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Biomedical subjects

E F Ullman

Publications and source records attributed to E F Ullman.

35 records · Page 2Linked to original sources
PubMed1980

Monitoring of phospholipid vesicle fusion by fluorescence energy transfer between membrane-bound dye labels.

A sensitive method which utilizes fluorescence energy transfer to assay Ca2+ -or Mg2+ -mediated fusion of phospholipid vesicles is reported. More than 85% quenching results when phosphatidylserine vesicles labelled with dansyl phosphatidylethanolamine (donor) are fused with vesicles labelled with rhodamine phosphatidylethanolamine (acceptor) in the presence of 5 mM CaCl2 or 10 mM MgCl2. Higher concentrations of divalent cations are required to obtain maximal quenching when phosphatidylserine is partially replaced with phosphatidylethanolamine or phosphatidylcholine. The rate of vesicle fusion is dependent upon the concentrations of both cation and vesicles. Maximum quenching occurs within 5 min using phosphatidylserine vesicles and 5 mM Ca2+, but quenching is incomplete even after 20 h with 0.8--2 mM Ca2+. This probably reflects the heterogeneous size distribution of these vesicles, since the extent of fusion was found to correlated with vesicle size. Binding of antibody to membrane-localized phenobarbital hapten effectively blocks Ca2+ -mediated vesicle fusion. This effect can be inhibited by preincubation of the antibody with phenobarbital. Leakage of tempocholine from intact vesicles induced by 5 mM Ca2+ occurs even when fusion is prevented by bound antibody. This demonstrates that fusion is not a necessary requirement for Ca2+ -induced leakage.

Antibodies↗
PubMed1980

Specific antibodies to theophylline for use in a homogeneous enzyme immunoassay.

Bovine serum albumin conjugate of 1-methyl-3-(3'-carboxypropyl)xanthine elicits highly specific anti-theophylline antibodies when injected into sheep. When used in a homogeneous enzyme immunoassay for theophylline these antibodies show insignificant cross-reactivity (less than 1%) to 1-methyl- and 1,3-dimethyluric acid, 3-methylxanthine, caffeine, and theobromine. In contrast, immunogens prepared from the C-8 functionalized drug afford antibodies which show more serious cross-reactivity to these compounds. Plausible rationale for attachment of the drug to carrier proteins through its N-3 position which furnished specific antibodies are given.

Animals↗
PubMed1979

Mechanism of inhibition of malate dehydrogenase by thyroxine derivatives and reactivation by antibodies: homogeneous enzyme immunoassay for thryroxine.

Pig heart mitochondrial malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) is about 90% inhibited upon labelling an average of two amino groups per subunit with an active ester of thyroxine. Inhibition is probably associated primarily with thyroxine binding to one specific group which is normally unreactive but becomes activated upon noncovalent binding of thyroxine derivatives to the enzyme. Enzyme inhibition is due to a decrease in the rate of association of NAD. Antibodies to thyroxine induce a slow conformational change with partial reversal of inhibition of more heavily labelled conjugates. The antibody-induced activation is not cooperative and does not require bivalent association of the antibody. Activation can be blocked by the presence of free thyroxine and is the basis for a clinically useful assay for serum thyroxine.

Animals↗
PubMed1979

Fluorescence protection immunoassay: a new homogeneous assay technique.

We describe a "fluorescence protection immunoassay," in which formation of an immune complex of a fluorescer-labeled antigen sterically protects the fluorescer from binding by antibodies to it. Competitive binding of unlabeled antigen by its antibody prevents formation of the fluorescer-labeled antigen immune complex, and allows anti-fluorescein to quench the fluorescence by binding to the fluorescer. This phenomenon is the basis of a new homogeneous assay technique that requires no separation step. The steric exclusion of anti-fluorescein from fluorescein-labeled human IgG immune complexes was altered by changing the molecular dimensions ob both antifluorescein and the immune complex. The assay did not require highly purified fluorescein-labeled human IgG. An assay is demonstrated in which was used a fluorescein-labeled human IgG conjugate containing IgG that was only 10% pure. Measurement of IgG in human serum samples correlated well with results by radial immunodiffusion. The method is applicable to the assay of both proteins and analytes of low molecular mass.

