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Biomedical subjects

A Faber

Publications and source records attributed to A Faber.

12 recordsLinked to original sources
PubMed1992

Human neutrophil cytosolic phospholipase C: partial characterization.

The activity of neutrophil cytosolic phospholipase C on PIP2 and PI was compared employing [3H]inositol-labeled heat-inactivated membranes of differentiated HL-60 cells, into which tracer [32P]PIP2 was incorporated. Hydrolysis of PIP2 did not require Ca2+ and was stimulated when the content of PIP2 in the membrane was increased by incorporation of unlabeled inositol lipid. At equal concentrations of PI and PIP2 in the membrane, hydrolysis of PIP2 was faster and no evidence of competition between the two substrates was obtained. Incorporation of PI into PE-[32P]PIP2 vesicles, accelerated PIP2 hydrolysis also at conditions that favor hydrolysis of PI. Partial purification of neutrophil cytosolic PLC on Q Sepharose, phenyl Sepharose and heparin-Agarose columns is described. From heparin-Agarose column, two PLC activity peaks exhibiting different substrate specificities were eluted. The elution profile of the main PLC species from Superose 12 gel filtration column was compatible with an approx. 150 kDa protein.

Cell Fractionation↗
PubMed1991

Arachidonate supports hydrolysis of phosphatidylinositol by neutrophil cytosolic phospholipase C: relation to NADPH oxidase.

NADPH oxidase is a superoxide-generating, membrane-bound complex activated in stimulated phagocytes or in a reconstituted system consisting of membranes, cytosolic components and arachidonate or SDS. To delineate mechanism of oxidase activation in the cell-free system, hydrolysis of phosphoinositides in the combined membrane-cytosol oxidase mixture was investigated. Arachidonate promoted hydrolysis of membrane-[3H]-phosphatidylinositol by cytosolic phospholipase C. PI hydrolysis was similarly supported by other unsaturated fatty acids and by SDS. Unlike activation of the NADPH oxidase, PI hydrolysis required the presence of calcium ions. Implications of these findings to the mechanism of NADPH oxidase activation are discussed.

Arachidonic Acid↗
PubMed1990

A protein of neutrophil granules interferes with activation of NADPH oxidase in a cell-free system.

A soluble extract of neutrophil granules interfered with activation of the NADPH oxidase in a cell-free system. The extract had no effect on superoxide production by preactivated enzyme. The inhibitory activity was retained during dialysis and was lost upon exposure to proteinase K indicating that the active substance was a protein. The inhibitor exhibited a high stability at elevated temperatures. Chromatography of granules extract on ion exchangers implied that the inhibitor was a positively charged protein eluting from S Sepharose cation exchanger above 0.4M concentration of NaCl.

Cell Extracts↗
PubMed1990

Large-scale purification of factor VIII by affinity chromatography: optimization of process parameters.

The optimization of a new process for the extraction of human coagulation factor VIII (FVIII) from plasma with the tailor-made affinity matrix dimethylamino-propylcarbamylpentyl-Sepharose CL-4B (C3-C5 matrix) is described. First, plasma is applied to DEAE-Sephadex A-50 anion exchanger in order to separate a number of proteins, including coagulation factors II, IX and X (prothrombin complex), from FVIII. Subsequently, the unbound fraction of the ion exchanger, containing FVIII, is contacted with the C3-C5 affinity matrix. Optimization of the FVIII affinity chromatographic procedure is accomplished in terms of the ligand density of the matrix, adsorption mode (batch-wise versus column-wise adsorption and matrix to plasma ratio), and conditions of pH and conductivity to be applied on washing and desorption. In scale-up experiments, by processing 20 l of plasma, the recovery (340 U VIII:C/kg plasma) and the specific activity (s.a.) (1.2 U VIII:C/mg protein) are better than those obtained by cryoprecipitation (recovery 300 U VIII:C/kg plasma, s.a. 0.3 U VIII:C/mg protein). The newly developed process using the specially designed C3-C5 affinity matrix has potential application in the process-scale purification of FVIII.

Buffers↗
PubMed1989

Affinity purification of plasma proteins: characterization of six affinity matrices and their application for the isolation of human factor VIII.

For the purification of coagulation factor VIII, (1,1'-carbonyl-diimidazole [CDI]-activated) Sepharose CL-4B was functionalized with two aminoalkyl and four aminoalkyl-carbamylalkyl ligand-spacer combinations. The affinity matrices were contacted with human plasma. All affinity matrices showed complete adsorption of factor VIII (greater than 90%) and three aminoalkyl-carbamylalkyl Sepharoses gave factor-VIII recoveries of 50-65% and a factor-VIII preparation with a specific activity of 1-2 U factor VIII/mg of protein. Furthermore, no fibrinogen, immunoglobulin G and albumin could be detected in the isolated factor VIII. Optimal results were obtained using the di-methyl-aminopropyl-carbamyl-pentyl-Sepharose affinity matrix.

