PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 6166677

Fluid-phase SC5b-8 complex of human complement: generation and isolation from serum.

S Bhakdi·M Roth
View original source ↗Export metadata ↓

Abstract

A rapid, simple method for isolating SC5b-9 generated in human serum, and SC5b-8 generated in C9-depleted serum is described. The procedure leads to approximately 30% recovery of either complex and consists of polyethyleneglycol precipitation followed by 1 DEAE ion exchange chromatography and a sucrose density gradient ultracentrifugation. The SC5b-8 complex is a water-soluble macromolecular of alpha-electrophoretic mobility. Apart from the absence of C9, it exhibits an identical SDS-gel electrophoresis polypeptide pattern as SC5b-9. A sedimentation coefficient of 19 to 20S and an effective molecular radius of approximately 10 nm were determined by sucrose density gradient centrifugation and gel filtration, respectively. From these values, a m.w. of 800 to 850,000 is tentatively assigned to the SC5b-8 complex. The S-protein was not dissociable from either SC5b-8 or SC5b-9 through treatment with desoxycholate and no alterations in the hydrodynamic properties of either complex were discernible after action of this detergent.

Explore related subjects

Alpha-GlobulinsBlood ProteinsCentrifugation, Density GradientChemical PrecipitationComplement Membrane Attack ComplexComplement System ProteinsHumansImmunoelectrophoresis, Two-DimensionalProtein BindingJournal Article

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

S Bhakdi, M Roth. 1981. Fluid-phase SC5b-8 complex of human complement: generation and isolation from serum.. https://pubmed.ncbi.nlm.nih.gov/6166677/

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2007

A functional SNP in ITIH3 is associated with susceptibility to myocardial infarction.

Myocardial infarction (MI) results from complex interactions of multiple genetic and environmental factors. To disclose genetic backgrounds of MI, we performed a large-scale, case-control association study using 52,608 gene-based single-nucleotide polymorphism (SNP) markers, and identified a candidate SNP located on chromosome 3p21.2-p21.1. Subsequent linkage-disequilibrium mapping indicated very significant association between MI and a SNP in exon 2 of the inter-alpha (globulin) inhibitor 3 gene (ITIH3; chi(2) = 24.88, P = 6.1 x 10(-7), 3,353 affected individuals versus 3,807 controls). In vitro functional analyses showed that this SNP enhanced the transcriptional level of the ITIH3 gene. Furthermore, we found expression of the ITIH3 protein in the vascular smooth muscle cells and macrophages in the human atherosclerotic lesions, suggesting ITIH3 SNP to be a novel genetic risk factor of MI.

Alpha-Globulins↗
PubMed2006

The presence of tryptase-positive and bikunin-negative mast cells in psoriatic skin lesions.

Human mast cells are well known to produce a serine protease, tryptase, which appears to play a pathogenic role in various skin inflammations. It was previously reported that a rat homologue of bikunin may inhibit tryptase activity. Various type of cells (i.e. keratinocytes) are able to produce this protein inhibitor, it still remains unclear if bikunin is present in dermal inflammatory milieu, in which mast cells, through secretion of tryptase, play an inflammatory role. Therefore, the purpose of the present study was to exploit expression and production of bikunin in dermis and dermal constituents. We first compared the dermal mast cells in psoriatic lesions with those in lesional skin of atopic dermatitis or of chronic eczema by use of immunoelectron microscopy and immunohistochemical analyses using antibodies to bikunin and tryptase. Then, we tested what kinds of cytokines may regulate the de novo synthesis of bikunin. To do so, RNA was extracted from a human mastocytic cell line, HMC-1, reverse-transcribed, and semiquantitative RT-PCR was performed using primers specific for bikunin. With immunoelectron microscopy, bikunin was found to localize on the cell membrane, while tryptase was in the secretary granules of the mast cells. In psoriatic lesions, around 70% of dermal mast cells were positive for both tryptase and bikunin, and the remaining was mostly positive for tryptase, but the expression of bikunin was under the detection limit of the experimental setting. This observation was seen in only psoriatic lesions, even in almost cured lesions, while in atopic dermatitis or chronic eczema only mast cells doubly positive for bikunin and tryptase were seen. In HMC-1, bikunin was constitutively expressed at an mRNA level, which was upregulated by stimulation with interleukine-4, but was suppressed by interferon-gamma. Bearing in mind the concept that in psoriasis local cytokine milieu is shifted toward a Th1 pattern (predominant secretion of interferon-gamma), tryptase-positive, bikunin-negative mast cells may be induced.

Alpha-Globulins↗
PubMed2006

Production of recombinant human alpha1-microglobulin and mutant forms involved in chromophore formation.

Alpha(1)-Microglobulin, a 26 kDa lipocalin present in plasma and tissues, carries a set of unknown chromophores, bound to C34, K92, K118 and K130, which cause its charge and size heterogeneity. In man, the protein is found in two forms, full length and lacking the C-terminal tetrapeptide LIPR (t-alpha(1)-microglobulin), both which are heme-binding and the latter with heme-degrading properties. We report cloning and overexpression of full length alpha(1)-microglobulin (wt protein), t-alpha(1)-microglobulin (wtdeltaLIPR) and the mutants C34S, K(92,118,130)T and C34S/K(92,118,130)T, the latter subsequently abbreviated as K(3)T and C34S/K(3)T, in Escherichia coli. After purification and refolding from inclusion bodies, all proteins were correctly folded as determined by far-UV circular dichroism and radioimmunoassay. As revealed by gel filtration, recombinant alpha(1)-microglobulins had lower tendencies to form dimers than human plasma or urine analogues. All alpha(1)-microglobulin forms displayed higher amounts of the chromophore than bovine serum albumin but significantly lower than the human urine or plasma counterparts. Differences in the absorbance and fluorescence profiles are consistent with a model where the chromophore is formed by a series of reactions with heme or other chromophore precursors and where C34 is essential for binding of the ligand, K92, K118 and K130 are involved in transformation into the chromophore and LIPR inhibits the latter reaction.

Alpha-Globulins↗