Structural and functional studies in C1q deficiency

The sera of two brothers were found totally lacking hemolytic C activity. One of them, a 16-yr-old male, presented a severe lupus-like syndrome, whereas the other was apparently healthy. Immunochemical quantitation of C components in both sera showed depressed levels of C1q, whereas the levels of C1r, C1s, and C1 inhibitor were elevated. C4, C3, C5, factor B, and beta 1H levels were in the normal range. Hemolytic C1 activity was totally lacking. C4 titers were elevated (150% of normal). C2 hemolytic activity was about one-third of normal, and the titers of the terminal components C3-C9 were also reduced in the two siblings. Double immunodiffusion against anti-C1q antiserum showed a partial loss of C1q antigenic determinants in the two siblings. Furthermore, the C1q of both siblings was unable to interact with immunoglobulins or to associate with C1r and C1s. Addition of purified human C1q to the sera restored their total C and C1 hemolytic activity. The dose response to the C1q addition was linear, indicating that the functional deficiency was not due to the presence of a serum inhibitor. Although antigenically deficient in comparison with normal C1q, the abnormal C1q appeared to have a larger m.w., as determined by gel chromatography. Investigation of other members of this family suggests a genetically linked disorder, because four out of six siblings had the same dysfunctional C1q in their serum.     

Binding and activation of the first component of human complement on artificial matrices

Artificial sorbents that comprise macroporous glass covered by the copolymer of N-vinylpyrrolidone and N-substituted acrylamide have been synthesized. Aminoethanol is bound to acrylic acid residue in one sorbent (AE-glass), whereas the other sorbent involves immunoglobulin G with the hexamethylenediamine spacer (IgG-glass). C1q binds specifically to IgG-glass with Ka 4,07(+/- 0,32) X 10(7) M-1. Free energy of the C1q binding to IgG-glass is twice higher than that of its binding to monomeric IgG. This evidences that one C1q molecule associates with two IgG molecules of the sorbent. A weak nonspecific sorption of C1q to AE-glass was found. Both specific (on IgG-glass) and nonspecific (on AE-glass) sorption of the first component of complement activate the classical pathway in human serum as manifested in the consumption of the C4, C2, C3 and C5 components. IgG-glass was employed for C1q isolation from human serum by affinity chromatography, whereas unbound part of serum may be used as a reagent R1q. The yield of highly purified C1q after IgG-glass affinity chromatography and gel filtration on Sephacryl S-300 is 63,6%.     

Differences between newly formed and recycled free small ribosome subunits in liver cytoplasm.

1. Ferritin has been isolated from the serum of four patients with iron overload by using two methods. 2. In method A, the serum was adjusted to pH 4.8 and heated to 70 degrees C. After removal of denatured protein, ferritin was concentrated and further purified by ion-exchange chromatography and gel filtration. In most cases, only a partial purification was achieved. 3. In method B, ferritin was extracted from the serum with a column of immuno-adsorbent [anti-(human ferritin)] and released from the column with 3M-KSCN. Further purification was achieved by anion-exchange chromatography followed by the removal of remaining contaminating serum proteins by means of a second immunoadsorbent. Purifications of up to 31 000-fold were achieved, and the homogeneity of the final preparations was demonstrated by polyacrylamide-gel electrophoresis. 4. Serum ferritin purified by either method has the same elution volume as human spleen ferritin on gel filtration on Sephadex G-200. Serum ferritin has a relatively low iron content and iron/protein ratios of 0.023 and 0.067 (mug of Fe/mug of protein) were found in two pure preparations. On anion-exchange chromatography serum ferritin has a low affinity for the column when compared with various tissue ferritins. Isoelectric focusing has demonstrated the presence of a high proportion of isoferritins of relatively high pI. 5. Possible mechanisms for the release of ferritin into the circulation are briefly discussed.     

Purification and characterization of subcomponent C1q of the first component of mouse complement.

