The arrangement of proteins in the human erythrocyte membrane

Phillips, David R.; Morrison, Martin

doi:10.1016/0006-291x(70)91007-7

Cited by 330 publications

(62 citation statements)

References 19 publications

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“…Radioactivity of 3H, (----) radioactivity of 1311 Lactoperoxidase has a molecular weight of 78 000 and does not dissociate into subunits. Since the iodination catalyzed by the enzyme takes place via the usual enzyme * substrate complex [13,14], only those proteins which are exposed on the surface of the particle should be labelled [15]. Those proteins buried in the interior of the particle would be inaccessible to the enzyme.…”

Section: Results and Discussion Proteins Iodinated In The 50-s Subunitmentioning

confidence: 99%

Molecular Morphology of Ribosomes

Michalski

Sells

Morison

1973

European Journal of Biochemistry

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Using the lactoperoxidase-catalyzed iodination system as a probe for exposed proteins, the location of ribosomal proteins on 50-S subunits of Escherichia coli was examined. These studies demonstrated that a t least three of the proteins in the 50-S ribosome are iodinated and may be termed exposed proteins. Most of the proteins, on the other hand, are not iodinated and can be classified as buried or partially buried proteins. Unfolding of the 50-5 ribosome by the removal of Mg2+ resulted in a decreased organization of the ribosome structure and allowed all the proteins except one to be iodinated.The Escherichia coli 50-5 subunit is composed of approximately 34 distinct proteins [l]. The studies of Traut et al. [I] have suggested that, except for two of these proteins, the remaining exist in one copy per ribosome. A question which remains unresolved is the arrangement of these proteins within the subunit structure. Specifically, in the present study the topography of the 50-S subunit has been investigated to establish which proteins are buried and which are exposed or on the surface of the ribosome. To answer this question, advantage has been taken of the lactoperoxidase-catalyzed halogenation system which, under the conditions employed, is a macromolecular probe for exposed proteins in organized macromolecular systems [Z]. MATERIALS AND METHODSThe methionine and arginine-required mutant of E. coli strain K711 (RCrel) was used throughout these studies. The bacteria were grown at 37 "C with shaking in minimal media [3] supplemented with I1 mM glycerol 0.33 mM L-methionine and 0.24mM L-arginine. Bacteria were grown to an absorbance a t 575 nm of 0.1 and incubated for three generations in the presence of tritiated tyrosine to label all proteins uniformly with tritium. The 30-S and 50-S ribosomal subunits were isolated as previously described [4]. The cells, collected by centrifugation, were washed with 10 mM Tris-HC1,6 mM 2-mercaptoethanol, 30nM ammonium chloride buffer pH 7.4, supplemented with 10 nM magnesium acetate and resuspended in 3 ml of Tris-mercaptoethanol-NH4ClEnzymes. Lactoperoxidase (EC 1.11.1.-).32 Eur. J. Uiochem., Vo1.33 buffer plus 1OmM Mg2+. Cells were broken in a French pressure cell a t 6000 to 8000 lb/in2 and the debris removed as previously described [4]. The ribosomes were pursed as outlined earlier [4]. To fractionate monosomes into subunits, approximately 125 Azao units (one A,,, unit is equal t o an absorbance of I a t 260 nm in a cuvette of light path 1 cm) were layered on 27 ml of a linear 5 to ZOO/, sucrose gradient in Tris-mercaptoethanol-NH4Cl buffer plus 10 mM Mg2+ and centrifuged a t 21500 rev./& for 10 h in an SW 25.1 rotor. Gradients were collected through an ISCO ultraviolet Monitor (Instruments Specialties Company) and the peaks corresponding to 30-S and 50-S ribosomal subunits collected. The 50-S fraction was placed in a length of dialysis tubing and concentrated by packing the tubing in fine granular sucrose. The concentrated sample was dialyzed against 50 mM Tris-acetate, 10 m...

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Section: Results and Discussion Proteins Iodinated In The 50-s Subunitmentioning

confidence: 99%

Molecular Morphology of Ribosomes

Michalski

Sells

Morison

1973

European Journal of Biochemistry

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“…(a) Surface Ig were labeled with a25I by the lactoperoxidase method (31,32). 10 million cells were labeled, washed extensively with phosphate-buffered saline (PBS), and lysed with PBS containing 0.5% sodium deoxycholate, 0.1% Nonidet P-40, 0.1% NaNa, 1 mM phenylmethylsulfonylfluoride, 2 mM e-aminocaproic acid, and 1 mg/ml soybean trypsin inhibitor for 30 rain at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Differential regulation of membrane and secretory mu chain synthesis in human beta cell lines. Regulation of membrane mu or secreted mu.

