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Biomedical subjects

E T Maggio

Publications and source records attributed to E T Maggio.

10 recordsLinked to original sources
PubMed1985

Demonstration of both immunologically unique and common antigenic determinants in Dirofilaria immitis and Toxocara canis using monoclonal antibodies.

Using an ELISA system, the immunological cross-reactivity between D. immitis and T. canis has been, for the first time, examined on an individual clone-by-clone basis. This study offers a definitive demonstration of the presence of immunologically distinguishable species-specific non-crossreactive antigens and the presence of cross-reactive antigens in antigenic extracts obtained from adult D. immitis and T. canis.

Animals↗
PubMed1980

A solid-phase fluoroimmunoassay for the quantitation of antibodies to Toxoplasma gondii.

A solid-phase fluoroimmunoassay (FIAX¿) for the detection and quantitation of human antibodies to Toxoplasma gondii is described. The method is a modification of the procedure of Walls and Barnhart [1] which differs from the earlier test in that it uses a dual rather than single surface StiQTM Sampler, having both an antigen and a control surface. Each sampler is carried through four steps: reaction with diluted serum, a buffer wash, reaction with FITC-labeled goat anti-human IgG, and a final buffer wash. The fluorescence of each surface is measured using a dedicated surface-reading fluorometer. Serum titers are interpolated from a standard curve. The antibody titers of 233 sera were determined by both the indirect immunofluorescent antibody (IFA) and the FIAX¿ assays. The titers determined by the two tests agreed within one four-fold dilution for 226 (97%) of the sera. For replicates (n = 21) of the sera with low, mid and high antibody titers the coefficients of variation were 13.6, 10.0, and 12.3% respectively.

Antibodies↗
PubMed1980

A simplified solid-phase immunofluorescence assay for measurement of serum immunoglobulins.

A simple 2-step solid-phase fluoroimmunoassay for the determination of serum immunoglobulins is described. The method employs a stabilized solid-phase immunoadsorbent consisting of antigen immobilized on a cellulose acetate/nitrate disc attached to a plastic StiQTM sampler. The polymeric disc serves both as a substrate for immobilization and as a highly uniform surface upon which very precise fluorescence determinations may be made. The assays rely upon the reaction of a precise and limiting amount of mono-specific fluorescent antibody with the specific antigen present in a test sample. Residual unreacted fluorescently labeled antibody is allowed to bind to the immunoadsorbent; non-specifically bound antibody is removed in a wash step. The amount of labeled antibody bound is inversely proportional to the amount of antigen present in the test sample. The fluorescence of the bound labeled antibody is measured using a FIAX surface-reading fluorometer. Comparison of this new procedure with the commercially available (4-step) FIAX assay, which requires generation of immunoadsorbent in situ, demonstrated excellent correlation between the two methods. The new 2-step procedure provides results in a much shorter period of time and requires one-half to one-eighth of the operator time required for the other available solid-phase fluoroimmunoassays.

Fluorescent Antibody Technique↗
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↗
PubMed1977

Properties of a CH3-blocked creatine kinase with altered catalytic activity. Kinetic consequences of the presence of the blocking group.

Steady state kinetic parameters for rabbit muscle creatine kinase (EC 2.7.3.2) and this enzyme stoichiometrically blocked at the iodoacetamide-sensitive cysteinyl residue with a CH3S-group have been measured at 30+/-0.1 degrees, pH 9.00, using Mg(II) as the required metal ion cofactor. The double reciprocal plots for the CH3S-blocked enzyme with MgATP as the variable substrate are biphasic, each curve showing a break at approximately 1.9 mM MgATP, and suggest the possibility of negative cooperativity in metal-nucleotide binding. Furthermore, extrapolated lines at high MgATP concentrations intersect on the abscissa, indicating loss of synergism in binding of substrates. In contrast, observed Michaelis constants for creatine are, within experimental error, the same for both native and blocked enzymes. The maximal velocity of the CH3S-blocked enzyme is found to be 28.1% of the value of the native enzyme. Product inhibition patterns for both native and blocked enzyme are also compared. Again, these patterns indicate that the CH3S-blocking group alters the nucleotide binding site more than the guanidino substrate binding site. Calculations using the methods of Chou and Fasman (1970) Biochemistry 13, 211-222) lead to the prediction that the active cysteinyl residue occurs at the beginning of a beta turn which separates two portions of beta sheet structure of the enzyme, and so may be in a position to mediate conformational changes in the protein.

Adenosine Diphosphate↗
PubMed1975

Mandelate racemase from Pseudomonas putida. Magnetic resonance and kinetic studies of the mechanism of catalysis.

