The purification and properties of the second component of human complement.

The second component of human complement (C2) was purified by a combination of euglobulin precipitation, ion-exchange chromatography, (NH4)2SO4 precipitation and affinity chromatography. The final product was homogeneous by the criterion of polyacrylamide-gel electrophoresis and represents a purification of about 4000-fold from serum with 15-20% yield. Component C2 comprises a single carbohydrate-containing polypeptide chain, with an apparent mol.wt. of 102000; alanine is the N-terminal amino acid. The molecule is rapidly cleaved by activated subcomponent C1s with the loss of haemolytic activity to yield two fragments with apparent mol.wts. of 74000 and 34000. These fragments are not linked by disulphide bonds and can be easily separated. A second protein isolated during the purification of component C2 was identified by its haemolytic and antigenic properties as complement Factor B, the protein serving an analogous function to component C2 in the alternative pathway. The protein, which is also a single carbohydrate-containing polypeptide chain, has an apparent mol.wt. of 95000 and threonine as N-terminal amino acid. The amino acid analyses of component C2 and Factor B are compared.     

Genetic variation in haemolytic activity in Atlantic salmon (Salmo salar L.)

Spontaneous and antibody-dependent haemolytic activity against sheep red blood cells (SRBC) has been studied in serum from Atlantic salmon, Salmo salar L. Considerable increase in haemolytic activity was detected when SRBC were sensitized with haemolysin produced in Atlantic salmon. Haemolytic activity was sensitive both to divalent cations and to heat treatment. Significant reduction in haemolytic activity was detected after absorption with SRBC, indicating the presence of natural antibody against SRBC in Atlantic salmon serum. Persistent haemolytic activity of unsensitized SRBC in serum absorbed to remove natural antibodies was found, which suggests the activation of the alternative complement pathway, while the observed increased haemolytic activity in the presence of specific antibody against SRBC suggests activation of the classical complement pathway. Spontaneous and antibody-dependent haemolytic activity were both analysed in absorbed and non-absorbed sera in a family material of Atlantic salmon. The material consisted of 574 fish belonging to 57 fullsib groups within 20 paternal halfsib groups. Fish with signs of sexual maturity generally showed reduced haemolytic activity. Statistically significant effect of sire on the spontaneous haemolytic activity of both absorbed and nonabsorbed serum, and on antibody-dependent activity, provides evidence of significant additive genetic variation in both the alternative and the classical complement activation in Atlantic salmon. Neither on a phenotypical nor on a family basis were the two traits statistically correlated. The estimated heritabilities were 0.2–0.3 with a standard error of approximately 0.1.     

An altered heart-labile enterotoxin (LT') produced by Escherichia coli serogroup O55 strain.

An Escherichia coli serogroup O55 strain produced heat-labile enterotoxin only, which exerted unusual effects on cell cultures; it caused elongation of CHO and HeLa cells but no changes in Y-1 cells. Injection of this substance, designated LT', into mouse foot pad and rabbit skin caused a well-expressed necrotic effect beside the LT-like activity. LT' showed no cytotoxic effect and failed to produce mouse lung oedema. The strain was not haemolytic. According to Sephadex G-100 fractionation, the LT' had a high molecular weight. The LT' and the necrotic activities could not be separated by fractionation. Neutralization experiments suggested an antigenic relationship between LT and LT'. The antigenic deficiency of LT' was closely related to the common antigenic component of LT and choleragen. The necrotic effect caused by crude LT' was neutralized only by the homologous serum.     

Activity of radiation degradation products of vitamins A and E to haemolyse erythrocyte

Exposure of vitamin A acetate in freely dissolved state to γ-radiationin vitro caused a dose dependent degradation accompanied by the formation of new products. The radiation degradation products were separated by chromatography using step gradient elution. The parent molecule, vitamin A acetate, induced negligible haemolysis of erythrocytes. In contrast, the polar products formed by irradiation were found to be potent haemolysing agents. A highly polar product, eluted with methanol revealed maximum haemolytic activity. Acetylation of these products resulted in loss of their haemolytic properties. Similarly, vitamin E acetate, a known stabilizer of the biomembranes, after irradiation yielded products which caused haemolysis of erythrocytes. It was demonstrated that irradiation introduces hydroxyl groups which impart haemolytic properties to the radiation degradation products of vitamin A     