1. Mouse C1q, a subcomponent of the first component of complement, has been purified in a highly haemolytically active form by a combination of precipitation with EGTA, ion-exchange chromatography and gel filtration. Yields ranged from 3 to 5 mg/200 ml of serum, and the activity of final preparations was in the range of 2 X 10(13)-4 X 10(13) C1q effective molecules/mg. 2. The molecular weight of mouse C1q was 439 500 +/- 1586, as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. 3. Mouse C1q was shown to be composed of non-covalently linked subunits, all being in the molecular-weight range 45 000-46 000, and three covalently linked chains each having a molecular weight of approx. 23 000 as determined on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate by using non-covalently and covalently linked subunits of human C1q as markers with known molecular weights calculated theoretically previously [Porter & Reid (1978) Nature (London) 275, 699-704]. 4. Mouse C1q contained hydroxyproline, hydroxylysine, a high percentage of glycine and approx. 9% carbohydrate. The absorption coefficient and nitrogen content of C1q were also determined.     

Isolation and function of a human endothelial cell C1q receptor

It has been shown previously that cultured human venous and arterial endothelial cells (EC) bind C1q in a time- and dose-dependent manner. Cultured human endothelial cells express an average number of 5.2 x 10(5) binding sites/cell. In the present study the putative receptor for C1q (C1qR) was isolated from the membranes of 1-5 x 10(9) human umbilical cord EC by affinity chromatography on C1q-Sepharose. During isolation, C1qR was detected by its capacity to inhibit the lysis of EAC1q in C1q-deficient serum. The eluate from C1q-Sepharose was concentrated, dialysed and subjected to QAE-A50 chromatography and subsequently to gel filtration on HPLC-TSK 3000. C1qR filtered at an apparent molecular weight of 60 kDa. Purified C1qR exhibited an apparent molecular weight of 55-62 kDa in the unreduced state and a molecular weight of 64-68 kDa in reduced form. Two IgM monoclonal antibodies (mAb) D3 and D5 were raised following immunization of mice with purified receptor preparations. Both monoclonal antibodies increased the binding of (125)I-C1q to endothelial cells but F(ab')(2) anti-C1qR mAb inhibited the binding of a(125)I-C1q to EC in a dosedependent manner. The D3 mAb recognized a band of 54-60 kDa in Western blots of membranes of human EC and polymorphonuclear leukocytes. Previously, the authors showed that C1q induces the binding of IgM-containing immune complexes to EC. Therefore, it was hypothesized that during a primary immune response generation of IgM-IC may occur, resulting in binding and activation of C1, dissociation of activated C1 by C1 inhibitor and subsequent interaction of IgM-IC bearing C1q with EC-C1qR.     

魔芋葡苷聚糖（KGM）凝胶为亲和层析载体与Sepharose4B的比较 …

将ＫＧＭ和Ｓｅｐｈａｒｏｓｅ ４Ｂ凝胶在相同条件下活化、偶联接上染料Ｃｉｂａｃｒｏｎ Ｂｌｕｅ Ｆ３ＧＡ制成了ＫＧＭ和Ｓｅｐｈａｒｏｓｅ ４Ｂ染料亲和吸附剂,并用来与牛血甭白蛋白（ＢＳＡ）作用,每毫升ＫＧＭ亲和吸附剂可吸附ＢＳＡ ２８ｍｇ,用ＮａＳＣＮ洗脱时间为８４．５％,而Ｓｅｐｈａｒｏｓｅ ４Ｂ染料样和吸附剂每毫升可吸附ＢＳＡ １５．３ｍｇ,用ＮａＳＣＮ洗脱时间收迷８１．７％,并对两种凝胶的染     

Absence of induction of IL-1 production in human monocytes by complement fragments

