Components of human complement system. Testing and isolation of C3 and C5

Modified reagents for testing the hemolytic activity of human complement components, C3 and C5, have been obtained. These reagents were obtained by treatment of human blood serum pools with a saturated solution of KBr (reagent R3) or 2 M KSCN and denaturated yeasts (reagent R5). These reagents were found to be rich in the serum factor obtained through the use of DEAE-cellulose DE-52 and containing the active component of the complement (C4). To test the sensitivity and specificity of the above reagents, components C3 and C5 were purified. After this procedure these components emerged as hemolytically active, electrophoretically and immunophoretically homogeneous components, C3 and C5. DEAE-cellulose DE-52, DEAE-Sephacel, Hydroxylapatite and Ultra-gel AcA-34 were used consecutively as purification agents. The activity yields of components C3 and C5 with regard to the initial serum levels were 31% and 18%, respectively.     

Isolation and partial characterization of canine epidermal growth factor/urogastrone.

Canine epidermal growth factor (EGF)/urogastrone was partially purified from dog urine by fractional precipitation with (NH4)2SO4, ion-exchange chromatography with DEAE-cellulose DE-52, gel filtration with Sephadex G-50, and a second DE-52 chromatography, to yield receptor-competing activity equivalent to 13 micrograms of standard mouse EGF/litre of starting urine. The purification was monitored by a competitive radioreceptor assay using fixed monolayers of A431 cells. The partially purified canine EGF/urogastrone demonstrated a growth-stimulating activity in 3T3 mouse fibroblast cells as potent as mouse EGF. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed one major peptide component with an Mr similar to that of mouse EGF, and two minor peptides of slightly higher Mr. The major peptide component was isolated after reduction and its amino acid composition was determined.     

C1q: isolation from human serum in high yield by affinity chromatography and development of a highly sensitive hemolytic assay.

C1q, a subcomponent of the first component of complement, has been isolated from human serum in fully hemolytically active form by affinity column chromatography and gel filtration with Bio-Gel A-5M. The affinity column was prepared by covalent coupling of purified human IgG to CNBr-activated Sepharose 4B. Final yields of C1q ranged from 25 to 40% with 650- 890-fold purification based on recovery of hemolytic activity. The preparations were free of contaminating serum proteins as judged by SDS-polyacrylamide gel electrophoretic and immunochemical criteria. The final C1q preparations were also devoid of any demonstrable C1q-inhibitor activity. A C1q-depleted reagent (C1qD) was obtained from the nonabsorbed protein containing fractions of the human IgG-Sepharose 4B affinity column and utilized in conjunction with sensitized sheep erythrocytes (EA) for the detection and quantitation of C1q hemolytic activity. Employing optimal quantities of C1qD in the hemolytic assay mixture, the highly purified C1q preparations contained 0.5 to 1 x 10(13) effective molecules/mg and 0.5 to 1 x 10(12) effective C1q molecules/ml of human serum. This assay would therefore reproducibly detect less than 1 ng of C1q hemolytic activity.     

Fractionation of serum transcobalamins on charged cellulose filters.

A simple and rapid fractionation procedure of the three transcobalamins, TCI, TCII, and TCII, of human serum was achieved by filtration through a stack of charged cellulose filters composed of one cellulose-nitrate and three DEAE-cellulose (DE-81) disks. A reaction mixture containing microliter amounts of serum was incubated with excess of 57Co B12 of high specific activity, diluted with 0.1 M sodium borate buffer (pH 8.5), and passed through the filter stack by applying vacuum. Under these conditions TCII is selectively and quantitatively adsorbed to the cellulose-nitrate filter while both TCI and TCIII adsorb to the DE-81 filters. In the second step TCIII is selectively desorbed from the latter filters by a 0.05 M monopotassium phosphate solution of pH 4.6. Using sera of different distribution of transcobalamins the data obtained were comparable to those determined by the more laborious methods employing DE-52 column chromatography combined with procedures to remove TCII.     