Haemolytic factor from the crop of Rhodnius prolixus: Evidence and partial characterization

A haemolytic factor, which lysed sheep red cells in an isotonic buffer, was found in the crop of all larval stages and adult Rhodnius prolixus. Little or no haemolytic factor occurred in unfed insects but haemolytic activity increased for 2–4 days after feeding. From the 4th day on, the activity declined gradually. Fifth-instar larvae fed on whole blood, erythrocytes and haemoglobin produced large quantities of haemolytic factor, while those fed on plasma and erythrocyte stroma did not. The haemolytic factor was purified approximately 1200-fold by a two-step procedure: (1) Bio-Gel P-6 Gel-Filtration and (2) SP-Sephadex chromatography. Purified haemolytic factor was heatstable (100°C, 10 min), dialysable, inactivated by trypsin treatment, and could be recovered in the supernatant after addition of ethanol. It was concluded that the haemolytic factor is a peptide displaying a basic character.     

Biosynthesis of the first component of complement by human fibroblasts

1. Haemolytic activity corresponding to that of the first component of complement (C1) was synthesized and secreted by all nine human fibroblast cell lines examined. No activity was found in the culture media of a variety of other human cell lines. 2. The component-C1 haemolytic activity secreted by the fibroblast lines behaved in an identical manner, in most respects, with that of the component-C1 haemolytic activity of human serum. The component-C1 haemolytic activity secreted by fibroblasts, however, was less susceptible to inhibition by rabbit fragment F(ab′)₂ anti-(human subcomponent C1q) than was the component-C1 haemolytic activity of human serum. 3. Biosynthesis of fibroblast component-C1 haemolytic activity was inhibited by the presence of cycloheximide and regained on its removal. 4. Incorporation of radioactivity into proteins secreted by the fibroblasts and release of component-C1 haemolytic activity by the fibroblasts both increased in a linear manner until several days after the cultures had reached a state of confluent growth. 5. Radioactivity was incorporated into subcomponents C1q, C1r and C1s, as judged by the formation of specific immunoprecipitates and by absorption with immune aggregates. 6. The immunoprecipitates formed by using antisera against subcomponents C1r and C1s were run on polyacrylamide gels in sodium dodecyl sulphate, and this provided convincing physiochemical evidence for the biosynthesis of these subcomponents de novo. 7. The results obtained with immunoprecipitates formed by using anti-(subcomponent C1q) suggest that subcomponent C1q may be synthesized and secreted by fibroblast cell lines in vitro, in a form with a higher molecular weight than that of subcomponent C1q which is isolated by conventional techniques of protein fractionation from fresh serum.     

Effect of Human Serum Albumin Upon the Permeabilizing Activity of Sticholysin II, a Pore Forming Toxin from Stichodactyla heliantus

Sticholysin II (St II) is a haemolytic toxin isolated from the sea anemone Stichodactyla helianthus. The high haemolytic activity of this toxin is strongly dependent on the red cell status and the macromolecule conformation. In the present communication we evaluate the effect of human serum albumin on St II haemolytic activity and its capacity to form pores in the bilayer of synthetic liposomes. St II retains its pore forming capacity in the presence of large concentrations (up to 500 μM) of human serum albumin. This effect is observed both in its capacity to produce red blood cells haemolysis and to generate functional pores in liposomes. In particular, the capacity of the toxin to lyse red blood cells increases in the presence of human serum albumin (HSA). Regarding the rate of the pore forming process, it is moderately decreased in liposomes and in red blood cells, in spite of an almost total coverage of the interface by albumin. All the data obtained in red cells and model membranes show that St II remains lytically active even in the presence of high HSA concentrations. This stubbornness can explain why the toxin is able to exert its haemolytic activity on membranes immersed in complex plasma matrixes such as those present in living organisms.     

