Detection of membrane-associated antigens on lymphoid cells by antibody coupled to staphylococcal protein A.

A simple technique is described for the detection of membrane-associated antigens on lymphoid cells. It is based on the observation that the protein A component of staphylococci binds to the Fc pieces of IgG molecules. Lymphocytes from various sources (mouse, rat, and human tissues) were incubated with hyperimmune antisera directed against surface determinants. Subsequent treatment with a suspension of staphylococci containing protein A permitted visualization of both the presence and distribution of determinants on the cell membrane. The method had comparable sensitivity to the fluorescent sandwich technique and could be used to detect a variety of membrane antigens on both T cells and B cells.     

Validation of an in vitro method for incorporating labelled cholesterol into human erythrocytes.

At least 85% of the cholesterol of human erythrocytes can be replaced by isotopically labelled cholesterol, by incubation of the cells with the plasma lipoprotein HDL3 into which labelled cholesterol has been incorporated from Celite. With erythrocytes containing 40% labelled cholesterol, the reactivity of the incorporated labelled cholesterol was the same as that of the remaining native cholesterol with regard to: oxidation by cholesterol oxidase for both intact erythrocytes and cell ghosts, extraction by cholesterol depleted plasma, and exchange for HDL3 cholesterol. Also, the reactivity of the cholesterol in the cells in which labelled cholesterol was incorporated was the same as in untreated erythrocytes.     

Evidence for two uroporphyrinogen decarboxylase isoenzymes in human erythrocytes

In animals and plants, uroporphyrinogen decarboxylase catalyzes the stepwise decarboxylations of uroporphyrinogen, the precursor of heme and chlorophyll. To better understand its metabolic roles, we characterized the enzyme purified to electrophoretic homogeneity (about 11,000-fold) from human erythrocytes by a novel uroporphyrin-sepharose affinity chromatographic method. Native polyacrylamide disc gel electrophoresis of the purified enzyme preparation showed two bands detected by staining either for protein or with uroporphyrin-I. Each individual protein eluted from the gel when subjected to re-electrophoresis on SDS-polyacrylamide gel, appeared as a single protein band with molecular masses of approximately 54,000 and approximately 35,000 daltons respectively. Both proteins were able to catalyze all four decarboxylation steps, though the ratios of enzyme activity using octa-, hepta-, hexa- to pentacarboxylic porphyrinogen substrates were distinctly different. Also, their kinetic analysis with heptacarboxylic porphyrinogen-I substrate provided distinctly different apparent Michaelis constants. This provides the first evidence that decarboxylations of uroporphyrinogen to coproporphyrinogen are catalyzed by two isoenzymes.     

Evidence that calcium acts as an intracellular messenger for adrenergic responses in human erythrocytes

Adrenergic stimulation of membrane protein phosphorylation has been studied in human erythrocytes. The adrenergic enhancement in phosphorylation of band 2 could be mimicked by the calcium-specific ionophore A23187 in the presence of 10 micron extracellular calcium. Experiments with the potassium ionophore, valinomycin, showed that potassium efflux was not the primary effector of the response. Trifluoperazine, an inhibitor of the Ca2+-dependent regulatory protein, calmodulin, inhibited phosphorylation stimulation by either norepinephrine or the calcium ionophore. The norepinephrine response was observed in the absence of extracellular calcium, implicating Ca2+ released from cellular bound pools in mediating the response.     

Detection of the low-density-lipoprotein receptor with biotin-low-density lipoprotein. A rapid new method for ligand blotting.

A new technique has been developed to identify low-density-lipoprotein (LDL) receptors on nitrocellulose membranes, after transfer from SDS/polyacrylamide gels, by ligand blotting with biotin-modified LDL. Modification with biotin hydrazide of periodate-oxidized lipoprotein sugar residues does not affect the ability of the lipoprotein to bind to the LDL receptor. Bound lipoprotein is detected with high sensitivity by a streptavidin-biotin-peroxidase complex, and thus this method eliminates the need for specific antibodies directed against the ligand. The density of the bands obtained is proportional to the amount of pure LDL receptor protein applied to the SDS/polyacrylamide gel, so that it is possible to quantify LDL receptor protein in cell extracts. Biotin can be attached to other lipoproteins, for example very-low-density lipoproteins with beta-mobility, and thus the method will be useful in the identification and isolation of other lipoprotein receptors.     

A membrane blotting method for following the time course of protein radioiodination using Iodobeads.

A method for monitoring and optimizing the incorporation of radioiodide into protein is described. The use of immobilized chloramine-T (Iodobeads) combined with timed blots on nitrocellulose forms the basis of an efficient assay system. Results with Iodobeads and with the commonly utilized soluble form of chloramine-T are compared.     

Glucose induces lipid peroxidation and inactivation of membrane-associated ion-transport enzymes in human erythrocytes in vivo and in vitro.