The ability of C fragments to induce IL-1 production in human monocytes was examined by using various approaches to carefully exclude the role of contaminating endotoxin. The presence of IL-1 activity in monocyte supernatants and lysates was assayed by the augmentation of PHA-induced proliferation of murine thymocytes. SRBC were opsonized with IgM rabbit antibodies and various human C components to prepare EAC reagents that contained less than 25 pg LPS/ml of EAC at 5 x 10(8) cells/ml. EAC1q, EAC4b, EAC4b2aoxy, EAC4b2aoxy C3b, EAC4b2aoxyC3bi, and EAC4b2aoxyC3d all failed to induce IL-1 production when incubated at 10- to 100-fold excess with adherent human monocytes. Similarly, LPS-free purified C3a, C5a, and C5a des Arg all showed no IL-1-inducing activities at concentrations up to 25 micrograms/ml. However, the same C5a preparations were active on human monocytes in the induction of chemotaxis, and C3a and C5a both induced skin-blueing in guinea pigs. Fragment Ba and Bb preparations purified by gel filtration chromatography contained approximately 100 pg LPS/micrograms Ba or Bb. These Ba and Bb preparations at 10 and 50 micrograms/ml, respectively, induced IL-1 production in the presence of 5 micrograms/ml polymyxin B (PMB). However, Ba and Bb preparations purified by affinity chromatography and HPLC contained lower levels of endotoxin contamination and displayed IL-1-inducing activities at Ba and Bb concentrations of 50 and 100 micrograms/ml, respectively, that were almost completely inhibited by PMB. To explore further the role of contaminating endotoxin, a Bb preparation was adsorbed with PMB-4B in the presence of a dialyzable detergent to remove LPS bound to the Bb. This LPS-free Bb preparation failed to induce IL-1 production while maintaining intact enzymatic activities. These results indicate that various solid phase or soluble C fragments, including C3b, iC3b, C3d, C3a, C5a, Ba or Bb do not induce IL-1 production in human monocytes in the absence of contaminating endotoxin.     

A simple one-step hemolytic assay for C2 with C2-deficient human serum.

A simple one-step procedure has been developed for the molecular titration of C2 by utilizing the ability of the test material to restore the hemolytic activity of human serum selectively deficient in C2 (C2D serum). In this assay, equal volumes of EA (10(8) cells/ml), C2D serum (1/20), and a suitable dilution of a source of C2 were incubated at 37 degrees C for 60 min and the fraction of cells lysed was used to calculate the effective molecules of C2/ml test material. The assay can be used to titrate C2 in human, guinea pig, rat, mouse and rabbit sera, but not C2 in dog serum. The assay is simple and reproducible, and comparable in sensitivity to the conventional two-step assay with EAC14 cells and Cgp-EDTA.     

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.     

Characterization of C1q found in a patient with hypocomplementemic vasculitis-urticaria syndrome

C1q, a subcomponent of the first complement component, of a 60-year-old female patient with hypocomplementemic vasculitis-urticaria syndrome (HVUS) was characterized. The C1q-precipitin activity (C1q-p) could not be detected by a routine method with 0.6% agarose in 10 mM Na-phosphate buffer containing 10 mM EDTA (pH 7.2). Hemolytic activity of her serum complement (CH50) and levels of C1 and C4 were significantly reduced at the exacerbation stage, but levels of other complement components were almost within the normal range throughout her clinical course. The specific activity of C1q at her exacerbation stage was significantly low, and its elution position on Sephacryl S-300 column was spread toward the low molecular weight in comparison with that of normal plasma. Molecular weights of the delayed fraction of C1q were estimated to be approximately 300,000 on the Sephacryl and 440,000 by the polyacrylamide gel electrophoresis (PAGE) containing sodium dodecyl sulfate (SDS) followed by immunoblotting, respectively. On reduction of her plasma, two bands with molecular weights equivalent to those of B and C chains of the normal C1q in an approximate molar ratio of 2:1 were immunostained. Plasma at her exacerbation stage showed only one precipitation line against anti-human C1q-antiserum which was completely fused with that formed between purified normal human C1q and the same antiserum. The probable structural change of the hypofunctional C1q in the case of this HVUS is discussed.     

