Biomedical subjects

G A Cumme

Publications and source records attributed to G A Cumme.

16 recordsLinked to original sources

PubMed1995

Characterization of the interaction of alkaline phosphatase with an activity inhibiting monoclonal antibody by progress curve analysis.

Using the enzyme activity inhibiting monoclonal antibody IB 10B8 against alkaline phosphatase of calf intestine (AP), the interaction of a macromolecular antigen with the antibody was studied with different reaction conditions and with different conformations of the antigen, i.e. using (i) different pH values, (ii) different temperatures, (iii) different substrate saturation of the enzyme, (iv) different glycosylphosphatidyl-AP (GPI-AP) aggregates, and (v) membrane-bound species. In the case of antibody excess and negligible substrate consumption enzymic product formation proceeds according to [P] = a + b x t - c x exp(-d x t). By direct progress curve fitting and secondary data evaluation using nonlinear regression, omitting numerical derivation and graphic techniques, kinetic constants of the immune reaction have been estimated. The method does not require any artificial labelling nor any separation of bound and free entities. (i) Upon increasing pH from 9.8 to 11.0, the dissociation constant of the enzyme-antibody complex is increased strongly, mainly due to the decreasing association rate constant. (ii) A temperature increase from 25 degrees C to 37 degrees C produces a marked increase of both the association and dissociation rate constant. (iii) To differentiate between the interaction of the antibody with the free (E) and substrate-bound (ES) enzyme, experiments were done at different substrate concentrations. The results were fitted to a model allowing determination of association and dissociation rate constants of the free and substrate-bound enzyme. The inverse variation of association and dissociation rate constants caused by substrate binding produces a marked increase of the dissociation constant of the antibody-enzyme complex. The antibody-bound enzyme shows a nearly three-fold higher Km value and a six-fold lower catalytic constant as compared to the free enzyme. (iv) Investigations of the interaction of the antibody with anchorless AP, different hydrophobic aggregates of purified GPI-AP (fractions II-V). (v) Membrane-bound GPI-AP show that the epitopes of all species are fully accessible to the antibody and not cryptic. Surprisingly the insertion of the GPI-moiety into the membrane and the aggregation of the different GPI-AP fractions II-V seem to improve antibody binding. Such improvement of binding was not found in control experiments with Fab, indicating only for the bivalent antibody a stronger interaction with the multivalent antigen than with the monovalent antigen.

Alkaline Phosphatase↗

PubMed1990

Determination of rate constants of monoclonal antibodies to enzymes.

A novel graphical method for determining rate constants of the immune reaction of enzyme-inhibiting or -activating antibodies has been evaluated. The experimental determination of kinetic constants does not require purification of antibody and enzyme nor any separation step of bound and free entities. The resultant enzyme activity is used as a measurement of the extent of enzyme-antibody complex formation. The plot ln magnitude of v - v infinity versus t (v = enzyme activity at time t, v infinity = enzyme activity at infinite time) yields straight lines in the case of antibody excess. Slope and vertical intercept of that primary plot can be used for secondary plots to obtain association (k1) and dissociation (k-1) rate constant, the dissociation constant KD of the complex and the residual enzyme activity of the complex (g/f). The suitability of the graphical method has been established experimentally using the mab IB 10B8 (2) which inhibits alkaline phosphatase activity. With the homogeneous assay in the presence of substrate as well as a microassay in the absence of substrate, k1 and g/f were found to be 4.68 x 10(7) M-1 min-1; 3.74 x 10(7) M-1 min-1 and 0.035; 0.107 respectively. For k-1 and KD, only crude estimates could be derived. The method has been tested for model discrimination using computer simulated data.

Alkaline Phosphatase↗

PubMed1986

Turnover of phosphomonoester groups and compartmentation of polyphosphoinositides in human erythrocytes.

The turnover of phosphomonoester groups of phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] was investigated in human erythrocytes by short-term labelling with [32P]Pi. The procedure applied ensured a quantitative extraction of erythrocyte polyphosphoinositides as well as their reliable separation for the determinations of pool sizes and specific radioactivities. The pool sizes of phosphatidylinositol (PtdIns), PtdIns4P and PtdIns(4,5)P2 are 25, 11 and 44 nmol/ml of cells respectively. Under steady-state conditions, the phosphorylation fluxes from [gamma-32P]ATP into PtdIns4P and PtdIns(4,5)P2 are in the ranges 14-22 and 46-94 nmol X h-1 X ml of cells-1 respectively. Only 25-60% of total PtdIns4P and 6-10% of total PtdIns(4,5)P2 take part in the rapid tracer exchange, i.e. are compartmentalized. In isolated erythrocyte ghosts, the turnover of PtdIns4P approximately corresponds to that in intact erythrocytes, although any compartmentation can be excluded in this preparation. Under the conditions of incubation employed, the turnover of PtdIns(4,5)P2 is more than one order of magnitude smaller in isolated ghosts than that obtained for intact erythrocytes.

Adenosine Triphosphate↗

PubMed1986

Two-stage median-based methods for characterizing enzyme inhibition.