Erythrocytes of diabetic subjects (non-insulin dependent) were found to have eight- to ten-fold higher levels of endogenously formed thiobarbituric acid reactive malonyldialdehyde (MDA), thirteen-fold higher levels of phospholipid-MDA adduct, 15-20% reduced Na(+)-K(+)-ATPase activity with unchanged Ca+2-ATPase activity, as compared with the erythrocytes from normal healthy individuals. Incubation of normal erythrocytes with elevated concentrations (15-35 mM) of glucose, similar to that present in diabetic plasma, led to the increased lipid peroxidation, phospholipid-MDA adduct formation, reduction of Na(+)-K(+)-ATPase (25-50%) and Ca+2-ATPase (50%) activities. 2-doxy-glucose was 80% as effective as glucose in the lipid peroxidation and lipid adduct formation. However, other sugars, such as fructose, galactose, mannose, fucose, glucosamine and 3-O-methylmannoside, and sucrose, tested at a concentration of 35 mM, resulted in reduced (20-30%) lipid peroxidation without the formation of lipid-MDA adduct. Kinetic studies show that reductions in Na(+)-K(+)-ATPase and Ca+2-ATPase activities precede the lipid peroxidation as the enzyme inactivation occur within 30 min of incubation of erythrocytes with high concentration (15-35 mM) of glucose, while lipid peroxidation product, MDA appears at 4 hr and lipid-MDA adducts at 8 hr. The lipoxygenase pathway inhibitors, 5,8,11-eicosatriynoic acid and Baicalein (5,6,7-trihydroxyflavone), reduced the glucose-induced lipid peroxidation by 30% and MDA-lipid adduct formation by 26%. Indomethacin, a cyclooxygenase pathway inhibitor, had no discernible effect on the lipid peroxidation in erythrocytes. However, the inhibitors of lipid peroxidation, 3-phenylpyrazolidone, metyrapone, and the inhibitors of lipoxygenase pathways did not ablate the glucose-induced reduction of Na(+)-K(+)-ATPase and Ca+2-ATPase activities in erythrocytes. Erythrocytes produce 15-HETE (15-hydroxy-eicosatetraenoic acid), which is augmented by glucose. These results suggest that the formation of lipoxygenase metabolites potentiate the glucose-induced lipid peroxidation and that the inactivation of Na(+)-K(+)-ATPase and Ca+2-ATPase occurs as a result of non-covalent interaction of glucose with these enzymes.     

Characterization of the spontaneous murine B cell leukemia (BCL1). III. Evidence for monoclonality by using an anti-idiotype antibody.

We have raised an anti-idiotypic antibody against the cell surface IgM of the murine BCL1 tumor cells. This antiserum reacts exclusively with the IgM expressed on the tumor cells and detects a unique population of cells in the spleen and blood of the tumor-bearing mice. When these cells are stimulated in vitro with LPS, they secrete an IgM bearing the same idiotype as the cell surface Ig. These results are discussed in terms of a model for the immunotherapy of a chronic lymphocytic leukemia-like syndrome in mice.     

Interaction of dichlone with human erythrocytes. I. Changes in cell permeability

The uptake of the fungicide dichlone (2,3-dichloro-1,4-naphthoquinone) by human erythrocytes was extremely rapid, reaching a maximum within 5 min of treatment. Most of the dichlone taken up was present in the interior of the cell; only a small fraction of the pesticide (less than 5%) was bound to the cell membrane. Dichlone (3 · 10^?5M-10^?4M) induced a rapid loss of intracellular potassium from the erythrocytes; the leakage of K⁺ varied with the fungicide concentration as well as with cell concentration. Pretreatment of the cells with glutathione was able to reduce potassium loss. Cells exposed to dichlone showed increased osmotic fragility. Dichlone also inhibited Na⁺-K⁺ ATPase, which is associated with active ion transport. However, the leakage of potassium in dichlone-treated cells does not appear to be related to the interference with active ion transport. An extensive loss of potassium within a relatively short time after treatment suggests that dichlone produces its effect by increasing passive cation permeability, probably as a result of direct action on the membrane structure. Dichlone was able to induce hemolysis, but only at concentrations higher than those which resulted in K⁺ loss. The loss of hemoglobin appeared to be mainly due to osmotic swelling of the treated cells. Exposure of red cells to dichlone also resulted in a rapid and extensive formation of methemoglobin as well as a denaturation of hemoglobin. Thus, dichlone not only may be capable of lowering the capacity of erythrocytes to transport oxygen but also alters their permeability.     

Bioelectrochemical immunoassay for human chorionic gonadotrophin in serum using an electrode-immobilised capture antibody

An amperometric technique for the quantification of an enzyme immunoassay which utilises a capture antibody covalently attached to a carbon electrode is described. The electrode is used both to separate the assay and to monitor the activity of the bound enzyme label. A ‘two-site’ immunometric assay with monoclonal antibodies directed against human chorionic gonadotrophin (HCG) was used as the model system. The activity of the enzyme bound to the electrode is determined electrochemically by the use of an electron transfer mediator (dimethylaminomethyl ferrocene) permitting rapid quantification of the analyte without the need for a separate incubation step to measure enzyme activity. The sensitivity of the assay is 9mIU HCG ml⁻¹ in serum (1st International Reference Preparation). The correlation between the amperometric measurement of serum HCG and data for an immunoradiometric assay was r = 0·988. The assay is rapid requiring a total assay time of 20 min per sample, which includes 15 min for antibody—antigen binding.     

