title: Pyridoxal phosphate. An anionic probe for protein amino groups exposed on the outer and inner surfaces of intact human red blood cells.
abstract: Pyridoxal phosphate is a potent probe for exploring the "sidedness" of proteins in the membrane of the intact red blood cell. It reacts with amino groups with a high degree of specificity, forming a Schiff's base that can be fixed as an irreversible bond upon reduction with NaBH4; its binding site can be identified by use of [3-H]pyridoxal phosphate or NaB3-H4; it can be used as a surface probe under conditions of minimal penetration, or it can be used as a probe for proteins on the inside of the membrane under conditions of substantial uptake. Pyridoxal phosphate uptake involves a rapid and a slow component. The former represents the binding to the outer surface of the membrane; it is not substantially affected by pH and temperature, but is reduced considerably by pretreatment of cells by 4,4-diisothiocyano-2,2-stilbenedisulfonic acid, a specific inhibitor of anion transport. The slow component represents penetration into the cell; it is blocked by high pH, low temperature, or pretreatment with the disulfonic stilbene. Pyridoxal phosphate itself is also an effective and specific inhibitor of the permeation of other anions. Under conditions of minimal uptake, the only labeled proteins are three glycoproteins and a protein of apparent molecular weight 95,000. Under conditions of substantial uptake into the cell, the other major protein bands seen by staining on acrylamide gels after electrophoresis are labeled. It is concluded that virtually all of the major membrane proteins interact with pyridoxal phosphate from one side of the membrane or the other. The differences in labeling under conditions of minimal or maximal uptake can, therefore, be attributed to the sidedness in the distribution of the membrane proteins rather than to differences in their reactivity.
mesh_terms: D000596:Amino Acids / Q000032:analysis; D001881:Borates / Q000378:metabolism; D002352:Carrier Proteins; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D004912:Erythrocytes / Q000378:metabolism*; D006023:Glycoproteins; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D010504:Periodic Acid / Q000494:pharmacology; D010761:Phosphorus Radioisotopes; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D011733:Pyridoxamine / Q000378:metabolism*; D012545:Schiff Bases; D013267:Stilbenes / Q000494:pharmacology; D013431:Sulfates / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014316:Tritium
chemical_list: D000596:Amino Acids; D001881:Borates; D002352:Carrier Proteins; D006023:Glycoproteins; D006454:Hemoglobins; D010761:Phosphorus Radioisotopes; D012545:Schiff Bases; D013267:Stilbenes; D013431:Sulfates; D013462:Sulfur Radioisotopes; D014316:Tritium; D010504:Periodic Acid; D011732:Pyridoxal Phosphate; D011733:Pyridoxamine