Hendershot¹,

Levitt²

1982

The Journal of Experimental Medicine

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Regulation of membrane and secretory mu synthesis was examined in human lymphoblastoid cell lines representing various stages of differentiation. Immunoglobulin phenotype was determined by surface and cytoplasmic staining with fluorochrome-conjugated antibodies and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of anti-mu precipitable cellular products. The thymidine analogue, 5-bromo-2'-deoxyuridine (BUdR), which inhibits differentiation-specific proteins in a variety of systems, was used to examine regulation of immunoglobulin synthesis. We found that BUdR had a differential effect on membrane (mum) and secretory (mus) type mu heavy chains. Ig production in pre-B and plasma cell-like lines, which make mus, was unaffected by BUdR. However, surface expression of IgM (mum) in B cell lines was drastically inhibited at similar doses of BUdR without diminishing total Ig or protein synthesis. Examination of labeled mu chains from control and BUdR-treated B cell lines by SDS-PAGE revealed the production of two sizes of mu (mum and mus) in control cells and only the smaller size (mus) in BUdR-treated cells. This size difference could not be attributed to alterations in glycosylation of the molecules. These data show that BUdR inhibits the production of membrane mu chains without diminishing secretory mu chain synthesis in the same cell. Our findings suggest that thymidine-rich regions of the genome are involved in the regulation of mum vs. mus during B cell differentiation.

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“…Total HSV-1 purified from 20 roller bottles was surface-labeled with iodine-125 (125I) in the presence of lactoperoxidase and H202, as described by Philips and Morrison (9). The envelope was separated from the surface-labeled virion by treatment with Nonidet P-40 (NP-40), described elsewhere (8).…”

mentioning

confidence: 99%

Identification of virion polypeptides in hamster cells transformed by herpes simplex virus type 1.

Gupta¹,

Rapp²

1977

Proc. Natl. Acad. Sci. U.S.A.

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ABSTRACr Ten polypeptides were detected on the surface of the virion of herpes simplex virus type 1. Of these ten polypeptides, three were detected in hamster cells transformed by herpes simplex type 1. Virus-specific antigens have been detected on the plasma membrane of cells productively infected with herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) (1-3). Hamster embryo fibroblast cells transformed by inactivated HSV-1 or HSV-2 also show the continued presence of virus-specific antigens on the cell surfaces but do not release virus particles (4-6). Reed et al. (7) have reported the presence of a group of undefined virion-associated antigens, designated CP-1, on the surfaces of hamster cells transformed by inactivated HSV-1. A major difficulty associated with these experiments was that the antigens were detected by immunofluorescence methods, which do not characterize the particular virion polypeptide present in the test cell.In this report we describe the characterization of herpesvirus polypeptides present in the HSV-1-transformed cells. The search for the virion polypeptides was made logical by the observation that rabbit antiserum to a tumor derived by the inoculation of HSV-1-transformed cells into a syngeneic hamster is capable of strongly neutralizing HSV-1 but not HSV-2. Thus, it was possible to immunoprecipitate the labeled virion surface protein antigens with this rabbit antitumor serum and to identify the antigens by polyacrylamide gel electrophoresis. Using this technique, we found evidence for the presence of three virion-associated proteins in the HSV-1-transformed cells.MATERIALS AND METHODS Cells and Virus. HSV-1 (strain Patton) was grown in Vero cells in medium 199 supplemented with 10% fetal calf serum. The purification of virus from infected cells by dextran gradient centrifugation has been described (8). The purified virus was found by electron microscope examination to be 95-97% enveloped and to contain no cellular contamination.Preparation of Surface-Labeled Virion Envelope. Total HSV-1 purified from 20 roller bottles was surface-labeled with iodine-125 (125I) in the presence of lactoperoxidase and H202, as described by Philips and Morrison (9). The envelope was separated from the surface-labeled virion by treatment with Nonidet P-40 (NP-40), described elsewhere (8). Briefly, was added to the surface-labeled virus to a concentration of 1%; the mixture was incubated in ice for 15 min and then centrifuged at 100,000 X g for 90 min at 40. The supernatant was removed and used as virion surface antigen.Chromatography on Concanavalin A-Sepharose Column. A 3-ml column of concanavalin A bound to Sepharose 4B Abbreviations: HSV, herpes simplex virus; NP-40, Nonidet P-40; NabodSO4, sodium dodecyl sulfate.(Pharmacia Fine Chemicals) was prepared and equilibrated with buffer I (0.02 M Tris.HCI, pH 7.5,0.25 M NaCl, and 1 mM each MgCl2, MnCl2, and CaCl2). A 50-,l aliquot of surfacelabeled virion envelope antigen (3 X 105 trichloroacetic acidinsoluble cpm) in phosphate-buffered saline containing 1...

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The arrangement of proteins in the human erythrocyte membrane

Cited by 330 publications

References 19 publications

Molecular Morphology of Ribosomes

Molecular Morphology of Ribosomes

Differential regulation of membrane and secretory mu chain synthesis in human beta cell lines. Regulation of membrane mu or secreted mu.

Identification of virion polypeptides in hamster cells transformed by herpes simplex virus type 1.

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