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O J Bjerrum

Publications and source records attributed to O J Bjerrum.

At least 37 records · Page 2Linked to original sources
PubMed1984

Characterization of an active hydrophilic erythrocyte membrane acetylcholinesterase obtained by limited proteolysis of the purified enzyme.

Purified human erythrocyte membrane acetylcholinesterase was subjected to limited proteolysis with papain. This treatment generated a hydrophilic form of the enzyme as determined by charge-shift crossed immunoelectrophoresis and by binding to phenyl-Sepharose. The hydrophilic enzyme was stable and its activity was independent of the presence of amphiphiles. Electroimmunochemical analysis showed no antigenic difference between the two enzyme forms. Although the proteolytic treatment only brought about a small change in molecular weight, marked differences in the hydrodynamic properties were encountered. The Stokes radius decreased from 8.2 to 5.9 nm and the sedimentation coefficient increased from 6.3 to 7.0 S. The results are consistent with the view that a short hydrophobic peptide responsible for the amphipatic character of acetylcholinesterase is removed by the treatment with papain.

Acetylcholinesterase↗
PubMed1984

A detergent-induced charge shift as a prerequisite for the electrochemical analysis of the adenine nucleotide translocator, a basic membrane protein.

The adenine nucleotide translocator is a hydrophobic, basic protein of the inner mitochondrial membrane which is solubilized by the non-ionic detergent Triton X-100. For immunochemical characterization of this membrane-protein by crossed immunoelectrophoresis a charge shift of the protein-Triton X-100 micelle by the introduction of an ionic detergent (deoxycholate) was necessary as a prerequisite to avoid unspecific precipitation of the protein. Beside the charge shift of the protein-detergent micelle, the selection, concentration and ratio of the detergents used and the choice of the agarose with different degrees of electroendosmosis should be carefully considered. The principle derived from these results provides a new methodological possibility for the immunochemical characterization of hydrophobic, basic membrane proteins.

Animals↗
PubMed1984

The effect of Tween 20 on indirect immunoperoxidase staining of blood group antigen A in human urothelium.

The effect of the nonionic detergent Tween 20 on background staining, sensitivity, and specificity in the indirect immunoperoxidase staining for blood group antigen A was investigated histologically and spectrophotometrically. Pretreatment of dewaxed formalin-fixed Paraplast-embedded tissue sections from human ureters with 2% Tween 20 and dilution of the first and second layer antisera with 0.05 or 2% Tween 20 significantly reduced background staining of the urothelial cell cytoplasm, ureteral stroma, and musculature. Spectrophotometrical analysis of tissue sections from hypernephroma (rich in cytoplasm), cervix (fibrous stroma), and myometrium (musculature) underlined the histological results with a significant reduction of the maximum absorbance of Tween 20-modified indirect immunoperoxidase-stained tissue sections. Sensitivity, evaluated histologically by the endpoint titers of urothelial cell membrane staining, endothelial cell staining, and focal cytoplasmic staining of urothelial cells, was not influenced by the Tween 20 treatment. The specificity was improved as the staining was highly reduced or absent in control sections subjected to Tween 20.

ABO Blood-Group System↗
PubMed1983

Degradation of transmembrane proteins in Ca2+-enriched human erythrocytes. An immunochemical study.

Apart from causing the formation of gamma-glutamyl-epsilon-lysine cross-linked polymers, exposure of human erythrocytes to Ca2+ and ionophore A23187 leads to a breakdown of the two major transmembrane proteins, i.e. the anion-transporting band 3 and glycophorin. This apparently proteolytic phenomenon was examined by crossed immunoelectrophoretic techniques. The main product of the cleavage of band 3 had a chain weight of about 55,000 and showed good precipitation with the antibody raised against the intact protein. The degradation of glycophorin was more rapid and, when complete, gave rise to small fragments which were barely precipitated with antiglycophorin antibody. Incubation of the cells with pepstatin or N-ethylmaleimide prior to and during Ca2+ loading prevented the breakdown of both transmembrane proteins. Histamine, a competitive inhibitor of the transglutaminase-catalyzed formation of gamma-glutamyl-epsilon-lysine cross-links in Ca2+-enriched erythrocytes, also provided some protection, suggesting that the breakdown of the two transmembrane proteins might in some manner be related to the transglutaminase-dependent polymerization process. Pathophysiological implications of the proteolytic event, which would distort the normal interaction of membrane proteins with the cytoskeleton, are discussed.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1983

IgG rheumatoid factor-secreting lymphocytes in rheumatoid arthritis: evaluation of a haemolytic plaque-forming cell technique.