Partial purification and characterization of the soluble phosphatidate phosphohydrolase of rat liver.

A method is described by which the Mg2+-stimulated phosphatidate phosphohydrolase can be purified from the soluble fraction of liver from ethanol-treated rats. The increase in specific activity was about 416-fold. This involved purification by adsorption on calcium phosphate, chromatography on DE-52 DEAE-cellulose, separation on Ultrogel AcA-34 and chromatography on CM-Sepharose 6B. The effects of phosphatidylcholine, phosphatidate and Mg2+, Mn2+ and Zn2+ on the activity are described. Inhibitor studies indicate that the phosphohydrolase contains functional thiol groups and arginine residues.     

Neutral maltase/glucoamylase from rabbit renal cortex.

Maltase activity (EC 3.2.1.20) was solubilized from rabbit kidney brush-border membrane by using 1.0% Triton X-100 and purified 230-fold with an overall recovery of 30%. The purification procedure makes use of heat precipitation, chromatography on DE-52 DEAE-cellulose and gel filtration on Sephacryl S-300. Rabbit kidney brush border exhibited glucoamylase activity with a maltase/glucoamylase ratio of 1.5:1 to 2.0:1. During purification the maltase and glucoamylase activities behaved identically. The Mr of the complex is 590,000, and it appears to be composed of eight identical subunits linked by disulphide bridges.     

Cathepsin B from human renal cortex.

Cysteine-proteinase activity was observed in homogenates of human-cadaver renal cortex. This activity co-purified with renin enzymic activity until separation by aminohexyl-Sepharose--pepstatin affinity chromatography. The cysteine proteinase was purified 1780-fold after the following successive chromatographic procedures: Sephadex G-75, DEAE-cellulose DE-52, and an organomercurial affinity resin. The proteinase activity was dependent upon activation by thiol-containing compounds such as dithiothreitol, as well as by EDTA, and was inhibited by the thiol-group-specific alkylating reagents iodoacetic acid and N-ethylmaleimide. DE-52 cellulose chromatography resolved the cysteine proteinase into two components. On the basis of molecular size (26 000 daltons), activity as a function of pH, stability as a function of pH, substrate specificity and thermal lability, the major component (95%) has been identified as cathepsin B. The DE-52 cellulose elution pattern of the minor component (5%) is suggestive of cathepsin H [Schwartz & Barrett (1980) Biochem. J. 191, 487-497] Enzymic activity was determined with synthetic substrates, in particular alpha-N-benzoyl-DL-arginine 2-naphthylamide (Bz-Arg-NNap), thus precluding the detection of cathepsin L [Kirschke, Langner, Wiederanders, Ansorge, Bohley & Broghammer (1976) Acta Biol. Med. Germ. 35, 285-299]. Inhibition by dimethyl sulphoxide was observed in the determination of Km = 7.0 +/- 0.4 mM for the substrate Bz-Arg-NNap, and care must therefore be taken in the preparation of substrate solutions.     

嗜碱细菌环状糊精葡糖基转移酶的纯化和性质

嗜碱细菌52—2除去菌体的培养液经硫酸铵沉淀和DEAE－纤维素离子交换柱层析,得到凝胶电泳均一的环状糊精葡糖基转移酶,纯化了11.5倍,酶活力回收为5.7％。用浓度梯度PAGE测分子量为151700。酶反应最适温度为65℃,50℃以下比较稳定。酶反应最适pH为7．0,在6．0～9.0范围内稳定。Zn²⁺、Hg²⁺、Pb²⁺、Al³⁺、Cu²⁺、Ag⁺和Fe²⁺强烈抑制酶活力。紫外光谱在270nm和244nm处分别有最大和最小吸收。荧光光谱的最大激发波长和发射波长分别为283nm和335nm。用NBS、NEM、NAI、DEP和EDC对酶进行了化学修饰,初步推测组氨酸和色氨酸残基可能为酶活力必需基因,羧基与酶活力有一定关系。     

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.     

