ABSTRACT
Factor VIII delta II is a genetically engineered deletion variant of factor VIII expressed by recombinant Chinese hamster ovary cells, in which a major portion of the central (B) domain and a part of the light chain (Pro771-Asp1666) are missing. After immunoaffinity purification, the kinetics of thrombin cleavage of the novel molecule was analysed by SDS/PAGE, Western blotting and N-terminal amino acid sequencing. Thrombin first cleaves factor VIII delta II at Arg740-Ser741 to generate the 90-kDa heavy chain and an 80-kDa fusion polypeptide consisting of the remaining portion of the B domain and the 73-kDa light chain. The 90-kDa fragment is further cleaved, giving rise to 50-kDa and 40-kDa fragments while the 80-kDa fragment generates a 71/73-kDa doublet. The 71/73-kDa doublet, 50-kDa and 40-kDa fragments were further analysed by N-terminal amino acid sequencing and found to correspond to the predicted amino acid sequences. Our study shows that, in spite of the 900 amino acid deletion present in factor VIII delta II, the essential structural elements required for thrombin activation are conserved.
Subject(s)
Factor VIII/chemistry , Factor VII/chemistry , Peptide Fragments/chemistry , Thrombin/metabolism , Amino Acid Sequence , Animals , Blotting, Western , Cell Line , Chromatography, High Pressure Liquid , Chromosome Deletion , Electrophoresis, Polyacrylamide Gel , Factor VII/genetics , Factor VII/isolation & purification , Factor VIII/genetics , Factor VIII/isolation & purification , Genetic Variation , Humans , Kinetics , Molecular Sequence Data , Molecular Weight , Peptide Fragments/genetics , Peptide Fragments/isolation & purification , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , TransfectionABSTRACT
The phosphotriester method for the stepwise synthesis of deoxyoligonucleotides has been employed using HPLC-grade silica gel (Porasil B) as the solid support. The procedure results in a convenient flow-through system for the synthesis of oligomers where all the reaction steps including the zinc bromide method of detritylation are compatible with the selected support. Deoxyoligonucleotides of 25-30 nucleotides in length can be synthesized in high yields utilising stable phosphotriester intermediates. Ease of handling of the solid support allows convenient synthesis of mixed oligonucleotide sequences.
Subject(s)
Oligodeoxyribonucleotides/chemical synthesis , Oligonucleotides/chemical synthesis , Base Sequence , Chromatography, High Pressure Liquid , Indicators and Reagents , Organophosphates , Silica Gel , Silicon DioxideABSTRACT
By means of nyctometry we examinated in a group of normal persons and in a group of juvenile diabetics if the adaptability of the adaptation system in possible under various conditions of initial adaptation. It appears, that nondiabetics possess a sufficiently reactive functional system, while in diabetics, we found a defective functional reaction of the adaptation system because of beginning retinopathy.
