Measurement of hemolytic complement and the third component of complement in nonhuman primates.

Complement, determined by hemolytic assay, and the third component of complement (C3), determined by radial immunodiffusion assay, were measured in nine nonhuman primate species. The species studied were the titi (Callicebus mollach). The sooty mangabey (Cercocebus atys), the thick-tailed galago or bushbaby (Galago crassicaudatus panganiensis), the crab-eating monkey (Macaca fascicularis), the rhesus monkey (Macaca mulatta), the bonnet monkey (Macaca radiata), the stumptailed macaque (Macaca speciosa), the yellow baboon (Papio cynocephalus), and the black-and-red tamarin (Saguinus nigricollis). Both sheep and bovine erythrocytes were used in the hemolytic complement assays. With the sheep erythrocyte system, sera from four species (yellow baboon, sooty mangabey, bonnet monkey, black-and-red tamarin) had similar titers with both antibody sensitized and non-sensitized erythrocytes. In contrast, the titers obtained using sensitized bovine erythrocytes was always higher than the values obtained using non-sensitized bovine erythrocytes. In all species, the titers for non-sensitized sheep erythrocytes was higher than the titer for non-sensitized bovine erythrocytes. When the species were compared for cross reactivity using the radial immunodiffusion assay for human C3, the rhesus monkey showed the strongest cross reaction; the thick-tailed galago, a prosimian, showed no detectable cross reactivity; and the other species examined showed intermediate degrees of reactivity.     

Heat-induced thiol-disulfide interchange reaction on the third component of human complement, C3

The third component of human complement, C3 is composed of two disulfide-bridged polypeptide chains of Mr 120,000 (alpha chain) and Mr 70,000 (beta chain). C3 has a thioester bond that serves as a binding site for targets when C3 is activated. Heat treatment of C3 induces autolytic peptide bond cleavage at the thioester site in the alpha chain as well as rupture of the thioester bond. The alpha chain fragments are linked to each other and beta chain via disulfide bonds. This study, however, documented that prolonged heating gave rise to liberation of several fragments including beta and the larger fragment of alpha chain. Using a fluorescent thiol reagent and [14C]iodoacetamide, we analyzed thiol residues present on each fragment, and elucidated that the thiol residue exposed by rupture of the thioester bond shifts in turn to another fragment resulting in the liberation of the fragments. The results were compatible with those on C4, and suggested that the generated thiol residue induces thiol-disulfide interchange reaction. On heating of plasma, fragments of C3 were not released, while the cleavage of the alpha chain occurred more effectively. The heated C3 (56 degrees C, 15 min) became insusceptible to C3b inactivator (I) and factor H, suggesting that additional conformational change is accompanied with cleavage of the thioester bond.     

An adenosinetriphosphate-activated hemolytic system. II. Utilization of adenosinetriphosphate in the reaction

Failure to demonstrate ATP¹ utilization in an ATP-activated hemolytic system had been previously reported. In the present study ATP utilization is shown to be associated with the hemolytic reaction and also with the ATP-dependent inactivation of the hemolytic factor of the system by crude, washed, human red cell stroma. Using crude stroma, relatively large ATP utilization occurs and continues, but at a decreasing rate, after inactivation of the hemolytic factor is complete. With purified stroma there is very slight uptake of ATP by the stroma in the presence of hemolytic factor and Mg⁺⁺. This uptake can only be demonstrated by radioactive ATP. Both C¹⁴- and P³²—labelled nucleotide were used for this purpose. In the presence of an excess of stroma the uptake seems to be dependent on the amount of hemolytic factor used. Evidence is given contraindicating the possibility that this uptake is non-specific.     

Morphology and distribution of type II supraependymal cells in the third ventricle of the guinea pig.

The distribution and morphology of phagocytic (Type II) supraependymal cells residing within the third ventricle of the guinea pig were investigated by scanning electron microscopy. Type II supraependymal cells were restricted to nonciliated regions of the ventricle. They were most numerous on the choroid plexus, abundant within the infundibular recess and were present on the ventricular floor in the region of the median eminence. Morphologically, they were characterized by a soma from which pseudopodia-like processes extended to the subjacent ependyma. Type II cells varied in configuration according to their location. Those residing on the choroid plexus typically had irregular somas and possessed processes that generally terminated in finger-like extensions. In contrast, cells on the ventricular floor and within the infundibular recess were stellate and possessed processes that terminated in fan-like cytoplasmic expansion. There were no differences noted in the frequency, distribution or morphology of Type II supraependymal cells in male and female animals. Furthermore, cell frequency did not appear to vary in relation to the estrous cycle. The data suggest that the pleomorphism exhibited by Type II supraependymal cells may reflect adaptations to diverse environmental conditions present within different regions of the third ventricle.     

Human anaphylatoxin (C3a) from the third component of complement. Primary structure.

C3a anaphylatoxin is derived from the third component (C3) of the blood complement system. Selective proteolysis of C3 by activated proenzymes indigenous to blood generates the C3a fragment. Human C3a was isolated from inulin-activated serum containing 6-aminohexanoic acid, according to recently published procedures (Hugli, T. E., Vallota, E., and Müller-Eberhard, H. J. (1975) J. Biol. Chem. 250, 1472-1498). The human C3a fragment is a highly cationic molecule exhibiting an approximate molecular weight of 9000 and composed of 77 amino acid residues. It consists of a single polypeptide chain containing 8% cysteine and lacks both tryptophan and carbohydrate. A tentative primary structure for the human C3a molecule, deduced from overlapping peptides obtained after cyanogen bromide cleavage, tryptic and chymotryptic digestion, is: See article. Two cystelhylcysteine sequences were established at positions 22, 23 and 56, 57 in human C3a. The 6 half-cystine residues in C3a are all interconnected through three disulfide linkages intersecting in a disulfide knot. The functionally amino acid residues distributed among 14 residues at the COOH-terminal end of C3a. This unusually cationic COOH-terminal region of C3a is presumed to play an important role in the interaction of this protein molecule with cellular receptors. A comparison between the linear sequence of human C3a and the NH2-terminal sequences of light and heavy chains of human immunoglobulin indicates that limited identity exists.