Haemolytic and bactericidal activity of serum from the ring dove (Streptopelia risoria) after treatment with exogenous prolactin

The purpose of this study was to elucidate the effect of exogenous prolactin on the haemolytic and bactericidal capacity of serum obtained from ring doves (Streptopelia risoria) previously injected with either bovine serum albumin or saline solution. Haemolytic activity was measured in CH-50 units (which represents the capacity of serum complement to lyse 50% of sheep red blood cells in the presence of specific antibody) and the bactericidal activity was estimated from the number of colony-forming units ofStaphylococcus aureus which survived after 24 h of incubation in the presence of serum. The results indicated that: (1) bovine serum albumin stimulated both haemolytic and bactericidal activity, the highest values occurring 24 h and 4 days after administration, respectively. (2) Prolactin induced an increase in the haemolytic activity of complement. (3) The administration of bovine serum albumin to animals previously treated with prolactin produced a greater stimulation than either bovine serum albumin or prolactin alone.Abbreviations BSA bovine serum albumin - CFT complement fixation test - CFU colony-forming units - CH-50 units, the reciprocal of the complement dilution with 50% lysis of the hemolysin-treated erythrocytes - IU international units - PBS phosphate-buffered saline solution - SS saline solution     

A rapid and sensitive analytical procedure for human plasminogen subspecies. Combination of high-performance affinity chromatography and specific monitoring of proenzymes

Human plasminogens specifically separated by high-performance affinity chromatography were specifically detected by a newly devised, on-line monitoring system, in which the proenzymes were activated by urokinase and plasmin activity thus generated was assayed. Presence of Lys-plasminogen as a constituent in the blood was demonstrated by both chromatographic patterns and biochemical experiments. This system made it possible to estimate rapidly not only Glu-plasminogen but also Lys-plasminogen in the plasma without any pretreatment. Its utility as a tool for clinical analysis of fibrinolytic system was suggested.     

Human 3-methyladenine-DNA glycosylase: Demonstration of a stimulatory factor

Cultured human lymphoblasts contain a component that stimulates 3-methyladenine-DNA glycosylase, resulting in increased removal of 3-methyladenine from alkylated DNA. Increased release of other methylated bases was not detected by high-pressure liquid chromatography. Separation of the component and 3-methyladenine-DNA glycosylase during enzyme purification results in a loss of glycosylase activity. Stimulation of glycosylase activity can be demonstrated by recombination of the separated component with a partially purified lymphoblast enzyme fraction.     

Isolation and characterization of C1q, a subcomponent of the first component of complement, from human and rabbit sera

1. C1q, a subcomponent of the first component of complement, has been isolated, in a haemolytically active and soluble form, by ion-exchange chromatography and gel filtration, from human and rabbit sera. Yields ranged from 10 to 25mg/litre of serum and the activity of final preparations was consistently in the range 5x10(3)-15x10(3) C1qH(50) units/mg. 2. The molecular weights of human and rabbit subcomponent C1q were 409600 and 417600, as determined by sedimentation equilibrium studies. 3. Subcomponent C1q from both species was shown to be composed of non-covalently linked subunits of approximately 57000 molecular weight as determined by gel-filtration or sedimentation equilibrium studies in 5.3m-guanidinium chloride. Reduction or oxidation of human and rabbit subcomponent C1q yielded three chains each having a molecular weight of approximately 23000 and which differed slightly in amino acid composition but markedly in carbohydrate content. The oxidized chains were separated, on a preparative scale, by ion-exchange chromatography in 8m-urea on DEAE-cellulose. 4. Both human and rabbit subcomponent C1q contained hydroxyproline, hydroxylysine, a high percentage of glycine and approximately 8% carbohydrate. Glutamic acid and aspartic acid were the free N-terminal amino acids of human subcomponent C1q whereas only serine was found in rabbit subcomponent C1q. 5. Collagenase digestion of human or rabbit subcomponent C1q caused a rapid loss of haemolytic activity which correlated with the breakdown of collagenous regions in the molecule.     

Increased erythrocyte adenosine deaminase activity without haemolytic anaemia

A 4-fold increase of red blood cell adenosine deaminase (ADA) activity was found in a patient without haemolytic anaemia, but with mild anisopoikilocytosis. High-performance liquid chromatography showed a 40% reduction of adenosine-5'-triphosphate (ATP) while all the other nucleotides were in normal ranges. The patient's parents (first cousins) and a brother displayed the same enzyme activities as the controls. This observation suggests that mild increases of ADA activity is neither a marker for congenital hypoplastic anaemia as previously reported nor associated with haemolytic anaemia.     