C1q,a collagen-like complement subcomponent,in dermatosparactic cattle: its extracellular modification is not affected by lack of procollagen N-terminal proteinase (pN-proteinase)

C1q, a collagen-like complement protein, was purified from the serum of a ddermatosparactic calf which lacks procollagen N-terminal proteinase (pN-proteinase). The specific hemolytic activity of the serum Clq from the dermatosparactic animal was identical to that of C1q from a normal calf. Gel-filtration of serum from dermatosparactic calf, on Sepharose 6B, showed the presence of C1q-antigenic material at only one position which was identical to the elution position of normal bovine C1q. No differdence, under dissociating conditions, could be seen in the size of the chains of C1q in specific immunoprecipitates isolated from the sera of dermatosparactic and normal animals, as judged by polyacrylamidegel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). The C1q from the dermatosparactic animal showed the same N-terminal amino acid and typtic-digest peptide pattern on HPLC as C1q from the normal calf. These results strongly suggest that pN-proteinase is not involved in the extracellular processing of C1q.     

猪C1q的分离纯化与鉴定

猪Ｃ１ｑ由猪血清通过ＰＥＧ沉淀优球蛋白、低离子强度透析沉淀、ＩｇＧ－Ｓｅｐｈａｒｏｓｅ４８亲和层析、ＳｅｐｈａｄｅｘＧ－２００凝胶层析等步骤分离纯化,每３００ｍｌ血清可制得９．８ｍｇＣ１ｑ,产率为４６．７％。纯化的猪Ｃ１ｑ在ＳＤＳ－ＰＡＧＥ上显示出三条染色带,分别在２９、２６、２２ｋＤ处,薄层扫描结果表明纯度达９１％;纯化的Ｃ１ｑ保持了较高活性,终浓度为４μｇ／ｍｌ时仍可使致敏的绵羊红细胞出现明显的凝集现象。猪Ｃ１ｑ－ＥＬＩＳＡ结果表明,动物Ｃ１ｑ代替人Ｃ１ｑ应用于临床检测是可行的。     

Radioreceptor assay for lactogenic hormones based on membranes from rat mammary tumour

A highly sensitive radioreceptor assay (RRA) for human prolactin (hPRL) based on membrane preparations obtained from chemically induced rat mammary tumour is described. The binding of 125I-labelled, highly purified pituitary human prolactin was specific for lactogenic hormones and depending on time, temperature, and concentration of receptor protein. Optimal specific receptor binding (18-20%) was obtained by incubation at 21 degrees C for 18 h. The prolactin receptor was shown to have a single "class" of binding sites with an affinity constant (Ka) of 6.0 X 10(10) mol-1. The binding capacity was 8-33 fmol/mg membrane protein. The sensitivity of the radioreceptor assay was 0.5 ng/ml ovine prolactin (NIH-PS-10) or 0.84 ng/ml human prolactin (NIH-VLS-4). The receptor binding activity of various purified prolactin preparations from different species was comparable to the biological hormone activities, indicating that this in vitro assay system measures values which are biologically relevant.     

Binding of the pentamer/hexamer forms of mannan-binding protein to zymosan activates the proenzyme C1r2C1s2 complex, of the classical pathway of complement, without involvement of C1q