Partial purification and properties of frog liver tyrosine aminotransferase.

Hepatic tyrosine aminotransferase of the frog Rana temporaria was partially purified by (NH4)2SO4 fractionation and successive chromatography on DEAE-cellulose DE-52, Ultrogel AcA-34, DEAE-cellulose DE-52 again and, finally, hydroxyapatite. During the last step, the enzyme activity separated into two fractions; traces of a third fraction were also found. The major form was purified 6000-fold to a specific activity of 200 units/mg of protein; it was about 50% pure by electrophoretic criteria. It had mol.wt. about 85 000 as determined by gel filtration on a Sephadex G-100 column. It was not activated by added pyridoxal 5'-phosphate. The enzyme was, however, inactivated by the pyridoxal phosphate reactants canaline and amino-oxyacetate. The enzyme was specific for 2-oxoglutarate as the amino group acceptor. Homogentisate inhibited the enzyme and adrenaline was an activator; both effects were seen at low concentrations of the effectors. The relationship between initial rate and tyrosine or 2-oxoglutarate concentration was abnormal and complex. Form-2 enzyme had similar or identical molecular weight, cofactor requirements, oxo acid specificity and kinetics.     

Purification, characterization and identification of an agglutinin in human serum

A serum protein named agglutinin is able to induce mitochondria to agglutinate. The protein has been purified from human serum by chromatography on DE-52. Sephadex G-200 and immunoglobulin-Sepharose 4B columns. Agglutinin is a glycoprotein that migrates electrophoretically as a gamma-globulin. Its molecular weight was determined to be 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Monospecific antiserum prepared against the agglutinin was found to be identical with anti-beta 2-glycoprotein I and agglutinating activity could be adsorbed on anti-beta 2-glycoprotein I-Sepharose 4B columns. Thus, the agglutinin has been identified as beta 2-glycoprotein I. The reaction between mitochondria and agglutinin shows positive cooperativity, which is independent on the stage of purification of agglutinin. The agglutinating activity could be diminished (inhibited) by acidic non-soluble lipids such as oleic acid, phosphatidic acid, phosphatidyl serine and phosphatidyl inositol.     

A phospholipid-stimulated protein kinase from Dictyostelium discoideum.

A protein kinase with unusual characteristics has been found in Dictyostelium discoideum. This kinase can use histone H1 as exogenous substrate, and the activity is stimulated by phospholipids, but not by Ca2+. This enzyme has been partially purified by using chromatography on DEAE-cellulose DE-52, spermine-agarose and phosphatidylserine-polyacrylamide. The protein kinase activity is very labile, even in the presence of protease inhibitors, making further purification difficult. In the activity-containing fractions, an endogenous protein of 140 kDa is labelled in vitro with [gamma-32P]ATP under conditions in which intramolecular rather than intermolecular reactions are favoured. This protein is labelled only in the presence of phospholipids, but not of Ca2+. We propose that the 140 kDa phosphoprotein might be the autophosphorylated enzyme.     

Purification of the proteolytically solubilized, active catalytic subunit of adenylate cyclase from ram sperm. Inhibition by adenosine

