Regeneration of the Surface Glycoproteins of a Transplantable Mouse Tumor Cell After Treatment With Neuraminidase

Hughes, R. Colin; Sanford, Barbara H.; Jeanloz, Roger W.

doi:10.1073/pnas.69.4.942

Cited by 71 publications

(18 citation statements)

References 14 publications

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“…Since some of these agents have deleterious effects on the cell, cell survival may depend on the mitigation of these surface effects, either by their reversal or repair. Treatment of cultured cells with hydrolytic enzymes results in loss of antigens and of viral or other receptors on the cell surface that, upon subsequent incubation of the cells, return to their initial titer within a few hours (1)(2)(3)(4). In such studies, it is not clear whether the reappearance of the surface properties represents repair above and beyond normal membrane turnover.…”

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confidence: 94%

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Regeneration of Cation-Transport Capacity in HeLa Cell Membranes After Specific Blockade by Ouabain

Vaughan

Cook

1972

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

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The cardiac glycoside, ouabain, inhibits alkali-cation transport in HeLa cells. It binds to 0.75 X 106 sites per cell, and the half-time for its dissociation is 16 hr. After partial blockade by ouabain, the cell generates new ouabain-binding sites, with total restoration of transport in 10% of a cell cycle (-3 hr). This recovery requires protein snythesis and appears to be a response to altered cell-electrolyte content, since growth of cells in media with low K + concentration enhances the titer of the transport enzyme in a fashion similar to the effect of ouabain. Totally blocked cells do not recover.The interaction of any exogenous chemical with a cell begins at the cell surface; in fact many biologically active chemicals penetrate no further into the cell. Since some of these agents have deleterious effects on the cell, cell survival may depend on the mitigation of these surface effects, either by their reversal or repair. Treatment of cultured cells with hydrolytic enzymes results in loss of antigens and of viral or other receptors on the cell surface that, upon subsequent incubation of the cells, return to their initial titer within a few hours (1-4). In such studies, it is not clear whether the reappearance of the surface properties represents repair above and beyond normal membrane turnover. The rate of bulk turnover of surface proteins, carbohydrates, and lipids does not seem to be stimulated after treatment with hydrolytic enzymes (4, 5).We investigated membrane repair more specifically by use of alkali-cation transport (Na+-K+-Mg2+-ATPase) in HeLa cells. Our rationale for this selection was as follows: (i) the proper functioning of the cation-transport system, in association with maintenance of normal cell electrolyte composition, is recognized as a principal factor in the regulation of cell volume (6, 7). Also, K+ is a cofactor for several intracellular enzymes, and for their activity intracellular K+ must be maintained at appropriate concentrations (see, e.g., refs. 8-10). Thus, the integrity of the alkali-cation transport system is of known and demonstrable importance to cell viability. (ii) Alkali-cation transport can be specifically blocked by cardiac glycosides (11-13). In HeLa cells, ouabain, in low concentrations, binds tightly to the cells and is an effective inhibitor. (iii) By the use of [3H Jouabain it is possible to monitor the extent of binding of the inhibitor, while at the same time the activity of alkali-cation transport can be assayed by measurement of transport of labeled cations. In sum, these characteristics enable us to make specific measurements of an essential enzymatic function localized at the cell surface and subject to specific inhibition.In this paper, we show that subtotal blockade of transport

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confidence: 94%

“…In such studies, it is not clear whether the reappearance of the surface properties represents repair above and beyond normal membrane turnover. The rate of bulk turnover of surface proteins, carbohydrates, and lipids does not seem to be stimulated after treatment with hydrolytic enzymes (4,5).…”

mentioning

confidence: 99%

Regeneration of Cation-Transport Capacity in HeLa Cell Membranes After Specific Blockade by Ouabain

Vaughan

Cook

1972

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

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“…1 c, e) . No other changes were detectable after this trypsin treatment, although more extensive digestion will remove up to 40% of the incorporated sugars (unpublished data and references 15,16) . A similar difference between normal, trypsinised, and transformed cells was detected after labeling with [ 14 C]leucine (data not shown) .…”

