higher temperatures. Germinating spores survived exposure to the lethal temperature of 50 degrees C when they had been preexposed to 45 degrees C; this thermal protection depends on the synthesis of heat shock proteins, since protection was abolished by cycloheximide. During the heat shock response mitochondria also discontinue normal protein synthesis; synthesis of the mitochondria-encoded subunits of cytochrome c oxidase was as depressed as that of the nucleus-encoded subunits.  相似文献   

10.
1. A monoclonal antibody (TG7A) reacts with a Mr 90,000 mammalian protein, accumulating during virus infection and heat shock. 2. This protein is encoded by a member of the Mr 90,000 heat shock gene family present in a range of organisms form yeast to man. 3. The antibody also recognises a Mr 94,000 protein in E. coli which similarly accumulates in virus infection and heat shock. 4. This protein has been identified as the Lon protease of E. coli. 5. The shared epitope and similar stress inducibility of the two proteins suggests that a functional and/or evolutionary relationship exists between them.  相似文献   

11.
The grpE protein of Escherichia coli. Purification and properties   总被引:31,自引:0,他引:31  
The grpE gene of Escherichia coli was first identified because a mutation in it, grpE280, prevented bacteriophage lambda DNA replication in vivo. Subsequent work resulted in the identification of the grpE protein in two-dimensional gels and its classification as a heat shock protein. Here we report the purification of the grpE protein. We show that overproduction of grpE occurs in dnaK 103 bacteria which do not produce a functional Mr 72,000 dnaK protein. The grpE protein was purified from this strain primarily by its specific retention on a dnaK affinity column. The interaction between these two proteins, which is stable in the presence of 2 M KCl, allowed other proteins to be washed from this column. grpE was then eluted by ATP, which disrupts the interaction. During purification, grpE activity was monitored by its ability to complement an in vitro lambda dv DNA replication system dependent on the lambda O and lambda P proteins. The effect of ATP on the dnaK-grpE complex was also observed during sedimentation of the two proteins in glycerol gradients. Purified grpE protein has a Mr of approximately 23,000 under both denaturing and native conditions, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sedimentation, respectively. However, in the presence of dnaK under native conditions, grpE cosediments with dnaK. When ATP is added to the gradient, the complex is disrupted, and the two proteins sediment independently as monomers.  相似文献   

12.
Translation in vitro of mRNA and immunoprecipitation with specific rabbit antisera showed that the unglycosylated precursor polypeptides of the mouse Mac-1 and lymphocyte function associated antigen (LFA-1) alpha subunits are 130,000 Mr and 140,000 Mr, respectively. Furthermore, polysomes purified by using anti-Mac-1 IgG yielded a similar major product of translation in vitro of Mr = 130,000. The Mac-1 and LFA-1 alpha subunit translation products are immunologically noncross-reactive, showing that differences between these related proteins are not due to post-translational processing. Mac-1 and LFA-1 alpha subunits could only be in vitro translated from mRNA from cell lines the surfaces of which express the corresponding Mac-1 and LFA-1 alpha-beta complexes, showing tissue-specific expression is regulated at the mRNA level. The glycosylation of Mac-1 was examined by both translation in vitro in the presence of dog pancreas microsomes and by biosynthesis in vivo and treatment with tunicamycin, endoglycosidase H, and the deglycosylating agent trifluoromethane sulfonic acid. High mannose oligosaccharides are added to the Mac-1 alpha and beta polypeptide backbones of Mr = 130,000 and 72,000, respectively, to yield precursors of Mr = 164,000 and 91,000, respectively. The alpha and beta subunit precursors are then processed with partial conversion of high mannose to complex type carbohydrate to yield the mature subunits of Mr = 170,000 and 95,000, respectively.  相似文献   