A haemolytic plaque-forming cell (PFC) assay detecting human B lymphocytes secreting IgG rheumatoid factor (RF) was established using sheep erythrocytes (SRBC) sensitized with rabbit IgG, developing rabbit anti-human IgG, and complement. IgG-RF PFC were only demonstrated with IgG-depleted guinea-pig serum as the source of complement. Cells spontaneously secreting IgG-RF were found among synovial fluid mononuclear cells (mean, 134/10(6)) and synovial tissue mononuclear cells (mean, 1775/10(6)) from patients with rheumatoid arthritis, whereas few were recorded among blood lymphocytes (mean, 3/10(6)). The experiments revealed that the RF-IgG PFC were protein-synthesizing B lymphocytes. The antibody specificity of the secreted IgF-RF was verified by the inhibitory effect of exogenous human and rabbit IgG on PFC formation.

Animals↗
PubMed1983

Quantitative enzyme antigen immunoassay of acetylcholinesterase in amniotic fluid.

An enzyme immunoassay for acetylcholinesterase in amniotic fluid is described. Rabbit antiserum (IgG fraction) against human erythrocyte membrane acetylcholinesterase is first attached to microtitre plates. Samples containing acetylcholinesterase are added and the enzyme activity is then measured, with acetylthiocholine as substrate and Ellman's reagent as coupling salt. Results are comparable with those by qualitative determination of the enzyme after polyacrylamide gel electrophoresis, except for blood-contaminated amniotic fluids. One person can perform 200 enzyme analyses per day. Intra- and interassay coefficients of variation are less than 5% at concentrations from 15 to 450 arb. units/L. The mean catalytic concentration in 400 amniotic fluid samples was 21 arb. units/L (range 5 to 70 arb. units/L) with 1000 arb. units/L for a human serum pool as standard. All 39 cases of neural tube defects and two-thirds of cases with omphalocele and gastroschisis had abnormally high values, exceeding 75 arb. units/L.

Acetylcholinesterase↗
PubMed1983

Secretion of IgM-rheumatoid factor and IgM by blood lymphocytes in rheumatoid arthritis.

A direct plaque forming cell (PFC) assay for the detection of complement fixing IgM-rheumatoid factor (RF) secreting lymphocytes was evaluated. The specificity of the RF-PFC assay was demonstrated by the inhibitory action of exogenous human IgG. The sensitivity of the RF-PFC assay was similar to that of a reverse PFC assay detecting all cells secreting IgM (IgM-PFC). In blood from 75% of seropositive patients with rheumatoid arthritis (RA) RF-PFC were demonstrated, median value 6.8 (range: 0.4-1477) RF-PFC/10(6) mononuclear cells. Practically no RF-PFC were detected in seronegative RA patients and controls. Despite the fact that most IgM-RF undoubtedly is produced outside the blood, a positive correlation was found between the number of RF-PFC and the Waaler-Rose titer in serum. The number of circulating IgM secreting cells did not differ significantly between seropositive RA patients, seronegative RA patients and controls. Comparison of RF-PFC and IgM-PFC in seropositive patients revealed that in mean 7% of IgM-secreting cells in blood secrete IgM-RF.

Antibody Specificity↗
PubMed1983

Demonstration and partial characterization of chronic lymphocytic leukemia lymphocyte-associated antigens by crossed immunoelectrophoresis of Triton X-100 extracts.

Crossed immunoelectrophoresis was used to study the antigens of chronic lymphocytic leukemia lymphocytes. A reference pattern was obtained and 20 samples from 18 patients were compared with this pattern. Extensive variation in the expression of individual antigens was observed. The antigens in the reference pattern were further characterized by modifications of crossed immunoelectrophoresis, viz. labelling of intact lymphocytes by lactoperoxidase-catalyzed iodination, affinity crossed immunoelectrophoresis with phenyl-Sepharose and Lentil lectin-Sepharose, charge-shift crossed immunoelectrophoresis and postelectrophoretic incubation in (125I) Lentil lectin. Six antigens were identified as surface membrane glycoproteins, 5 as cytoplasmic proteins while 5 could not be classified. Two antigens were finally identified as HLA-ABC and HLA-DR by application of small amounts of monospecific rabbit antiserum and monoclonal antibody, respectively.