Components of human complement system. Testing and isolation of C4

A modified reagent for testing the hemolytic activity of human complement component C4 has been obtained. Reagent R4 was obtained by treatment of human blood serum pools with 0.075 M solution of hydrazine hydrate. This reagent was found to be rich in the serum fraction obtained by chromatography on DEAE-cellulose DE-52 and containing an active complement C3 component. To test the sensitivity and specificity of the reagent, component C4 was subjected to purification. This procedure resulted in a hemolytically active, electrophoretically and immunoelectrophoretically homogeneous component C4. DEAE-cellulose DE-52, DEAE-Sephacel, Ultragel AcA-34 and, again, DEAE-Sephacel were used consecutively as purification agents. The activity yield of component C4 with regard to the initial serum level was 20%.     

A one step separation of immunoglobulin g from bovine serum by pseudobioaffinity chromatography on histidine grafted to epoxy activated sepharose

The selective adsorption of proteins and macromolecules on activated matrix grafted with histidine has been shown to be dependent on certain separation parameters like, pH and buffer type. In the present study, the significant potential of the histidine ligand to separate bovine IgG from other bovine serum proteins has been demonstrated. The successful separation was carried out by pseudobioaffinty chromatography on histidine grafted-epoxy activated sepharose. The method was applied to sterile bovine serum. Bovine IgG was completely separated in the form of a single peak with 25 mM MES buffer containing 0.2 M NaCl at pH 5. The purity of the separated bovine IgG was determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Ouchterlony radial immunodiffusion (ODD) assay showed that bovine IgG was the main component present in the elution fraction.     

Cortisol and immune characteristics in rainbow trout (Oncorhynchus mykiss) selected for high or low tolerance to stress

The influence of exposure to stressors on cortisol and the non-specific immune traits lysozyme and serum haemolytic activity were examined in second generation rainbow trout ( Oncorhynchus mykiss ) selected for either high or low serum cortisol level following a confinement stress. Lysozyme and serum haemolytic activity were also assessed, together with levels of specific antibodies against Aeromonas salmonicida A-layer, Vibrio salmonicida O-antigen and Vibrio anguillarum O-antigen, following injection of vaccines against these pathogens.
Significant differences in mean cortisol levels between the two selection lines were observed, but in only one of two stress experiments was the 'high-stress' line found to have the higher cortisol level; in the other experiment the 'high-stress' line had significantly lower cortisol levels than the 'low-stress' line. Lysozyme levels were in four of four assessments higher in the high-stress line than in the low-stress line, whereas components of serum haemolytic activity tended to be lower in the high-stress line than in the low-stress line. Levels of specific antibodies against all three bacterial pathogens were elevated following the injection of the vaccines. Only antibody production against A. salmonicida A-layer was significantly different between the two lines, the higher production of antibody being in the high-stress line.     

Partial purification and characterization of platelet factors stimulating the multiplication of normal human glial cells

Multiplication-stimulating activity for human glial cells was purified from human outdated platelets. By ion exchange chromatography anionic activity was separated from cationic activity. The former could be further separated by Sephadex G-200 gel chromatography into two peaks, whose molecular weights were 40 000 and < 10 000. The cationic activity was partially purified by concanavalin A (ConA) Sepharose chromatography, hydroxylapatite chromatography and SDS-polyacrylamide gel electrophoresis. The cationic activity was heterogeneous as demonstrated by isoelectric focusing (Ip 9.5–10.4), gel filtration on Bio-Gel P-150 and SDS-polyacrylamide gel electrophoresis (mol. wt 26 000–33 000). Less than 50 ng/ml was required of the factor to give a glial cell stimulation corresponding to that afforded by 1 % of human serum. A thymidine-degrading enzyme, present in human platelets and to a low degree also in human serum, was found to interfere with the assay for multiplication-stimulating activity. The enzyme (probably a thymidine phosphorylase) converted [³H]thymidine to [³H]thymine, causing a reduced incorporation of ³H into cellular DNA. This difficulty was circumvented by use of an autoradiographic estimation (per cent labelled nuclei) of the multiplication-stimulating activity.     