Non-parametric linear regression is used for inhibition diagnostics and parameter estimation based on the velocity equation v = sKsV/(1 + sKS + iKI + isKIS) = sV(i)/(K(i) + s) = W(s)/(L(s) + i). From velocities measured as a function of inhibitor level i at different constant substrate levels s, median-based estimates of 1/W(s) and L(s)/W(s) are derived. Different diagnostic secondary plots are introduced, which take special shapes at limiting inhibition types. Two-stage median-based methods are presented for fitting the four-parameter equation to the observed velocities. These methods cause only little computational effort in contrast to one-stage median-based estimation which requires medians to be taken of solutions of impracticably large numbers of linear equation systems. Performance of the new methods is compared with that of the least-squares fit by application to simulated velocity data. For velocity data containing outliers, the median-based estimates are superior to the least-squares ones, whereas for normally distributed data the reverse is true.

Enzyme Inhibitors↗

PubMed1981

Dynamic properties of a phosphofructokinase/pyruvate kinase system. Experiments in vitro using the substrate-stat technique.

It is known from theoretical work that very simple enzyme systems may exhibit non-linear dynamic properties like those found in vivo. To realize such a system experimentally, an ATP-producing reaction (pyruvate kinase) was coupled with an ATP-consuming reaction (phosphofructokinase). The substrate-stat technique has been adapted to characterize each single enzyme as well as to follow up one enzyme in the coupled system. If the plots of both pyruvate kinase activity versus [ADP] and phosphofructokinase activity versus [ATP] are hyperbolic, the coupled system approaches a unique steady state as predicted from the single characteristics. Under assay conditions where phosphofructokinase is inhibited by ATP, its characteristics as found in a single assay and in the coupled system are different. For a system with ATP-inhibited phosphofructokinase, a paradoxical behaviour is predicted and is demonstrated experimentally. If the total pyruvate kinase activity is increased over a certain limit, the system is switched from a low [ATP], high-activity steady state into a high [ATP] low-activity steady state.

Adenosine Diphosphate↗

PubMed1981

Binding of magnesium and chloride ions to human hemoglobin A. Mg2+ concentrations in solutions simulating red cell conditions.

Ultrafiltration and direct determination of [Mg2+] by ion exchange were used to study the binding of Mg2+ and Cl- to isoionic human hemoglobin. A value of 58.8 +/- 1.7 l/mol was determined for the volume of hydrated hemoglobin from ultrafiltration of hemoglobin solutions containing 0.15-0.8 M glucose. In solutions with 5.7 mmol hemoglobin, 150 mmol KCl and 0.5-3.5 mmol MgCl2/l total water, 0.6 mol and 2 mol Cl- were bound/mol oxygenated and deoxygenated hemoglobin respectively. A value of about 11 l/mol was determined for the association constant of Mg2+ to hemoglobin monomer. Free Mg2+ concentrations were measured in hemoglobin solutions containing KCl, MgCl2, ATP and D-glycerate-2,3-bisphosphate at concentrations close to those of red cells. The experiments yielded 0.65 mmol/l free Mg2+ after oxygenation and 0.82 mmol/l after deoxygenation. The data indicate that only small changes of free Mg2+ levels in red cells are caused by physiological changes of pO2.

Chlorides↗

PubMed1980

[Calcium oxalate activity products and discriminant analysis of urinary parameters as basis for a calcium oxalate screening program (author's transl)].

The values of 7 urinary parameters in divided urine collections were measured in 20 patients with calcium oxalate stones and ten normal persons. On this basis the thermodynamic stone formation risk was calculated for the different urine collections. Stone formation risk in the 0-6 a.m. urine fractions appeared to be the most important. The risk of stone formation is significantly increased in the time from midnight to 6 a.m. To detect the risk situation the determination of oxalic acid, calcium, potassium and inorganic sulfate in night urine is adequate.

Calcium↗

PubMed1978

[Comparison of procedures for determining Michaelis-Menten parameters together with their standard deviations using simulated measurement series].

The direct non-linear fit of Cleland and different methods applying linear regression analysis to linearized forms of the Michaelis-Menten equation have been compared. Comparison was made with respect to bias and standard deviation of the estimated values of Km, Vm, deltak and deltav. The Lineweaver-Burk method, weighted by approximately v3, turned out to produce estimates nearly as reliable as the direct fit of Cleland if one does not know whether the velocities are subject to constant absolute or relative errors. The comparison was based on ca. 10000 simulated series of velocity measurements. Runs with fewer simulated measurement series showed that for comparing different evaluation methods, at least 1000 measurement series should be simulated in order to get reliable results.

Enzymes↗

PubMed1977

[An ion exchange method to determine free metal concentrations, adapted for use in biological fluids: methods and determination of (Mg2+)].

An ion exchange method for measuring concentrations of free (ionized) metal ions and its application to the determination of [Mg2+] is described. A surface sulfonated polystyrene material is used as the "twodimensional" cation exchanger. The sample-volume is 1 ml. About 50 determinations can be performed within 1 hr having a standard error of +/- (2-4)% in the optimal range of measurement. Advantages and disadvantages of the method compared with other ones are demonstrated and discussed. Free Mg2+ ion concentrations were measured in solutions containing pyrophosphate as well as haemoglobin and compared with those which were determined by equilibrium calculation or ultrafiltration.

Cation Exchange Resins↗

PubMed1976

[Program for the computation of concentrations of free metabolites, metal ions and their complexes in chemical balance].

Enzymic reaction velocities often depend primarily on the concentrations of single free and complexed species rather than on the total metal and metabolite concentrations. For determining single species concentrations from total concentrations and complex stability constants, a program is described. The program may not only calculate the single concentrations, but also estimate their uncertainty due to the unavoidable uncertainty of complex stability constants. Making use of the ICT 1900 magnetic drum storage, it may deal with quite large complex patterns as are found in vivo.

Chelating Agents↗

PubMed1973