Immunochemistry

Porter, Roy; Press, E M

doi:10.1146/annurev.bi.31.070162.003205

Cited by 54 publications

(17 citation statements)

References 43 publications

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“…The active site serine residue is circled and the residues that form the secondary substrate binding pocket are boxed. Amino acid residues underlined agree with the previously determined protein sequence (12). 10 20 [ 60 70 80 90 100 110 C2:…”

Section: Resultsmentioning

confidence: 52%

“…Two of the positively hybridizing clones, pC201 and pC204, contained inserts of -400 bp. Nucleotide sequence analysis by the method of Maxam and Gilbert (29) showed that both contained a C2 coding sequence that agreed with the known C2 amino acid sequence (12) in the region of the active site serine residue.…”

Section: Resultsmentioning

confidence: 57%

“…Partial amino acid sequence analysis (ref. 12; J. Gagnon, personal communication) has confirmed the extensive structural homology between C2 and factor B.…”

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confidence: 77%

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Isolation of cDNA clones for human complement component C2.

Bentley¹,

Porter²

1984

Proc. Natl. Acad. Sci. U.S.A.

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Two cDNA clones for complement component C2 have been isolated from a high-complexity human liver cDNA library by using a mixture of 64 synthetic oligonucleotides as a probe. The 400-base-pair insert of pC201 codes for a region containing the active site serine residue and the secondary substrate binding pocket of the serine protease. This part of C2 is 34% homologous to the corresponding region of the related serine protease factor B and additional similarity is evident from a number of conservative amino acid replacements in this region. The insert of pC201 was used as a specific probe in RNA transfer analysis to determine the size of the C2 mRNA as -2.9 kilobases. Southern blot analysis of genomic DNA of unrelated individuals identified a single C2 locus and showed no cross-hybridization with the factor B locus. (Mr 60,000) in association with activated C4 forms the C3 convertase of the classical pathway of the complement system (8). In structure, function, and mechanism of activation, C2 resembles factor B, which together with C3b forms the C3 convertase of the alternative pathway (8). Most of the amino acid sequence (9, 10) and gene structure (11) of factor B has been determined. Partial amino acid sequence analysis (ref. 12; J. Gagnon, personal communication) has confirmed the extensive structural homology between C2 and factor B.C2 is synthesized in the liver (13) and also in macrophages (14). It is present in low levels in human serum ("15 mg/liter) (8,15) compared to most of the other complement proteins-e.g., factor B (150 mg/liter) (16), C4 (400 mg/liter) (15), and C3 (1200 mg/liter) (15). It has been possible to isolate limited amounts of C2 protein for sequence analysis (17) but the yields obtained are low because the protein is unusually susceptible to proteolysis during isolation (8,18).To isolate the gene for C2 and to determine the extent of the homology between C2 and factor B and to map precisely the C2 locus relative to the C4 and factor B loci in the HLA region, cDNA clones corresponding to C2 mRNA were isolated and characterized for use as specific hybridization probes.Because of the low levels of C2 protein present in the blood, it was estimated that the level of C2 mRNA in the liver might be as low as 0.01%. Therefore, it was necessary to construct a cDNA library of high complexity from human liver RNA. C2 cDNA clones were identified by using a mixture of sixty-four 17-long oligodeoxyribonucleotides as a hybridization probe and were characterized by nucleotide sequence analysis. MATERIALS AND METHODSSynthesis of Oligodeoxyribonucleotides. A mixture of sixtyfour 17-long oligonucleotides of sequence 3' C-TA G-TAC-TT-T-TAC-TTA-A 5' was synthesized against the known C2 protein sequence Asp-His-Glu-Asn-Glu-Leu by using the solid-phase phosphotriester technique (19,20). The mixture was radioactively labeled by using T4 polynucleotide kinase and [y 32P]rATP to a specific activity of S x 108 cpm/,ug of DNA for use as a hybridization probe.Isolation and Purification of RNA. Total RNA was...

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Section: Resultsmentioning

confidence: 52%

Section: Resultsmentioning

confidence: 57%

See 1 more Smart Citation

Isolation of cDNA clones for human complement component C2.

Bentley¹,

Porter²

1984

Proc. Natl. Acad. Sci. U.S.A.

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“…Die beiden y-Globulinprobcn (die nach Rivanolfällung sowie die nach Trennung an der DEAE-Säule) jedes Serums wurden den nachfolgenden Behandlungen unterzogen: Jede Probe wurde zunächst mit Papain versetzt, wobei im Prinzip nach den Angaben von Porter [37,38] …”

Section: Papainisierungunclassified

“…Jüngste Forschungs arbeiten haben die Struktur dieser Bruchstücke aufgezeigt. Es ist heute bekannt, dass an die verschiedenen y-Globulin-Bruchstücke auch klinisch interessante Eigenschaften, etwa genetische Fak toren, antigenbindende oder Paraprotcin-Eigenschaften gebunden sind [1,6,8,9,10,11,12,13,16,18,28,38,39,40,52].…”

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