The serum lectin, mannan binding protein (MBP), was isolated in a yield of 40 micrograms/liter from pooled normal human serum by affinity chromatography on mannan-Sepharose, followed by gel-filtration and ion-exchange chromatography and finally by passage down an anti-IgM Sepharose column. A rabbit antiserum was prepared against the purified MBP and an enzyme-linked immunoassay developed that used both the specificity of the polyclonal antibody and the Ca+(+)-dependent carbohydrate binding property of MBP. Assay of the sera from 103 blood-donors showed a wide range of MBP levels, ranging from 0 to 870 micrograms/liter. MBP, after interaction with zymosan, caused efficient activation of a C1r2 125I-C1s2 complex that was prepared by incubation of 125I-C1s2 with serum, from a patient with a complete genetic deficiency of C1q, followed by gel-filtration on Sepharose 6B. The purified MBP is composed of a mixture of trimers, tetramers, pentamers, and hexamers of an approximate 90-kDa structural unit as judged by chromatography, SDS-PAGE and electron microscopy studies. Only the molecules in the pentamer/hexamer fraction, which have a similar overall structure to that of C1q, appeared to cause efficient, zymosan-dependent, activation of C1s within the C1r2C1s2 complex. The pentamer/hexamer form of MBP may therefore play an important role in antibody-independent activation of the C system during the early stages of certain infections.     

Characteristics of solubilized human-somatotropin-binding protein from the liver of pregnant rabbits.

A specific binding site for somatotropin was solubilized by 1% (v/v) Triton X-100 from a crude particulate membrane fraction of pregnant rabbit liver, partially purified and characterized. The solubilized binding site retained many of the characteristics observed in the original particulate fraction, indicating that extraction of the binding site with Triton X-100 does not cause any major changes in its properties. The binding of human 125I-labelled-somatotropin to the solubilized binding site is a saturable and reversible process, depending on temperature, incubation time, pH and ionic environment. Analysis of the kinetic data revealed a finite number of binding sites with an affinity constant of 0.32 x 10(10)M-1. The binding activity for human 125I-labelled-somatotropin was adsorbed to a concanavalin-A-Sepharose column and was dissociated from the column with alpha-methyl-D-glucoside, suggesting that the binding protein may be a glycoprotein. Using affinity chromatography on concanavalin-A-Sepharose, ion-exchange chromatography on DEAE-cellulose and gel filtration on Sepharose 6B, the binding protein was purified 1000-4000-fold from the original liver homogenate. When the partially purified preparation was chromatographed on Sepharose 6B, the binding protein eluted as a molecule with an apparent molecular weight of 200000, with a Stokes' radius of 4.9 nm. Sucrose-density-gradient centrifugation of the preparation showed that the sedimentation coefficient of the binding protein was 7.2S. Isoelectric focusing experiments revealed that a major part of the protein has an acidic pI (4.2-4.5). Exposure of the protein to trypsin decreased the binding activity for human 125I-labelled-somatotropin or bovine 125I-labelled-somatotropin, whereas ribonuclease, deoxyribonuclease, phospholipase C or neuraminidase had little or no effect.     

Murine C4-binding protein: a rapid purification method by affinity chromatography

A simple, 3-step method was described for purification of murine C4 binding protein (C4-bp), a recently recognized serum protein that functions as one of the regulatory proteins of the complement system. The method consists of 1) affinity chromatography using TNBS-BGG-conjugated Sepharose beads, 2) gel filtration on a Sepharose 6B column, and 3) heparin-Sepharose chromatography. By this method, milligram quantities of C4-bp can be easily purified by more than 500-fold from EDTA-serum of various mouse strains, and the whole purification process can be completed within 1 wk. The overall yield of C4-bp is about 15%. The C4-bp thus prepared is homogeneous as judged by SDS-polyacrylamide gel electrophoresis and immunelectrophoresis. The purified mouse C4-bp showed physicochemical properties very similar to those described for human C4-bp. Like human C4-bp, mouse C4-bp is composed of several apparently identical subunits of the m.w. of 80,000. However unlike the human counterpart, the subunits of mouse C4-bp are not linked by disulfide bonds but are connected by non-covalent forces that can be disrupted by SDS. The purified mouse C4-bp retained binding affinity for C4 and showed unaltered antigenicity. Immunization of rabbits with the purified mouse C4-bp resulted in the production of potent and monospecific antisera.     