13.
When primary cultures of chicken myoblasts were subjected to incubation at a temperature higher than their normal growing temperature of 36-37 degrees C, the pattern of protein synthesis was altered. This condition of heat shock induced a vigorous production of a number of proteins collectively known as 'heat-shock proteins'. The synthesis of heat-shock proteins was achieved without a significant decrease in the production of a broad spectrum of proteins by muscle cells. The synthesis of three major heat-shock polypeptides with Mr values of 81 000, 65 000 and 25 000 was observed in both mononucleated dividing myoblast cells and terminally differentiated myotubes. Two-dimensional electrophoretic separation of the heat-induced polypeptides synthesized by myogenetic cultures further established that same set of polypeptides with Mr of 65 000 (pI 6.0 and 5.5), 81 000 (pI 6.2) and 25 000 (pI 5.6 and 5.3) were produced in myoblasts and myotubes. The effect of the changes in pattern of protein synthesis on the mRNA and protein moieties of non-polysomal cytoplasmic mRNA-protein complexes (free mRNP) was examined. Free mRNP complexes sedimenting at 20-35 S were isolated from the post-ribosomal supernatant of both normal and heat-shocked myotube cultures by centrifugation in a sucrose gradient. A 10-20S RNA fraction isolated from these complexes stimulated protein synthesis in a cell-free system. The RNA fraction obtained from heat-shocked cells appeared to direct the synthesis of all three major heat-shock proteins. In contrast, synthesis of these polypeptides was not detected when RNA from free mRNP complexes of normal cells was used for translation. The free mRNP complexes of both normal and heat-shocked cells showed a buoyant density of 1.195 g/cm3 in metrizamide gradients. A large number of polypeptides of Mr = 35 000-105 000 were present in the highly purified free mRNP complexes isolated from the metrizamide gradient. Similar sets of polypeptides were found in these complexes from both normal and heat-shocked myotube culture. However, the relative proportion of a 65 000-Mr polypeptide was dramatically increased in the free mRNP complexes of heat-shocked cells. Two-dimensional gel electrophoretic analysis revealed that this polypeptide and the 65 000-Mr heat-shock polypeptide exhibit similar electrophoretic migration properties. These observations suggest that, following heat-shock treatment of chicken myotube cultures, the changes in the pattern of protein synthesis is accompanied by alteration of the mRNA and protein composition of free mRNP complexes.  相似文献   

14.
15.
A Elgavish  D J Pillion  E Meezan 《Life sciences》1989,44(15):1037-1042
[125I]VIP (vasoactive intestinal peptide) bound to apical membranes isolated from the bovine tracheal epithelium with a half maximal inhibition by unlabeled VIP (IC50) of 0.6 x 10(-9)M and binding was reversible. Glucagon did not affect [125I]VIP binding to the membranes. [125I]VIP was covalently cross-linked to tracheal membrane proteins using disuccinimidyl suberate. SDS-polyacrylamide gel electrophoresis of labeled tracheal membranes revealed one major [125I]-receptor complex of Mr = 71,000 to which binding of [125I]VIP was inhibited by 10 microM unlabeled VIP. These results are consistent with the presence of a specific, high-affinity receptor for VIP, with a Mr = 71,000, in apical membrane vesicles isolated from the bovine tracheal epithelium.  相似文献   

16.
The analysis of proteins synthesized in rat thymocytes and mouse teratocarcinoma PCC-4 Aza 1 and myeloma Sp2/0 cells after 1 h of treatment at 42 or 44 degrees C was carried out. Shock at 42 degrees C reduced the total synthetic rate of proteins in all three cell lines and induced "classical" heat-shock protein with a mass of 70 kDa (hsp 70). Heat shock at 44 degrees C resulted in almost complete inhibition of protein synthesis; only a small amount of hsp 70 was synthesized. Meanwhile a new 48-kDa polypeptide (pI = 7.5) was found in the cells exposed to severe heat shock. This protein was compared by peptide mapping with other known polypeptides of the same size: heat-shock protein from chicken embryo cells and mitogen-stimulated polypeptide from human lymphoid cells. The peptide maps were not identical. It was also shown that after a shock at 44 degrees C teratocarcinoma cells were able to accumulate anomalous amounts of hsp 70 despite hsp 70 synthesis inhibition. The data show that reaction of various cells to extreme heat shock depends heavily on cell type.  相似文献   

17.
Protein secretion from mouse blastocysts undergoing attachment and trophoblast outgrowth in vitro was assessed. When Day 5 blastocysts were cultured in serum-containing medium, secretion of several 'attachment-associated' proteins (PAS) was initiated within 24 h, coincident with attachment and outgrowth. Those proteins characteristic of the pre-attachment blastocyst disappeared or made-up only a small portion of the secretions once attachment began. The major secreted protein from attached embryos, PA1, is a 35,000-45,000 Mr acidic glycoprotein with multiple isoelectric forms. PA2, a group of basic 40,000 Mr proteins and PA3 a group of 72,000 Mr proteins were also produced during outgrowth. PAS were secreted during outgrowth on fibronectin-coated plastic in serum-free medium, but not by blastocysts held in a non-attachment state during culture in serum-free medium on uncoated plastic. In pre-attachment blastocysts, secreted proteins were produced by trophoblast vesicles, but not by isolated inner cell masses. Both trophoblast vesicles growing out in vitro and surgically isolated trophoblast from spreading blastocysts had secreted protein patterns qualitatively similar to those of intact blastocyst outgrowths. The results indicate that development of trophoblast protein secretion continues through the period of outgrowth and giant cell transformation. These changes are apparently dependent on attachment of the blastocyst to a suitable substrate, but not dependent on any other serum influence.  相似文献   

