ALBERT

Description: Sephacryl beads containing an immobilized aminopropylcobalamin- transcobalamin-II complex serve as foci for the adherence of L1210 murine leukemia cells. Bead-cell interaction does not occur when (A) nonderivatized beads are used; (B) transcobalamin-II is omitted or presaturated with cyanocobalamin in the preparation of the bead complex; (C) intrinsic factor replaces transcobalamin-II; and (D) the complex is removed from beads by photolysis. These observations suggest that adherence results from the ability of transcobalamin-II to form a bridge between immobilized cobalamin on the bead and receptors in the plasma membrane of the cell.

Description: Monocytes and macrophages have receptors for the iron-binding protein lactoferrin. Lactoferrin acts as a potent inhibitor of granulocyte- macrophage colony stimulating factor production when it binds to these cells. Using a rosette assay and immunofluorescence, we have shown that cultured leukemia cells, including the human erythroid leukemia cell line K562, also have lactoferrin binding sites. The number of binding sites on K562 cells was estimated using soluble 59Fe-lactoferrin. Inhibition studies demonstrate that lactoferrin binding sites are distinct and unrelated to receptors for transferrin or the Fc portion of IgG, which are present on K562 cells. However, electrostatic forces may be important for lactoferrin binding, since other polycationic proteins (eg, protamine) inhibit lactoferrin binding. Prior treatment of K562 cells with trypsin nearly abolishes lactoferrin binding. However, these cells recover their ability to bind lactoferrin when trypsin is removed. Unlike transferrin receptors, the expression of lactoferrin binding sites is not regulated by cellular iron status. Cytosine arabinoside arrests the proliferation of K562 cells and simultaneously leads to a reduction in lactoferrin surface binding, suggesting that lactoferrin binding may be dependent on cell proliferation.

Description: To determine the nature of binding of transcobalamin II (TC-II) to liver cells, we covalently coupled purified holo-TC-II to submicron latex minibeads using glutaraldehyde. Incubation of the probe with liver cell suspensions at 4 degrees C led to its binding by endothelial cells but not by hepatocytes or Kupffer cells, as visualized by scanning electron microscopy. At 37 degrees C, the probe was internalized by the endothelium through a system of coated pits and vesicles as shown by transmission electron microscopy. Inhibition studies by pre-incubation with excess native TC-II demonstrated the specificity of binding. Fractionation of these cell suspensions on metrizamide gradients yielded large cell (hepatocyte-rich) and small cell (endothelium-rich) fractions. The binding of the minibead probe occurred again exclusively on endothelial cells in the small cell fraction. 125I-labeled holo-TC-II also bound to the small cell but not to the large cell fraction. Binding was saturable (Ka, 0.225 X 10(9) mol/L-1) and receptor number was calculated to be 1.33 X 10(3) per cell. Time-dependent incubation of 125I-labeled TC-II with the endothelium-rich fraction led to its uptake, reaching a steady-state plateau at 4 degrees C. At 37 degrees C, however, the initial uptake was followed by gradual release of the label into the medium. We conclude that in the liver, holo-TC-II binds initially to endothelium, where it is internalized and is subsequently released probably to the interstitial space. Thus, the endothelium may play a fundamental role in the regulation of the uptake of TC-II by the liver.

Description: Monocytes and macrophages have receptors for the iron-binding protein lactoferrin. Lactoferrin acts as a potent inhibitor of granulocyte- macrophage colony stimulating factor production when it binds to these cells. Using a rosette assay and immunofluorescence, we have shown that cultured leukemia cells, including the human erythroid leukemia cell line K562, also have lactoferrin binding sites. The number of binding sites on K562 cells was estimated using soluble 59Fe-lactoferrin. Inhibition studies demonstrate that lactoferrin binding sites are distinct and unrelated to receptors for transferrin or the Fc portion of IgG, which are present on K562 cells. However, electrostatic forces may be important for lactoferrin binding, since other polycationic proteins (eg, protamine) inhibit lactoferrin binding. Prior treatment of K562 cells with trypsin nearly abolishes lactoferrin binding. However, these cells recover their ability to bind lactoferrin when trypsin is removed. Unlike transferrin receptors, the expression of lactoferrin binding sites is not regulated by cellular iron status. Cytosine arabinoside arrests the proliferation of K562 cells and simultaneously leads to a reduction in lactoferrin surface binding, suggesting that lactoferrin binding may be dependent on cell proliferation.

Description: Sephacryl beads containing an immobilized aminopropylcobalamin- transcobalamin-II complex serve as foci for the adherence of L1210 murine leukemia cells. Bead-cell interaction does not occur when (A) nonderivatized beads are used; (B) transcobalamin-II is omitted or presaturated with cyanocobalamin in the preparation of the bead complex; (C) intrinsic factor replaces transcobalamin-II; and (D) the complex is removed from beads by photolysis. These observations suggest that adherence results from the ability of transcobalamin-II to form a bridge between immobilized cobalamin on the bead and receptors in the plasma membrane of the cell.