Ram spermatozoa adenylate cyclase is insensitive to all usual regulatory processes. The purification of its active catalytic subunit was accomplished after proteolytic solubilization of a particulate fraction by alpha-chymotrypsin. The purification (26,000-fold from the particulate fraction or 125,000-fold from the whole-sperm proteins) was achieved by conventional procedures (DEAE-Trisacryl, Ultrogel AcA 34, DEAE-Sephacel, hydroxyapatite), in the absence of detergent, and with a yield of 5-10% and a final specific activity of 19 mumol cyclic AMP formed mg protein-1 min-1 at 30 degrees C in the presence of manganese as cosubstrate. The solubilized enzyme, stable at the beginning of the purification procedure, became unstable at the later stages. After the last step (chromatography on hydroxyapatite) half-lives of 27 min, 50 min and 160 min were obtained at 30 degrees C, 20 degrees C and 4 degrees C respectively. The enzyme was stabilized by addition of bovine serum albumin and Lubrol PX, 80% of the activity remaining after 24 h at 4 degrees C. The purified enzyme exhibited a Km value similar to that of the native enzyme (Km = 1.4 mM). Unlike the native enzyme, the purified enzyme has an absolute requirement for MnATP; no significant activity was recovered in the presence of MgATP. Adenosine inhibited the activity of both the native and purified forms of the enzyme to the same extent and in a non-competitive manner. This indicates that adenosine acts on the catalytic component itself and the inhibition site and the catalytic site are different. Data obtained with adenosine analogs indicate that adenosine interacts with the cyclase catalytic subunit with a 'P-site' specificity. The purified adenylate cyclase, which had an apparent molecular mass of 38 kDa on a high-performance liquid chromatography column [Stengel, D., Guenet, L. and Hanoune, J. (1982) J. Biol. Chem. 257, 10,818-10,826], gave a doublet of 36 kDa and 34 kDa on sodium dodecyl sulfate gel electrophoresis. This represents the smallest protein entity associated with adenylate cyclase activity so far reported.     

The purification and characterization of bovine C4, the fourth component of complement.

The fourth component of complement, C4, was isolated from bovine plasma in high yield, by using simple purification techniques. The protein, like human component C4, is a beta-globulin with a mol.wt. of about 200 000 and consists of three polypeptide chains, alpha, beta and gamma, with apparent mol. wts. of 98 000, 82 000 and 32 000 respectively. The chains of C4 have been separated by methods previously used for human C4. Their amino acid compositions are very similar to those of the human component, but differences in carbohydrate distribution have been observed. The haemolytic activity of bovine C4 is totally destroyed by incubation with bovine C1s, the activated subcomponent of the first component of complement. Component C4, treated in this way, was shown to be cleaved in the alpha chain, which was decreased in mol.wt. by about 9000, corresponding to the removal of subcomponent C4a.     

Functional and biochemical properties of the early classical complement system of mice

Mouse serum and EDTA plasma were subjected to low ionicity precipitation, gel filtration, and ion exchange chromatography in an attempt to purify C1, C4, and C2 to functional and chemical homogeneity. In marked contrast to human and guinea pig components, those of the mouse could not be separated by these techniques. Except for partial separation of C1 from C4 and C2 on DE-52 cellulose columuns with EDTA in the eluting buffers, there was no separation of those three components on ion exchange chromatographic columns. Sephadex G-200 gel filtration columns, or with precipitation of euglobulins from serum or plasma. Generation of EAC142 by incubation of EA in whole serum followed first order kinetics when mouse serum was used and second (or greater) order kinetics when human or guinea pig sera were used. Generation of EAC142 by incubation of EA in whole mouse serum followed by incubation in EDTA containing buffers resulted in rapid loss of all three activities from the cell. These experiments indicated that there were significant differences between the early classical C system of mice and those of human and guinea pig. In addition, they indicated that under a variety of in vitro conditions, murine C1, C4, and C2 behaved biochemically and functionally as a unit. The reasons for the major differences in behavior of the murine C components with not become clear until methods to stabilize their function are found so that they can survive multiple purification steps.     