18.
Affinity-purified medium T antigen encoded by NG59, a nontransforming mutant of polyomavirus, is specifically associated with a protein of 72,000 daltons (72K protein). Medium T antigens of wild-type polyomavirus and the transformation-competent mutant dl8 are not associated with the 72K protein. Instead, they form a complex with another protein of 61,000 daltons. Several lines of evidence suggest that the medium T antigen-associated 72K protein is equivalent to the abundant and constitutive 73K heat shock protein. First, on two-dimensional polyacrylamide gels the 72K protein migrated with the same pI (5.6) as did the 73K heat shock protein. Second, the 72K protein was immunoprecipitable with antibodies against heat shock proteins. Third, when digested with V8 protease, the 72K protein gave rise to the same pattern of fragments as did the 73K heat shock protein.  相似文献   

19.
Biochemical properties of the heat shock or stress proteins of mammalian cells have been investigated using two-dimensional gel electrophoresis and immunological techniques. Of the major mammalian stress proteins (Mr = 72,000, 73,000, and 90,000) and minor stress proteins (Mr = 80,000, 100,000, and 110,000), the 80- and 90-kDa proteins were found to be phosphoproteins in all cell types examined. The 100-kDa protein was found to incorporate phosphate in only some cell types examined. In studies of the metabolic incorporation of mannose into the stress proteins, only the 100-kDa protein was found to be a glycoprotein. Two of the stress proteins, the 80- and 100-kDa species, were found to be identical with the proteins induced in cells grown in the absence of glucose (i.e. the "glucose-regulated proteins"). These same two proteins also were induced in cells treated with the calcium ionophore A23187. To begin examining the intracellular location of these multiregulated proteins, immunofluorescence microscopy studies were carried out using a monoclonal antibody against the 100-kDa stress protein. The antigen was localized primarily with the Golgi apparatus and less prominently with the plasma membrane and nucleus. Heat shock treatment resulted in an increased number of the cells exhibiting a nuclear location of 100 kDa.  相似文献   

20.
Our previous research has shown that the Ca2+-dependent protease within platelets is activated when platelets aggregate, resulting in the production of three polypeptides (Mr = 200,000, 100,000, and 91,000). We have now shown that these three polypeptides arise from the hydrolysis of actin-binding protein. An antibody against actin-binding protein raised in rabbits was shown to be specific for actin-binding protein on immunoblots of total platelet proteins. This antibody reacted with additional polypeptides of Mr = 200,000, 100,000, and 91,000 on immunoblots of the proteins of thrombin-activated platelets. Actin-binding protein was purified from fresh, human platelet concentrate and hydrolyzed with platelet-derived Ca2+-dependent protease; hydrolysis resulted in the appearance of three polypeptides with molecular weights and isoelectric points identical to those of the three polypeptides generated within intact, aggregating platelets. Production of these polypeptides was inhibited by leupeptin and by the chelation of Ca2+. Hydrolysis of actin-binding protein was observed at micromolar Ca2+ concentrations, demonstrating that the level of Ca2+ in aggregated platelets is sufficient to account for the hydrolysis of actin-binding protein by the Ca2+-dependent protease. P235 was also purified and tested for its susceptibility to the protease. It was hydrolyzed by the Ca2+-dependent protease, and two polypeptides (Mr = 200,000 and 46,000) were produced. Antibodies against P235 raised in rabbits reacted only with P235 on immunoblots of total platelet proteins. These antibodies also reacted with polypeptides of Mr = 200,000 and 46,000 on immunoblots of thrombin-activated platelets. These data show that both actin-binding protein and P235 are cleaved during thrombin-induced platelet aggregation and suggest that the activation of the Ca2+-dependent protease may permit reorganization of the platelet cytoskeleton in aggregating platelets.  相似文献   

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