Abstract α1-Acid glycoprotein (orosomucoid), a human plasma globulin, is known to possess two major genetically transmitted variants. In this investigation the specific genetically determined amino acid substitution responsible for the occurrence of these proteins was localized to two heptapeptides by the peptide mapping technique. The variant peptides were subsequently isolated and purified from a peptic digest of the pooled desialyzed glycoprotein by column chromatography, and their sequences were established. It was found that 1 glutamine residue replaces 1 arginine residue, which is consistent with the difference in electrostatic net charge noted between the two aforementioned proteins and represents a single point mutation. The methods of investigation developed in this study will facilitate the identification of the specific amino acid substitutions responsible for the occurrence of the genetic variants of other highly conjugated glycoproteins.
The amino acid sequence of human plasma α 1 ‐acid glycoprotein, upon comparison with the sequences of other blood proteins, was shown to possess significant similarity with the immunoglobulins. Employing direct and corrected sequence identity, the average mutation value and two different computer comparisons for the evaluation of sequence similarity, the following two regions of this α‐globulin, which account for approximately half of the total amino acid sequence of the protein, were found to possess sequence similarity with the immunoglobulins. a) The region from residues 77 through 125 proved to be related to the variable region of several human H and L chains, and b) the region from residues 136 through 166 was found to be related not only to the constant region of a human and a mouse L chain but also to the third and fourth constant region of a rabbit and a human H chain, respectively. These results suggest that α 1 ‐acid glycoprotein is probably related to the immunoglobulins and further suggest that it possibly diverged from the immunoglobulin evolutionary tree prior to the formation of the primitive L chain.
A protein fraction that induces the resorption of bone explants in organ culture was isolated from the ascitic fluid of patients with advanced cancer metastatic to the peritoneal cavity. Partial purification was achieved by means of gel filtration, affinity chromatography, and ion-exchange chromatography. The isolated fraction, the components of which have an apparent molecular weight of 60,000, was found to be heterogeneous by disc gel electrophoresis and to be composed primarily of proteins with relatively acidic electrophoretic properties. The specific bone-resorptive activity of this protein fraction was greatly increased over that of the unfractionated starting material, and the activity could be completely destroyed upon incubation with pronase and on heating. As determined by immunoassay and extraction procedures with various solvents, the bone-resorptive action of the isolated fraction was not attributable to the presence of parathyroid hormone, prostaglandin E2 or vitamin D-like sterols. In parallel experiments the supernatants of phytohemagglutinin-stimulated normal human peripheral leukocytes were subjected to identical chromatographic techniques, and a proten fraction with a molecular weight of 60,000, which resembled the resorptive fraction isolated from cancer ascites fluid and which contained significant bone-resorptive activity, was also partially purified.
sequences Ala-Gly-Ser, Ala-Gly-Gly and Ser-Gly-Ala.These preliminary experiments indicate that similar degradation procedures, carried out on a larger scale, should yield peptides from bloodgroup-specific glycoproteins suitable for detailed sequence analysis.
A protein capable of stimulating bone resorption in vitro has been purified approximately 1250-fold from cancer ascites fluid. Purification was accomplished employing successive fractionation with ammonium sulfate, ion exchange, and Cibacron blue affinity chromatography, isoelectric focusing, and selective adsorption on hydroxylapatite. The bone-resorptive protein obtained by this procedure appeared homogeneous in polyacrylamide gels at pH 9.5, migrating with the mobility of an alpha 2-globulin, and in sodium dodecyl sulfate polyacrylamide gels from which an apparent molecular weight of 43,000 was calculated. The amino acid composition of the bone-resorptive protein distinguished itself by the absence of methionine and by its relatively high content of glycine (17%) and proline (11%). Furthermore, the protein possesses a single NH2-terminal amino acid residue (glycine). The ascites protein was found to contain 19% carbohydrate by weight including a high content of sialic acid (15 residues/mol) as compared to the other sugars (27 residues/mol). As to its biological properties, the homogeneous ascites glycoprotein proved to be as potent as parathyroid hormone in its ability to stimulate bone resorption in vitro.
Abstract There exists in the serum α-globulins an immunosuppressive factor(s) which was first described by Kamrin (1), and subsequently separated chromatographically by Mowbray (2), and by our laboratories (3, 4). This factor has been demonstrated to prolong skin grafts (3) and to inhibit antibody formation (5, 6). We have also found that this factor inhibits lymphocyte proliferation induced in vitro by both mitogens and antigens (4). We have recently found that the active factor is a polypeptide(s) which migrates electrophoretically with, and may be bound to, an α-globulin protein(s) (7). We have called the active material “immunoregulatory α-globulin” (IRA).4 The mechanism of action of this immunosuppressive factor remains to be determined. Studying mitogen stimulation of lymphocytes in tissue culture, we have observed that the IRA is not suppressive if this agent is added after the lymphocytes have become activated (8). This observation suggests that the IRA acts upon the early events involved in antigen recognition.