Adsorption↗
PubMed1988

Activation of complement by human serum IgA, secretory IgA and IgA1 fragments.

UNLABELLED: Activation of the complement (C) system by human IgA was studied. Both subclasses of IgA, IgA1 and IgA2, and secretory IgA were shown to activate C, as determined by deposition of C3 on glutaraldehyde-activated microwells coated with IgA. The activation of the C system occurred in the presence of MgEGTA and not in D-deficient serum. In addition to C3, deposition of properdin (P) but not of C4 was detected. These results indicate that C activation, as determined by measuring deposition of C3 and P, occurred by the alternative pathway (AP). The data further show that the major part of the hinge region, which is deleted in IgA2 as compared with IgA1 and which forms the major structural difference between the two subclasses, is not involved in C activation. Reduction and alkylation destroyed the ability of IgA to activate C, as has also been demonstrated for IgG. In order to define the C activating region of the IgA molecule, several fragments of IgA1 were tested. The four-chain molecules F(ab')2 and F(abc)2 were shown to activate the AP. No activation was observed with the two-chain fragments Fab and Fc. The Fc fragment of IgA also did not activate the CP, as does the Fc fragment of IgG. This indicates that activation of the AP of C by IgA is dependent on the presence of the F(ab')2 fragment. IN CONCLUSION: human IgA does activate C by the AP. This activation requires an intact F(ab')2 fragment.

Complement Activation↗
PubMed1986

Monoclonal antibodies against different domains of human IgA: specificities determined by immunoblotting and haemagglutination-inhibition.

The specificity of 14 monoclonal antibodies has been determined by immunoblotting (IB) and haemagglutination-inhibition (HAI) analysis using IgA1 and IgA2 myeloma proteins and eight different IgA1 fragments. Two antibodies probably recognized epitopes on the CH1 domain of IgA. They reacted with all Fab-containing fragments irrespective of whether these originated from the same or different IgA proteins. Seven antibodies were directed against epitopes on the CH2 domain. These antibodies were reactive with F(abc)2 fragments. They failed to react with Fab, Fab' and F(ab')2 fragments. Two out of these seven antibodies did not react with two-chain IgA half-molecules and Fabc fragments containing a single heavy and a single light chain. This suggests that these two antibodies recognized an epitope whose structure is dependent on disulfide linked heavy chains. Five other antibodies showed specificity for the CH3 domain. They were reactive with all CH3-containing molecules, irrespective of whether they comprised one or two alpha chains. Our study demonstrates that IB is an appropriate technique to determine domain specificity of monoclonal anti-immunoglobulin reagents. Although the IB tests were performed on denatured proteins the results agreed surprisingly well with those of the HAI analyses. Moreover, the IB technique could be used on fragments which could not be purified well enough for HAI analyses.

Antibodies, Monoclonal↗
PubMed1986

Production and characterization of pepsin fragments of human IgA1 to determine domain-specificity of monoclonal anti-IgA antibodies.

Eight human IgA1 myeloma proteins were analysed by SDS-PAGE. These experiments showed that purified IgA1 proteins comprise both fully S-S bonded and partly S-S bonded molecules. Pepsin digestion of the IgA1 proteins yielded three four-chain and two two-chain fragments. The four-chain fragments are likely to be derived from intact IgA through cleavage of its alpha chains at different sites: between the CH2 and CH3 domains or in the hinge region. The occurrence of F(abc) (ab') fragments, with alpha chains of different lengths, showed that the alpha chains of IgA can be cleaved independently at the hinge region site. The two-chain pepsin fragments must originate from IgA molecules, which lack inter-assay-chain disulphide linkages. The fragments F(abc)2 and Fabc tended to form dimers, probably through non-covalent interactions of their CH2 domains. An immunoblotting method was used to identify Fd-, CH2- and CH3-specific anti-IgA antibodies. The CH2-specific antibodies could be subdivided into antibodies recognizing an isotype present on both four-chain and two-chain molecules or on two-chain molecules only.

Antibodies, Monoclonal↗
PubMed1985

Binding of human IgA fragments to protein A-Sepharose studied with an ELISA method.

An enzyme-linked immunosorbent assay method was developed to investigate the binding of IgA fragments to protein A. The method proved to be specific and highly sensitive. Contamination with IgG did not interfere with the detection of IgA binding to protein A, and less than 10 ng of IgA could be detected. Four of nine IgA proteins tested bound to protein A to different extents. The binding was not disturbed by reduction and alkylation of the IgA proteins. Four-chain F(abc)2 and F(ab')2 fragments of the protein A-reactive IgA proteins also bound to protein A. On reduction and alkylation these fragments formed two-chain Fabc and Fab' fragments. Of these, Fabc did not bind, whereas both Fab' and IgA1-protease-produced Fab fragments did bind to protein A. These results demonstrate that the Fab fragment has a binding site for protein A. It is suggested that the protein A binding site is located on the CH1 domain of the IgA1 molecule. On Fabc fragments this binding site may be blocked because of structural alterations.

Binding Sites, Antibody↗