Chemistry and biology of the anaphylatoxin related serum peptide system. I. Purification, crystallization and properties of classical anaphylatoxin from rat serum

Abstract

Chemistry and biology of the anaphylatoxin related serum peptide system* I. Purification, crystallization and properties of classical anaphylatoxin from rat serum" Classical anaphylatoxin, which causes histamine liberation and lethal anaphylatoxin shock in the guinea pig, was purified from dextran-treated rat serum leading t o a 5000-fold increase in specific activity. It was crystallized in a molecular homogenous state. Rat anaphylatoxin consists of one peptide unit with a molecular weight of 9500, as determined by gel chromatography.First, evidence is presented that the anaphylatoxin-forming contact reaction of normal, fresh serum with hydrophilic, insoluble substances of high molecular weight, e.g. dextran, yeast or antigen-antibody complexes, does not lead to a stable anaphylatoxin molecule exclusively, but t o a group of peptides of similar physicochemical behavior. Only one of these peptides displays anaphylatoxin activity. The lack of significant chemotactic activity for neutrophil leucocytes is characteristic for this crystallized classical anaphylatoxin. This indicates that anaphylatoxin and chemotactic activities are two different biological principles. Abbreviations: CAT: Classical anaphylatoxin Complement components: Nomenclature according to Bull. WHO 1968. 39. 935, also given in Immunochernistry 1970. 7: 137 J.H. Wissler Eur. J. Immunol. 1972. 2: 73-83 del-de-Haen, Selze-Hannover, Germany and Fluka AG, Buchs SG., Switzerland. Proteins for column calibration were obtained in the highest purity grade available from Serva, and Sigma, St. Louis, U.S.A. Unless otherwise stated, these chemicals were used throughout. Trasylol (kallikrein inactivator) is a product of Bayer, Leverkusen, Germany. All kinds of Sephadex, Blue Dextran and Dextran 2000 (To. 4386 = 0.70, % , ca. 2 . l o 6 ) were obtained from Pharmacia AB, Uppsala, Sweden.Before use, all Sephadex sorts, especially CM-Sephadex C-50, were treated with hog serum for surface inactivation (minimum incubation time: 1 day).After removal of the excess of protein by rinsing of the gels with tap water, gels were regenerated by successive treatment with 0.1 N HCl and 0.1 N NaOH, as recommended by the seller (flotation method). For purposes of neutralization and for buffer solutions, only double distilled water was used. This is crucial for a successful purification procedure (see also under section 4, 5). Hydroxyapatite was prepared according t o Swingle and Tiselius [27], Hydroxyapatite was separated into crystal particles of homogenous particle size distribution according t o the method of Hamilton [28, 291. Particle size fractions of 5 -10 pm and 10 -I 5 pm in Martin's diameter [30] were obtained and used* throughout. Calcium phosphate gel was synthesized according t o the method of Keilin and Hartree [31]. Only gels of a minimum age of ten weeks were used.

Inability of Rat Anaphylatoxin to Induce Histamine Release in Rats

“…The purified anaphylatoxin with our method showed a single band both in PAGE and in SDS-PAGE (Fig. 5A and 5B), and the minimal concentration to contract guinea pig ileum was 1.1 X 10-7 M, which was similar to that of rat anaphylatoxin (1.7 x 10-7 M) previously reported (17,28).…”

“…Therefore, it is unlikely that the inability of rat anaphylatoxin to induce histamine release from rat connective tissue mast cells is due to its hydrolytic inactivation by the anaphylatoxin inactivator. Up to now, there has been only one report (17) dealing with the purification of rat anaphylatoxin, probably because of the difficulty in collecting a large volume of blood from rats. The purification procedure reported (17) is very laborious, involving eight complex steps.…”

Tumor necrosis factor/cachectin. Induction of hemorrhagic necrosis in normal tissue requires the fifth component of complement (C5).

“…In humans, this derivative is greatly reduced in its biological activity both in vivo and in vitro and it has completely lost spasmogenic activity (32) . In contrast, rodent C5a-des-arg retains much of the original activity of the parental C5a molecule (27) . It is possible, therefore, that C5a-des-arg behaves the same way as C5a in the HN response described here .…”

Biochemistry and Biology of a Leucotactic Binary Serum Peptide System Related to Anaphylatoxin

“…rat and guinea pig sera were used in the pre sent investigation. After removal of the activating substance, anaphyla toxin can be detected by various assays [44,45]: in vivo, intravenous injections of anaphylatoxin into guinea pigs results in lethal anaphyla toxin shock with formation of lung emphysema; in vitro, anaphylatoxin activity is demonstrable by contraction of guinea pig smooth muscle [for review, see 17].The presence of cocytotaxin, the second component of the serum peptide system can be demonstrated in combination with anaphylatoxin by a chemotactic activity assay (see I IB) for neutrophil and eosinophil leucocytes.The complex purification procedure of the two peptides, starting with modified serum, will not be described here in detail. Instead, summarizes the sequence and results of purification steps, including an aphylatoxin and chemotactic activity for neutrophils.…”