RESUMO
A procedure is presented for the preparation of a purified fraction of adult human hemoglobin (HbAo) from one unit of outdated blood. The entire process requires less than 16 h and gives a sterile, endotoxin-free solution of HbAo (approximately 30 g) in a yield of 50%. The solutions are isoionic with a conductivity of less than 15 mu mhos and less than 2 mmol total phosphorus per mol heme. The methemoglobin content is less than 1% and on storage at 4 degrees C rises less than 1% per month.
Assuntos
Substitutos Sanguíneos , Hemoglobina A/isolamento & purificação , Adulto , Células Sanguíneas , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Hemólise , Humanos , Focalização IsoelétricaRESUMO
The murine Cr2 gene encodes at least two related proteins. The first of these is predicted to include 1408 amino acids from a transcript including 4224 coding nucleotides. This protein is predicted to contain 21 60-amino acid repeats plus those residues encoding transmembrane and cytoplasmic regions for a total peptide m.w. of 155,307. The first six of these repeats are similar to human CR1 in sequence and organization. The second protein is encoded by an alternatively spliced Cr2 transcript that is lacking those sequences which encode the first six 60-amino acid repeats of the larger protein. This second, smaller protein, encoded by a transcript of 3096 coding nucleotides, is predicted to include 15 60-amino acid repeats, plus the transmembrane and cytoplasmic regions. This smaller protein contains 1032 amino acids for a peptide m.w. of 113,328. This second protein is extremely homologous in size and sequence to human CR2. Both proteins share the same signal sequence for membrane insertion. DNA sequence analysis, RNA protection studies and genomic phage mapping indicate the transcripts which encode these proteins are derived from the Cr2 gene via alternative splicing.
Assuntos
Receptores de Complemento/genética , Sequência de Aminoácidos , Animais , Antígenos de Diferenciação de Linfócitos B/genética , Sequência de Bases , Northern Blotting , Mapeamento Cromossômico , Clonagem Molecular , DNA/genética , Genes , Camundongos , Dados de Sequência Molecular , Peso Molecular , Família Multigênica , Sinais Direcionadores de Proteínas/genética , Splicing de RNA , RNA Mensageiro/genética , Receptores de Complemento 3dRESUMO
The murine Cr2 gene produces two distinct products in a variety of murine cell types. Both of these transcripts appear to initiate from the same position within the gene but vary from one another via an alternative splicing event within the coding exons. An analysis of those gene sequences that might control the cell specific expression of the Cr2 gene has identified a region of Cr2 5' of the transcription start site that is conserved in both the murine Cr2 and human CR2 genes. When this region was examined using the gel shift assay with nuclear extracts from cells expressing Cr2 (B cells) and those that do not (T cells and fibroblasts), at least four distinct proteins were identified that bound to at least three distinct sites. The DNA sequence recognized by two of these proteins is the octamer sequence recognized by a family of transcriptional regulators including the B cell specific Oct-2 protein. During an acute bacterial infection, the levels of Oct-2 and Cr2 mRNA are both depressed. This suggests that the Oct-2 protein directly controls the transcriptional activity of the Cr2 gene and that during such an infection, the levels of Ag receptors on B cells (Ig and complement receptors) are diminished.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Complemento/genética , Fatores de Transcrição/metabolismo , Animais , Antígenos de Diferenciação de Linfócitos B/genética , Sequência de Bases , Sítios de Ligação , Sequência Consenso , Regulação da Expressão Gênica , Fator C1 de Célula Hospedeira , Camundongos , Dados de Sequência Molecular , Fator 1 de Transcrição de Octâmero , Fator 2 de Transcrição de Octâmero , Oligodesoxirribonucleotídeos/química , Splicing de RNA , RNA Mensageiro/genética , Receptores de Complemento 3d , Sequências Reguladoras de Ácido Nucleico , Alinhamento de Sequência , Transcrição GênicaRESUMO
Pyridoxylated adult human hemoglobin (HbAo) was prepared using a one molar equivalent of pyridoxal 5-phosphate (PLP) per heme and reduced with either NaCNBH3 or NaBH4. A separate sample was pyridoxylated and passed through a mixed-bed ion exchange column without reduction. All three preparations had a P50 of 29 +/- 2 torr and a cooperativity of n = 2.4 +/- 0.1. These preparations, in both the oxy and deoxy forms, were then treated with 7 equivalents of glutaraldehyde per tetramer at pH 6.8 at 4 degrees C and at room temperature. The polymerization invariably reduced the P50 to 18 +/- 2 torr with Hill coefficients of less than 2. These solutions, with or without further reduction using NaCNBH3, all retained the PLP in differing amounts (2-3 moles/tetramer). Methemoglobin concentrations were increased during the polymerization reaction. The normal pyridoxylation procedure, using sodium borohydride reduction, resulted in a number of different molecular species. Polymerization with glutaraldehyde caused a further proliferation of molecular species that could not be separated by anion exchange chromatography or by isoelectric focusing. The extent of polymerization, estimated by gel exclusion chromatography and SDS polyacrylamide gel electrophoresis, was from 40 to 50%. Analysis of the reverse phase chromatograms, which separate the heme and the alpha- and beta-chains, showed extensive polymerization and distribution of the radioactively labeled PLP on the protein for all preparations. All of the polymerized and pyridoxylated samples were unstable, and showed different chromatographic patterns after storage at 4 degrees C for 1 month. Attempts to stabilize these preparations by further reduction with NaCNBH3 gave products with a lower P50 and lower cooperativity. When the reactions were conducted with a purified HbAo, heterogeneity was somewhat decreased compared to the normally used stroma-free hemoglobin, but a large number of molecular species were still formed.