Abstract
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.
Highlights
From the Laboratory of Cellular and Molecuhr Neurobiology,Department of Psychobiology and the §Department of Molecular Biology and Bwchemistry, University of California,Zrvim, California 92717
The factor could bepurified witha monoclonal antibody to serum albumin and its ability to elicit oscillatory opus oocytes even when diluted more than a million-fold [9]
Various commercial Here we describe the purification of the serum factor rehigh purity albumin preparations elicited oscillatory sponsible for eliciting oscillatory chloride currents in oocytes
Summary
Since Cohn fraction V is approximately 96% or higher pure albumin [28] we tested several other albumin preparations obtained from various vendors over a 3-year period To illustrate these results 12 different commercial albumin preparations were applied on a single oocyte to compare their potency for eliciting oscillatory currents. 3also contained albumin (see Fig. 2, gradient gel insets), the source of these different albumins, whether it was plasma no serum factor activity was detectable in these two fractions or serum, was not always available to us. Fresh uncoagulatedblood and plasma did not elicit oscillatory fold dilution This fraction (Ba5) was usedfor chloride currents in Xenopus oocytes (Fig. l), even at very further purification of the active component. Human plasma albumin (HPA, 1mg/ml) did not evoke oscillatory currents in oocytes, whereas human serum and human serum albumin (HSA, 1pg/ml) were effective
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