Aged↗
PubMed1982

Involvement of divalent cations in the complex between the platelet glycoproteins IIb and IIIa.

The major immunoprecipitate (No. 16) seen on crossed immunoelectrophoresis of Triton X-100-solubilized platelet proteins against whole platelet antibodies represents a complex containing the membrane glycoproteins IIb and IIIa. When EDTA is present during the solubilization, immunoprecipitate 16 as such is not observed, and two new arcs, termed 16a and 16b, appear. As with 16 these immunoprecipitates become radioactively labelled on lactoperoxidase-catalyzed iodination of platelets. Immunoprecipitate 16a showed partial immunochemical identity with 16, and was precipitated by an antibody raised against immunoprecipitate 16. The areas covered by immunoprecipitates 16, 16a and 16b were strongly reduced compared to normal with platelets from a patient with Glanzmann's thrombasthenia type II. Such platelets are known to contain reduced amounts of glycoproteins IIb and IIIa. The new arcs appearing when divalent cations are chelated by EDTA thus represent proteins derived from the immunoprecipitate 16 proteins, and divalent cations seem to be necessary to preserve the protein complex containing glycoprotein IIb and IIIa. The different complex formations between the components of immunoprecipitate 16 may reflect biochemical alterations of functional importance.

Blood Platelet Disorders↗
PubMed1982

Quantitative composition and characterization of the proteins in membrane vesicles released from erythrocytes by dimyristoylphosphatidylcholine. A membrane system without cytoskeleton.

Membrane vesicles were prepared by incubation of human erythrocytes with dimyristoylphosphatidylcholine [3] and isolated by isopycnic centrifugation on Dextran density gradients. Protein analyses were carried out with crossed immunoelectrophoresis and dodecylsulfate polyacrylamide gel electrophoresis. The right-side-out-oriented membrane vesicles contained membrane and cytoplasmic proteins of the erythrocyte but lacked cytoskeletal components. Comparison of proteins in vesicles and erythrocyte membranes showed that acetylcholinesterase was enriched two to six times in the vesicles relative to both membrane-spanning proteins, band 3, and glycophorin. Two further, hitherto unidentified, sialic acid-containing membrane antigens were found in the vesicles. Both faced the outside of the membranes and were enriched two to seven times. Ankyrin was not present in the membrane vesicles and spectrin could not be detected by dodecylsulfate polyacrylamide gel electrophoresis. We suggest that the redistribution of proteins in the vesicles reflects differences in their interactions with other membrane components and their relative mobility within the erythrocyte membrane.

Dimyristoylphosphatidylcholine↗
PubMed1982

Electroimmunochemical analysis of plasma membrane vesicles from Saccharomyces cerevisiae.

Plasma membrane vesicles of Saccharomyces cerevisiae were extracted with 1% (w/v) Triton X-100 and the solubilized proteins examined by crossed immunoelectrophoresis using rabbit antibodies against the vesicles. Solubilization was shown to be nonselective and 23 immunoprecipitates were observed reproducibly. Four glycoproteins were identified by interaction with concanavalin A and lentil lectin, either immobilized on agarose beads in an intermediate gel or incorporated in the free form in the first dimension gel. One glycoprotein was stainable by the periodic acid--Schiff procedure. None of the glycoproteins had their origin in the cell wall. Five amphiphilic proteins were identified on the basis of charge-shift and hydrophobic interaction crossed immunoelectrophoresis as well as [14C]Triton X-100 and Sudan black B binding. Three of the amphiphilic proteins were also glycoproteins. Based on the carbohydrate content and amphiphilic properties of the proteins, purification schemes using concanavalin A-Sepharose and phenyl-Sepharose were proposed. Trial separations using 1-mL columns were monitored by fused rocket and crossed immunoelectrophoresis.

Antibodies↗
PubMed1982

Monoclonal immunoglobulin of CLL lymphocytes detected by radioimmunofixation with 125I-protein A of Triton X-100 solubilized lymphocyte material.

