A factor that activates oscillatory chloride currents in Xenopus oocytes copurifies with a subfraction of serum albumin.
Vertebrate blood sera contain a factor that elicits oscillatory chloride currents in Xenopus oocytes through activation of the phosphatidylinositol second messenger system. This factor was purified from rabbit and human sera by a sequence of Blue-Agarose chromatography, concanavalin A affinity chromatography, and hydroxyapatite fractionation, yielding a single active protein band (67 kDa). This protein is a subfraction of serum albumin, as revealed by its molecular mass, isoelectric properties, peptide maps, amino acid composition, and NH2-terminal sequence. Moreover, the factor could be purified with a monoclonal antibody to serum albumin and its ability to elicit oscillatory currents was inhibited by several polyclonal-monospecific antibodies to serum albumin. Various commercial high purity albumin preparations elicited oscillatory currents in oocytes. The activity of albumin was partially reduced by charcoal absorption and was greatly diminished when crystalline albumin was extracted with dry methanol. However, the activity was resistant to extraction with chloroform/ether, disulfide cleavage, and denaturation with 8 M urea, 6 M guanidinium chloride, and 1% sodium dodecyl sulfate. Trypsin or lipase treatment substantially reduced the potency of the active albumin, but neither treatment alone abolished the factor even after prolonged digestion. In contrast to serum or serum albumin, freshly collected blood plasma or purified plasma albumin did not evoke oscillatory currents. This indicates that some of the plasma albumin changes during blood coagulation and acquires a "factor" that makes it capable of activating the phosphatidylinositol-Ca2+ system in Xenopus oocytes. The serum factor also activates the phosphatidylinositol system in a variety of mammalian cells, suggesting that the modified albumin may play a role in cellular events related to tissue repair following injury.
Department of Psychobiology, University of California, Irvine 92717.
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Tigyi
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Henschen
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Miledi
D000818:Animals; D000911:Antibodies, Monoclonal; D002712:Chlorides / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D008055:Lipids / Q000097:blood; D008970:Molecular Weight; D009865:Oocytes / Q000502:physiology*; D010449:Peptide Mapping; D010716:Phosphatidylinositols / Q000502:physiology; D011489:Protein Denaturation; D011817:Rabbits; D012709:Serum Albumin / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology*; D015398:Signal Transduction; D014982:Xenopus laevis / Q000502:physiology
D000911:Antibodies, Monoclonal; D002712:Chlorides; D008055:Lipids; D010716:Phosphatidylinositols; D012709:Serum Albumin