Processing and presentation of insulin. II. Evidence for intracellular, plasma membrane-associated and extracellular degradation of human insulin by antigen-presenting B cells

To study the biochemistry of processing of a soluble protein Ag by an APC, we investigated how 125I-labeled human insulin (HI) is processed in situ by TA3 mouse hybridoma B cells. Fractionation of TA3 cells into their extracellular, plasma membrane-associated and intracellular compartments coupled with the use of HPLC enabled us to analyze several peptides derived from each compartment. One HI peptide found in all three compartments is composed of residues A1-A14 disulfide-linked to B7-B26 (A1-A14/B7-B26). The presence of this peptide in the extracellular compartment likely resulted from digestion of HI by an enzyme(s) released from the APC. Extracellular processing of radiolabeled HI was inhibited completely by unlabeled HI and N-ethylmaleimide, an inhibitor of a previously described insulin-specific protease, partially by lysozyme but not by BSA or OVA. This suggests that the enzyme involved in the extracellular processing of insulin is relatively insulin-specific and gives rise to the A1-A14/B7-B26 peptide. The processing of HI both at the plasma membrane and intracellularly was inhibited by chloroquine, monensin, and NH4Cl, suggesting that both intracellular pH changes and endocytic and exocytic events may be required for these compartments to process insulin. Kinetic analyses revealed that the processing of insulin into the A1-A14/B7-B26 peptide is first detected at the plasma membrane then intracellularly and finally in the extracellular compartment. This unlabeled A1-A14/B7-B26 peptide was purified from the extracellular compartment of TA3 APC by HPLC; when presented by TA3 APC this peptide effectively stimulated pork insulin (PI/I-Ad) specific Th cells to secrete IL-2. These data, taken together with the identification of another processed insulin peptide, A7-A11/B7-B26, have enabled us to elucidate the first steps in the biochemical pathway(s) of processing of insulin as an Ag in a B cell APC.     

Inhibition of platelet aggregation by primary amines. Evidence for a possible role of membrane-associated calcium

The aggregation of human blood platelets by thrombin, adenosine diphosphate, wheat germ agglutinin or ristocetin was inhibited by primary amines. In general, thrombin-induced platelet aggregation was strongly affected by the amines while the effect was weak on cell aggregation by ristocetin. Usually, the diamines were stronger inhibitors of aggregation than the monoamines with cadaverine as the strongest and ethylamine as the weakest inhibitor. At concentration where platelet aggregation was inhibited, the amines neither displaced serotonin from serotinin-loaded platelets nor caused lysis of human red cells. The lectin activity of wheat germ agglutinin on human red cells was not affected by the amines indicating that the amines probably acted on platelets and not on the agglutinin. The clotting activity of thrombin on fibrinogen was partially inhibited by the amines while its esterolytic activity remained unaltered. The inhibitory action of the amines on platelet aggregation could be overcome with small amounts of calcium while other divalent cations tested had little effect. It is suggested that the amines affect platelet aggregation by interfering with the actions of membrane-associated calcium.     

Role of calpain in human sperm activated by progesterone for fertilization

We evaluated the activation of mu-calpain in progesterone-activated human sperm. Semen collected from fertile donors with informed consent was liquefied and subjected to percoll gradient centrifugation. After exposure to different concentrations of progesterone, the samples were used for immunostaining, SDS-PAGE and Western blot analysis. An increase of the intracellular free calcium concentration in the sperm following the addition of progesterone was observed using fura-2 AM. Immunostaining using an antibody against active mu-calpain produced 6 distinct staining patterns: (1) the acrosome, (2) an equatorial segment, (3) the whole head, (4) the neck, (5) the neck and tail or (6) unstained sperm. After addition of progesterone, the predominant type changed from the neck type (90%) to the neck and tail type (79%). Western blot analysis using a pro-mu-calpain and a mu-calpain domain III antibody revealed autodigestion of mu-calpain, indicating activation by progesterone. Using calpain-specific inhibitors it was shown that calpain activation contributes to sperm motility as well as to the acrosome reaction. These results suggest the possibility that activation of mu-calpain in human sperm by progesterone plays an important role in fertilization.     

A rapid one-step extraction procedure for the isolation of ubiquitin from human erythrocytes for antibody production.

A procedure is described that employs 5% perchloric acid extraction to isolate ubiquitin from human erythrocytes. The procedure is rapid and economical as it requires no specialized equipment. The extracted protein appeared to be highly purified as judged by electrophoresis and was identified as ubiquitin by immunoblotting and total amino acid analysis. The extraction yields about 78% of the ubiquitin in the hemolysate, which is a higher yield than is obtained with other procedures. The purified ubiquitin was used to make a polyclonal antiserum. As ubiquitin is a small and highly conserved protein, it is necessary to couple it to a larger immunogen to elicit an immune response. This ubiquitin antiserum was produced using an immunogen system that produces an immune response to the ubiquitin, but not to the carrier protein.