Antigen-Antibody Complex↗
PubMed1978

Inhibition of malate dehydrogenase by thyroxine and structurally related compounds.

The inhibition of pig heart mitochondrial malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37) by the thyroxine and structurally related compounds was studied to resolve a longstanding question about the exact nature of the inhibition. Thyroxine, in freshly prepared solution, was found to be a "pure" competitive inhibitor relative to the nucleotide cofactor. Upon standing in diffuse daylight, solutions of thyroxine showed increased ability to inhibit the enzyme, presumably as a result of oxidation of enzyme sulfhydryl groups by free iodine that is released photochemically. This behavior probably accounts for earlier reports of irreversible inactivation by thyroxine. Comment is made on the implications of these findings to the mechanism of thyroid hormmone action.

Animals↗
PubMed1978

Homogeneous enzyme immunoassay for cannabinoids in urine.

We describe a homogeneous enzyme immunoassay for measurement of cannabinoid metabolites, as well as delta9-tetrahydrocannabinol (I) in urine. Malate dehydrogenase from pig heart mitochondria was labeled with a derivative of I. The compound used to calibrate the assay was the I metabolite, 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid (II). With 15 microgram of II per liter of urine as the cutoff concentration, the assay can detect 25 microgram of II per liter with greater than 95% confidence. A positive response was obtained for urine specimens assayed within 30 min after exposure to cannabinoids. However, the persistence of metabolites of I in urine indicates that assay of this fluid is useful as an indicator of cannabinoid use but not as an indicator of intoxication.

Antibody Specificity↗
PubMed1976

Fluorescent excitation transfer immunoassay. A general method for determination of antigens.

A general immunochemical method for the assay of haptens and proteins has been devised and applied to morphine, a morphine-albumin conjugate, and human immunoglobulin G. A fluorescein-labeled antigen and a quencher-labeled antibody are employed. By use of fluorescein and rhodamine as the fluorescer and quencher, respectively, dipole-dipole-coupled excitation energy transfer can occur within the antigen-antibody complex. The resulting quenching of fluorescence can be inhibited by competitive binding with unlabeled antigen, Alternatively, separate antibody samples can be labeled with fluorescein and rhodamine, respectively. Unlabeled antigen causes aggregation of the separately labeled components with resultant quenching. Using the latter method, experiments suggest that up to about 20 anti-morphine antibody binding sites will associate with morphine-albumin conjugates. When an excess of the conjugate is present the antibodies appear to assemble in clumps on the protein surface. Mathematical analysis of the quenching of fluorescein-labeled morphine by rhodamine-labeled anti-morphine gives an approximate fit to the quenching data, but the calculations are very dependent on the assumptions used.

Animals↗
PubMed1975

Mechanism by which antibodies inhibit hapten-malate dehydrogenase conjugates. An enzyme immunoassay for morphine.

Antimorphine antibodies inhibit the activity of morphine conjugates of mitochondrial malate dehydrogenase. Conjugation of malate dehydrogenase through tyrosine and amino groups resulted in only moderate losses of enzyme activity. On conjugation through disulfide bonds the enzyme activity first increased but dropped sharply with increasing substitution. Only the former conjugates were inhibited by excess antibodies. The degree of inhibition (up to 86%) was directly related to the number of morphine residues bonded directly to amino groups. The maximum number of antibody binding sites that bind to enzyme was nearly equal to the number of haptens provided there were 16 or less haptens/enzyme. However up to 26 haptens/enzyme became completely bound by antibody on long incubation. Inhibition of enzyme activity was detectably reduced by 2 times 10 minus 9 M morphine or 2 times 10 minus 10 M codeine, thus providing a sensitive assay for these drugs. The data suggest that enzyme inhibition occurs by conformational freezing of the enzyme when antibody binds to a morphine residue attached to one specific amino group.

Animals↗