The interactions of mandelate racemase with divalent metal ion, substrate, and competitive inhibitors were investigated. The enzyme was found by electron paramagnetic resonance (EPR) to bind 0.9 Mn2+ ion per subunit with a dissociation constant of 8 muM, in agreement with its kinetically determined activator constant. Also, six additional Mn2+ ions were found to bind to the enzyme, much more weakly, with a dissociation constant of 1.5 mM. Binding to the enzyme at the tight site enhances the effect of Mn2+ on the longitudinal relaxation rate (1/T1p) of water protons by a factor of 11.9 at 24.3 MHz. From the frequency dependence of 1/T1p, it was determined that there are similar to 3 water ligands on enzyme-bound Mn2+ which exchange at a rate larger than or equal to 10-7 sec-1. The correlation time for enzyme-bound Mn2+-water interaction is frequency-dependent, indicating it to be dominated by the electron spin relaxation time of Mn2+. Formation of the ternary enzyme-Mn2+-mandelate complex decreases the number of fast exchanging water ligands by similar to 1, but does not affect tau-c, suggesting the displacement or occlusion of a water ligand. The competitive inhibitors D,L-alpha-phenylglycerate and salicylate produce little or no change in the enzyme-Mn2+-H2O interaction, but ternary complexes are detected indirectly by changes in the dissociation constant of the enzyme-Mn2+ complex and by mutual competition experiments. In all cases the dissociation constants of substrates and competitive inhibitors from ternary complexes determined by magnetic resonance titrations agree with K-M and K-i values determined kinetically and therefore reflect kinetically active complexes. From the paramagnetic effects of Mn2+ on 1/T1 and 1/T2 of the 13C-enriched carbons of 1-[13C]-D,L-mandelate and 2-[13C]-D,L-mandelate, Mn2+ to carboxylate carbon and Mn2+ to carbinol carbon distances of 2.93 plus or minus 0.04 and 2.71 plus or minus 0.04 A, respectively, were calculated, indicating bidentate chelation in the binary Mn2+-mandelate complex. In the active ternary complex of enzyme, Mn2+, and D,L-mandelate, these distances increase to 5.5 plus or minus 0.2 and 7.2 plus or minus 0.2 A, respectively, indicating the presence of at least 98.9% of a second sphere complex in which Mn2+, and C1 and C2 carbon atoms are in a linear array. The water relaxation data suggest that a water ligand is immobilized between the enzyme-bound Mn2+ and the carboxylate of the bound substrate. This intervening water ligand may polarize or protonate the carboxyl group. From 1/T2p the rate of dissociation of the substrate from this ternary complex (larger than or equal to 5.6 times 10-4 sec-1) is at least 52 times greater than the maximal turnover number of the enzyme (1070 sec-1), indicating that the complex detected by nuclear magnetic resonance (NMR) is kinetically competent to participate in catalysis. Relationships among the microscopic rate constants are considered.

Binding, Competitive↗
PubMed1975

Simple alkanethiol groups for temporary blocking of sulfhydryl groups of enzymes.

New reagents for the temporary blocking of active or accessible sulfhydryl groups of enzymes have been developed. These reagents, which are either alkyl alkanethiolsulfonates or alkoxycarbonylalkyl disulfides, rapidly and quantitatively place various RS- groups on the sulfhydryls to generate mixed disulfides. In all cases native enzymes can be regenerated with either dithiothreitol or beta-mercaptoethanol. In general the temporary blocking groups also afford total protection against normally inhibitory thiol blocking agents. When RS- groups were attached to rabbit muscle creatine kinase (EC 2.7.3.2), a trend toward lower residual activities with increasing bulk was observed. Treatment of the native creatine kinase with 14CH3HgC1 led to incorporation of greater than 1 equiv of CH3Hg- group per subunit. This CH3Hg- blocked enzyme was fully active, and the blocking group afforded no protection against iodoacetamide. These results suggest that CH3Hg- and the RS- groups are modifying two different sulhydryl groups on the enzyme. When papain (EC 3.4.4.10) was treated with excess methyl methanethiolsulfonate. complete and rapid inhibition was observed, and 1 equiv of CH3S- was incorporated/mol of active enzyme. Complete protection against normally inhibitory 5,5'-dithiobis(2-nitrobenzoic acid) was afforded by the temporary blocking group. When rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) was titrated with methyl methanethiolsulfonate, two sulfhydryl groups per subunit were found to be modified, one much more rapidly than the other. If one extrapolates the initial slope of the titration curve, the inactivation of the enzyme would be complete after modification of a single cysteinyl residue per subunit.

Alkanes↗