Immunoenzymic studies on testicular hyaluronidase from rhesus monkeys (Macaca mulatta)

Hyaluronidase from rhesus monkey testes was purified by detergent extraction, ammonium sulphate fractionation, Sephadex G-200 column chromatography and concanavalin A-Sepharose affinity chromatography. The purified hyaluronidase showed one protein band on acrylamide gel electrophoresis. Antibodies to the purified hyaluronidase were raised in rabbits and showed a single precipitin line by Ouchterlony gel diffusion. The enzyme had a molecular weight of 62,000. The Km was 0.5 mg/ml for hydrolysis of hyaluronic acid at 37 degrees C. The optimum pH for the enzyme was 5.0 but activity was present over a broad pH range. The hyaluronidase was inhibited by HgCl2, CuSO4, FeSO4 and p-chloromercuribenzoate all at a concentration of 2 x 10(-4) M. Cysteine protected the enzyme against HgCl2 inhibition.     

Two-step purification of mouse kidney ornithine decarboxylase

We developed a simple two-step purification procedure for ornithine decarboxylase (ODC, EC 4.1.1.17), consisting of DEAE-Cellulofine chromatography and affinity chromatography on a HO-101 monoclonal anti-rat liver ODC antibody-Affi-Gel 10 column. By this method, ODC was purified 1700-fold to homogeneity with about 80% yield from the kidney of ICR mice treated with testosterone enanthate. The final specific activity range between 1.0 x 10(6)-1.4 x 10(6) nmol/h.mg protein. On SDS-polyacrylamide gel electrophoretic analysis, the final preparations gave a major protein band of Mr 54,000 and a minor band of Mr 51,000. Although relative staining intensity of the two bands varied depending on preparations, both bands could be stained by immunoblotting and labeled by a preincubation with [14C]difluoromethylornithine (DFMO). On Oudin double diffusion immunoanalysis, a single fused precipitin line was formed between purified anti-mouse kidney ODC IgG and both the purified enzyme and crude mouse kidney extract. In contradiction with earlier reports, no significant difference was observed between mouse kidney ODC and rat liver ODC in either final specific activity or specific binding of labeled DFMO.     

Stepwise dissociation of subcomponents of C1, the first component of human complement, upon activation on an affinity sorbent

An affinity sorbent comprising macroporous glass coated with the polymer with the polymer with immobilized immunoglobulin IgG was used for the isolation from human serum of the first component of the complement and for its separation into subcomponents C1r, C1s and C1q by the one-step procedure. Serum C1 was quantitatively bound to the sorbent at 0 degrees C. The unbound part of the serum can be used as a R1 reagent for determining the hemolytic activity of C1. After activation of bound C1 by heating (30 degrees C, 40 min) the activated subcomponent C1r is eluted from the sorbent. Stepwise elution with EDTA at pH 7.4 or with EDTA + 1 M NaCl at pH 8.5 results in a selective and quantitative elution of the activated subcomponent C1s and subcomponent C1q. Stepwise elution of C1 subcomponents from the affinity sorbent after activation reflects the process of C1 breakdown following its activation on immune complexes.     

Purification of glucoamylase by acarbose (BAY g-5421) affinity chromatography

Aspergillus niger and Rhizopus sp. glucoamylases were purified on an affinity chromatography column from commercially available, impure enzyme preparations. Up to 2 mg of glucoamylase protein was bound without leakage to a 1-ml affinity gel column (0.7 X 2.5 cm) possessing a covalently linked acarbose ligand (1 mg acarbose/g wet gel), and the bound enzyme was specifically released by irrigation of the column with a solution of maltose. A complete cycle of purification was accomplished in about 8 h. Glucoamylases were recovered, in more than 80% yield, free of alpha-amylase activity and possessing specific activities comparable to those of preparations obtained by time-consuming, multistep procedures involving several ion-exchange and hydrophobic column fractionations. Thus, acarbose affinity chromatography provides a general method for the rapid and efficient purification of the glucoamylases, and seems to be ideally suited for scale-up for the commercial purification of these enzymes.