Description: To determine the nature of binding of transcobalamin II (TC-II) to liver cells, we covalently coupled purified holo-TC-II to submicron latex minibeads using glutaraldehyde. Incubation of the probe with liver cell suspensions at 4 degrees C led to its binding by endothelial cells but not by hepatocytes or Kupffer cells, as visualized by scanning electron microscopy. At 37 degrees C, the probe was internalized by the endothelium through a system of coated pits and vesicles as shown by transmission electron microscopy. Inhibition studies by pre-incubation with excess native TC-II demonstrated the specificity of binding. Fractionation of these cell suspensions on metrizamide gradients yielded large cell (hepatocyte-rich) and small cell (endothelium-rich) fractions. The binding of the minibead probe occurred again exclusively on endothelial cells in the small cell fraction. 125I-labeled holo-TC-II also bound to the small cell but not to the large cell fraction. Binding was saturable (Ka, 0.225 X 10(9) mol/L-1) and receptor number was calculated to be 1.33 X 10(3) per cell. Time-dependent incubation of 125I-labeled TC-II with the endothelium-rich fraction led to its uptake, reaching a steady-state plateau at 4 degrees C. At 37 degrees C, however, the initial uptake was followed by gradual release of the label into the medium. We conclude that in the liver, holo-TC-II binds initially to endothelium, where it is internalized and is subsequently released probably to the interstitial space. Thus, the endothelium may play a fundamental role in the regulation of the uptake of TC-II by the liver.

Description: Plasma membrane receptors for the serum cobalamin-binding protein transcobalamin II (TCII) were identified on human leukemia K562 and HL- 60 cells using immunoaffinity-purified human TCII labeled with [57Co]cyanocobalamin. The Bmax values for TCII receptors on proliferating K562 and HL-60 cells were 4,500 and 2,700 per cell, respectively. Corresponding dissociation constants (kd) were 8.0 x 10(- 11) mol/L and 9.0 x 10(-11) mol/L. Rabbit TCII also bound to K562 and HL-60 cells but with slightly reduced affinities. Calcium was required for the binding of transcobalamin II to K562 cells. Brief treatment of these cells with trypsin resulted in almost total loss of surface binding activity. After removal of trypsin, surface receptors for TCII slowly reappeared, reaching pretrypsin treatment densities only after 24 hours. Reappearance of receptors was blocked by cycloheximide. TCII receptor densities on K562 and HL-60 cells correlated inversely with the concentration of cobalamin in the culture medium. This suggests that intracellular stores of cobalamin may affect the expression of transcobalamin receptors. Nonproliferating stationary-phase K562 cells had low TCII receptor densities (less than 1,200 receptors/cell). However, the density of TCII receptors increased substantially when cells were subcultured in fresh medium. Up-regulation of receptor expression coincided with increased 3H-thymidine incorporation, which preceded the resumption of cellular proliferation as measured by cell density. In the presence of cytosine arabinoside, which induces erythroid differentiation, K562 cells down-regulated expression of TCII receptors. When HL-60 cells were subcultured in fresh medium containing dimethysulfoxide to induce granulocytic differentiation, the up- regulation of TCII receptors was suppressed. This event occurred well before a diminution of 3H-thymidine incorporation and cessation of proliferation. Thus, changes in the regulation of expression of TCII receptors correlate with both the proliferative and differentiation status of cells.

Description: Plasma membrane receptors for the serum cobalamin-binding protein transcobalamin II (TCII) were identified on human leukemia K562 and HL- 60 cells using immunoaffinity-purified human TCII labeled with [57Co]cyanocobalamin. The Bmax values for TCII receptors on proliferating K562 and HL-60 cells were 4,500 and 2,700 per cell, respectively. Corresponding dissociation constants (kd) were 8.0 x 10(- 11) mol/L and 9.0 x 10(-11) mol/L. Rabbit TCII also bound to K562 and HL-60 cells but with slightly reduced affinities. Calcium was required for the binding of transcobalamin II to K562 cells. Brief treatment of these cells with trypsin resulted in almost total loss of surface binding activity. After removal of trypsin, surface receptors for TCII slowly reappeared, reaching pretrypsin treatment densities only after 24 hours. Reappearance of receptors was blocked by cycloheximide. TCII receptor densities on K562 and HL-60 cells correlated inversely with the concentration of cobalamin in the culture medium. This suggests that intracellular stores of cobalamin may affect the expression of transcobalamin receptors. Nonproliferating stationary-phase K562 cells had low TCII receptor densities (less than 1,200 receptors/cell). However, the density of TCII receptors increased substantially when cells were subcultured in fresh medium. Up-regulation of receptor expression coincided with increased 3H-thymidine incorporation, which preceded the resumption of cellular proliferation as measured by cell density. In the presence of cytosine arabinoside, which induces erythroid differentiation, K562 cells down-regulated expression of TCII receptors. When HL-60 cells were subcultured in fresh medium containing dimethysulfoxide to induce granulocytic differentiation, the up- regulation of TCII receptors was suppressed. This event occurred well before a diminution of 3H-thymidine incorporation and cessation of proliferation. Thus, changes in the regulation of expression of TCII receptors correlate with both the proliferative and differentiation status of cells.