Section: Labeling With Carbohydrate Precursorsmentioning

confidence: 99%

Characterization of the External Proteins of Hamster Fibroblasts

Hynes

Humphryes

1974

The Journal of Cell Biology

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The results of metabolic labeling studies and enzymatic treatments followed by analysis on polyacrylamide gels show that the external proteins of hamster fibroblast cell lines, which have been identified by lactoperoxidase-catalyzed iodination, do not contain sulphated mucopolysaccharides or hyaluronic acid and are probably unrelated to collagen . Several of the iodinated species comigrate with carbohydrate-containing molecules . In particular, the major iodine-labeled polypeptide of normal fibroblasts appears to be a glycoprotein . This glycoprotein is absent or much reduced in virus-transformed cells, as detected both by iodination and by metabolic labeling . We conclude that the major iodinated polypeptide is not detected on transformed cells because it is absent rather than because it is masked . Approximate molecular weights of the external proteins are also reported .

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“…Others have studied the kinetics of regeneration of plasma membrane components after exposure of intact cells to hydrolytic enzymes (for review see 23; also references 17,22,24,28,35). In each study about 6-l0 h were required for full recovery of the parameter being measured (sialic acid residues, H-2 antigen, virus receptor, cholesterol exchange capacity, etc.).…”

Section: Other Studies Of Membrane Protein Turnovermentioning

confidence: 99%

“…Different techniques have been used (3,4,8,17,20,25,33) but most suffer from the necessity of isolating membranes of varying purity and yield as well as the problem of reutilization of label. Use of the enzymatic iodination technique to label only the externally disposed membrane proteins provided a reasonable solution to both problems.…”

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confidence: 99%

Externally disposed plasma membrane proteins. II. Metabolic fate of iodinated polypeptides of mouse L cells.

Hubbard¹,

Cohn²

1975

The Journal of Cell Biology

137

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The fate of the L-cell plasma membrane proteins labeled by enzymatic iodination was studied. The disappearance of label from growing cells exhibits a biphasic behavior, with 5-20% lost rapidly (t~ ~ 2 h) and 80-90% lost relatively slowly (t~ ~ 25-33 h). The loss is temperature dependent and serum independent, and is accompanied by the appearance of 51% [l~5l]monoiodotyrosine (MIT) in the medium by 47 h. A variable amount (1-14%) of acid-insoluble label can be recovered in the medium over 47 h. Sodium dodecyl sulfate (SDS)-polyacrylamide gel labeling patterns from cells cultured up to 48 h after iodination reveal no change in the relative distribution of radioactivity, indicating similar rates of degradation for most of the labeled membrane proteins.The fate of the labeled membrane proteins was studied at various times after phagocytosis of nondigestible polystyrene particles. Iodinated L cells phagocytose sufficient 1.1 /~m latex beads in 60 min to interiorize 15-30% of the total cell surface area. Electron microscope autoradiography confirmed that labeled membrane is internalized during phagocytosis. The latex-containing phagocytic vacuoles are isolated by flotation in a discontinuous sucrose gradient. 15-30% of the total incorporated label and a comparable percentage of alkaline phosphodiesterase I activity (PDase, a plasma membrane enzyme marker) are recovered in the phagocytic vacuole fraction. Lysosomal enzyme activities are found in the latex vacuole fraction, indicating formation of phagolysosomes. SDS gel analyses reveal that all of the radioactive proteins initially present on the intact cell's surface are interiorized to the same relative extent. Incorporated label and PDase activity disappear much more rapidly from the phagolysosomes than from the whole cell. In the phagolysosomal compartment, >70% of the TCA-precipitable labeled proteins and all of the PDase activity are lost rapidly (tv, = 1-2 h) but ~ 30% of the labeled proteins in this compartment are degraded with a 17-20-h half-life. The slowly degraded label is due to specific long-lived polypeptides, of 85,000 and 8,000--15,000 daltons, which remain in the phagolysosomal membrane up to 40 h after phagocytosis.

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Regeneration of the Surface Glycoproteins of a Transplantable Mouse Tumor Cell After Treatment With Neuraminidase

Abstract: ABSTRACT

Cited by 71 publications

References 14 publications

Regeneration of Cation-Transport Capacity in HeLa Cell Membranes After Specific Blockade by Ouabain

Regeneration of Cation-Transport Capacity in HeLa Cell Membranes After Specific Blockade by Ouabain

Characterization of the External Proteins of Hamster Fibroblasts

Externally disposed plasma membrane proteins. II. Metabolic fate of iodinated polypeptides of mouse L cells.

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