A sensitive radioimmunofixation method (detection limit 1 mg/1 for monoclonal IgM) utilizing 125I Protein A and immunofixation for demonstration of monoclonal immunoglobulin in Triton X-100 extracts of isolated chronic lymphocytic leukemia (CLL) lymphocytes is presented. Thirty-two untreated patients with clinically typical CLL were studied and the results compared to those obtained by immunofluorescence. Monoclonal IgM was detected with similar frequency by the two methods (69% and 75%), but IgD was detected less frequently by radioimmunofixation (25% and 40%). When the results obtained with the two methods were combined, all of the cases were immunoglobulin positive. Two complete IgM type M-components were demonstrated in two cases, an excess of seemingly free heavy chains was found in four cases, and an excess of seemingly free light chains in six cases.

Antibodies, Monoclonal↗
PubMed1981

An immunochemical approach for the analysis of membrane protein alterations in Ca2+-loaded human erythrocytes.

An increase in the intracellular concentration of Ca2+ in human erythrocytes results in the formation of gamma-glutamyl-epsilon-lysine cross-linked membrane protein polymers. Following solubilization of the membranes with SDS, these polymers can be isolated on a Lubrol-containing sucrose gradient. Immunoelectrophoresis of the polymeric material with a polyspecific rabbit antibody against human ghosts gave rise to a single, but heterogeneous, precipitate. The polymer was amphiphilic and, on addition to Triton-solubilized erythrocyte membrane proteins, it coprecipitated with spectrin. When the antihost antibody was absorbed with the polymer prior to cross immunoelectrophoresis of normal erythrocyte membrane proteins, the precipitates of glycophorin, acetylcholinesterase, and hemoglobin were normal, whereas the antibody titers against band 3 protein, spectrin, and ankyrin became reduced. Furthermore, a rabbit antibody raised against the isolated human polymer reacted selectively with the same three membrane proteins. No reactions occurred with lysate proteins.

Calcium↗
PubMed1980

Monitoring of detergent binding to amphiphilic proteins by means of micelles containing the hydrophobic dye Sudan Black B.

A simple method for detecting micellar binding of Triton X-100 to amphiphilic proteins is described. The hydrophobic dye Sudan Black B is incorporated into Triton micelles. Binding of the coloured micelles to serum apoliproteins, as well as to amphiphilic proteins, or erythrocyte and fat globule membranes renders these visible as dark bands after sucrose density gradient centrifugation. In contrast, the hydrophilic proteins present in lipoprotein-free serum do not show detergent binding. The method does not permit accurate quantification of detergent binding, but may serve as a pilot procedure for initial detection of amphiphilic proteins and for monitoring their isolation from crude solubilized membrane material. The sensitivity of the assay corresponds to that obtained with [3H]Triton X-100.

Animals↗
PubMed1980

Distribution of 'free' and HLA-associated human beta 2-microglobulin in some plasma membranes and biological fluids.

The distribution of free and HLA-associated human beta 2-microglobulin (beta 2m) in serum, urine, spinal fluid, parotid duct saliva, seminal fluid, amniotic fluid and whey and in membranes from thrombocytes, lymphocytes, neutrophils and fat globules from milk was studied by crossed radioimmunoelectrophoresis (CRIE). The hydrophobic domain of HLA was demonstrable in 'charge-shift CRIE' and by binding to phenyl-Sepharose in 'hydrophobic-interaction CRIE'. In 'lectin-affinity CRIE' with concanavalin A and Lens culinaris lectin Sepharose the carbohydrate moiety present on. HLA exhibited heterogeneity as judged by the appearance of two partly separated protein peaks. Except for isolated fat globule membranes, HLA-associated beta 2m was present on all cells investigated. 'Free' beta 2m did not contain a hydrophobic domain as assessed by charge-shift and hydrophobic-interaction CRIE. All body fluids contained beta 2m in its 'free' form only. In serum, besides the free beta 2m, 2% was present as HLA-associated beta 2m, which, however, did not contain a hydrophobic domain. A degradation product of 'free' beta 2m, with alpha-mobility, was observed in sera from patients with malignant disorders, rheumatoid arthritis or systemic lupus erythematosus.

Body Fluids↗