Simultaneous preparation from human placenta of several enzymes of glucose metabolism

A procedure for the simultaneous purification to homogeneity of hexokinase, phosphoglucomutase 1 and 2, aldolase, phosphoglucose isomerase and glucose-6-phosphate dehydrogenase from human origin has been developed. Human placenta homogenate was first chromatographed on DE-52 column which retains hexokinase and glucose-6-phosphate dehydrogenase while the other enzymes are recovered in the unabsorbed protein fraction. The other steps in the purification involve Matrex gel and specific affinity chromatography for the DE-52 retained enzymes and phosphocellulose and Matrex gel chromatography for the other enzymes. All the enzymes mentioned were obtained in one week, with recoveries from 14 percent for glucose-6-phosphate dehydrogenase to 75 percent for hexokinase. Thus, the procedures utilized seem to be useful in obtaining large amounts of enzymes in a a homogeneous form from an easily available human tissue.     

补体C3与BPI活性区融合蛋白（CB）的构建与表达

补体C3和杀菌通透性增加蛋白（BPI）对血液中的病原体均有黏附、促吞噬甚至杀灭作用,但两者的作用机制不同,制备两者活性区融合蛋白,可能具有更好的清除血液病原的作用。通过重叠延伸PCR融合人补体C3的补体受体Ⅰ、Ⅲ两个结合区,同时调取了杀菌通透性增加蛋白（BPI）活性区段rBPI,先后将补体C3活性区与BPI蛋白功能区基因克隆入原核表达载体pET28a中,获得融合蛋白（CB）表达载体pET28-CB,在大肠杆菌中进行了高表达产量、可溶性表达等条件的摸索,CB融合蛋白主要以包涵体形式表达,Western印迹证明CB具有C3的抗原活性,将包涵体蛋白变性与复性后,利用Ni2+固相化的螯合Sepharose Fast Flow亲和层析柱进行浓缩和纯化,最后得到了纯度较高的CB原核表达蛋白。CB融合蛋白的构建和高效表达、纯化为下步探讨其在促进血液病原清除上的功能鉴定和应用奠定了基础。     

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.     

The amino acid compositions of the tryptic, chymotryptic and peptic peptides from the L-2 light chain of rabbit skeletal muscle myosin.

The light chain fraction was separated from rabbit skeletal muscle myosin and four kinds of light chains, L-1, L-2, L-3 and L-4 in the fraction were further isolated by column chromatography using DEAE-cellulose DE-52. After amino-ethylation, the L-2 light chain was digested with trypsin. It was also digested with chymotrypsin and pepsin, respectively, after carboxymethylation. Each of the tryptic, chymotryptic and peptic peptides thus obtained was separated and purified and their amino acid compositions were analyzed.     

Purification of human vitamin K-dependent protein S and its limited proteolysis by thrombin

Vitamin K-dependent protein S exists in two forms in human plasma, namely as the free protein and in complex with C4b-binding protein [Dahlbäck & Stenflo (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2512-2516]. Now reported is a simple purification procedure for human protein S that includes barium citrate adsorption, DEAE-Sephacel chromatography and chromatography on Blue Sepharose. The yield was approx. 30% relative to the concentration of free protein S in plasma, which was found to be approx. 10 mg/l. Purified protein S migrated as a single-chain band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under non-reducing conditions and as a doublet of Mr approx. 85 000 and 75 000 on reduction. A third band of Mr 16 000 was observed after electrophoresis of 125I-labelled protein S and radioautography of reduced samples. This band appears to be disulphide-linked to the 75 000-Mr chain before reduction. Thrombin converted the 85 000-Mr chain of protein S into a 75 000-Mr chain and an 8000-Mr fragment, the latter again being detectable only by radioautography of reduced samples. The 16 000-Mr fragment was not observed, suggesting its degradation by thrombin. Under non-reducing conditions, no change in apparent molecular weight of thrombin-treated protein S was observed, indicating disulphide linkage of the fragments. Thrombin also affected the mobility of protein S on agarose-gel electrophoresis in the presence of Ca2+, suggesting a decreased affinity to Ca2+ of the cleaved form of protein S as compared with the undegraded molecule. After activation of the complement system in human serum, protein S was found to be a constituent part of the complex formed by C4b-binding protein and component C4b.