Purification and activity of two phospholipase enzymes from Naja nigricolis nigricolis Reinhardt venom

Two phospholipase enzymes NN1 and NN2 were purified from the venom of Naja nigricolis nigricolis Reinhardt to apparent homogeneity. NN1 was purified by a two-step anion-exchange chromatography on DEAE-cellulose column while NN2 was purified by a combination of anion-exchange chromatography and gel filtration on Sephadex G-150. The enzyme NN1 moved homogenously on acrylamide gel as a monomer with a molecular weight of 65 kDa while NN2 was a dimer of 71 kDa. Both enzymes were clearly separated. Both enzymes hydrolyzed L-alpha-phosphatidyl choline with activities of 345.5 for NN1 and 727.8 micromol min(-1) x mg(-1) for NN2. The dimeric 71-kDa enzyme has a higher haemolytic and anticoagulant activity than the monomeric 65-kDa enzyme. It is apparent that the dimeric enzyme has a more pronounced activity than the monomer has, thus toxic activity may be related to the hydrolysis of phospholipids.     

Protein kinase activities in mammalian blood fluid

Two protein kinase activities, one specific for phosvitin and another specific for histone, were detected in serum and plasma of calf as well as of human blood after precipitation with ammonium sulfate (40%) and chromatography on DEAE-Sephacel. The enzymes were separated by chromatography on phosphocellulose. The histone kinase is not related to the cyclic AMP-dependent protein kinase; it may derive at least partly from damaged cells. The phosvitin kinase activity carries characteristics of the so called casein kinase type II similar to that present at the surface of cells including blood cells.     

Mycoplasma growth factors in bovine serum fraction.

Mycoplasma growth factors in bovine serum fraction were separated by Sephadex G150 column chromatography and density ultracentrifugation. The major growth factor of bovine serum fraction eluted from the Sephadex column in the void volume. Its growth-supporting activity was greatly enhanced by the presence of bovine serum albumin in the mycoplasma culture media. Other investigators had previously identified the major growth factor in serum as an alpha-lipoprotein. Although density ultracentrifugation revealed the presence of traces of a high-density lipoprotein in bovine serum fraction, another, less dense component, isolated by ultracentrifugation (component 3) and containing cholesterol, cholesteryl esters, free fatty acids, triglycerides, and protein, but no lipoprotein, exhibited considerably more growth-supporting activity than did the high-density lipoprotein, thus indicating that at least two mycoplasma species do not require intact serum lipoprotein for growth. Both the high-density lipoprotein and component 3 exhibited maximum activity only in the presence of bovine serum albumin. A chloroform extract containing component 3 lipids combined with bovine serum albumin to form an effective, partially defined, less complex substitute for serum in mycoplasma culture media.     

Inhibition of serum complement haemolytic activity by lipid vesicles containing phosphatidylserine

The effect of artificial model membranes on the complement system was investigated. Incubation of the model membranes with human serum resulted in consumption of complement haemolytic activity when phosphatidylserine-containing vesicles were used. The activation of the complement system appeared to proceed through the alternative pathway. This conclusion was supported by the failure of [125I]Clq to bind to the membranes suggesting that the classical pathway was not involved. Although always obtained when phosphatidylserine was present in the model membranes, the activation of complement was enhanced by the contemporaneous presence of phosphatidylethanolamine. Liposomes prepared from lipid extracts of red blood cells were also able to stimulate a concentration-dependent activation of complement. Fresh, intact erythrocytes, however, could not initiate the same effects unless opsonized by antibodies. When artificially aged in vitro, red blood cells were lysed if incubated with normal human serum or with Clq-depleted serum. However, no lysis was obtained if the 'aged' erythrocytes were incubated with serum pretreated with ammonia to destroy the C3 component of complement. It is suggested that one of the mechanisms of macrophage recognition of senescent erythrocytes might be provided by the activation of the alternative pathway of complement if phosphatidylserine becomes exposed on the surface of the aging cells.