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G A Cumme

Publications and source records attributed to G A Cumme.

At least 19 recordsLinked to original sources
PubMed2001

Structural study on the carbohydrate moiety of calf intestinal alkaline phosphatase.

Surprisingly alkaline phosphatase (AP) (EC 3.1.3.1) of calf intestine is found in large amounts, e.g. 80%, within chyme. Most of the enzyme is present as a mixture of four differently hydrophobic anchor-bearing forms and only the minor part is present as an anchorless enzyme. To investigate whether changes in the N-glycosylation pattern are signals responsible for large-scale liberation from mucosa into chyme, the glycans of the two potential glycosylation sites predicted from cDNA were investigated by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry in combination with exoglycosidase treatment after tryptic digestion and reversed-phase chromatography. The glycans linked to Asn249 are at least eight different, mainly non-fucosylated, biantennary or triantennary structures with a bisecting N-acetylglucosamine. For the most abundant glycopeptide (40%) the following glycan structure is proposed: [carbostructure: see text]. The glycans linked to Asn410 are a mixture of at least nine, mainly tetraantennary, fucosylated structures with a bisecting N-acetylglucosamine. For the most abundant glycopeptide (35%) the following glycan structure is proposed: [carbostructure: see text]. For the structures the linkage data were deduced from the reported specificities of the exoglycosidases used and the specificities of the transglycosidases active in biosynthesis. The majority of glycans are capped by alpha-galactose residues at their non-reducing termini. In contrast to the glycans linked to other AP isoenzymes, no sialylation was observed. Glycopeptide 'mass fingerprints' of both glycosylation sites and glycan contents do not differ between AP from mucosa and chyme. These results suggest that the observed large-scale liberation of vesicle-bound glycosylphosphatidylinositol (GPI)-anchored AP from mucosa into chyme is unlikely to be mediated by alteration of glycan structures of the AP investigated. Rather, the exocytotic vesicle formation seems to be mediated by the controlled organization of the raft structures embedding GPI-AP. (c) 2001 John Wiley & Sons, Ltd.

Alkaline Phosphatase↗
PubMed2000

Gel chromatographic characterization of the hydrophobic interaction of glycosylphosphatidylinositol-alkaline phosphatase with detergents.

The interaction of a glycosylphosphatidylinositol (GPI) protein with different detergents was studied for the first time with a purified protein. Four differently hydrophobic fractions of GPI-alkaline phosphatase (GPI-AP) from calf intestine were used as model proteins. The mode of interaction was determined by investigating (i) the self-aggregation behaviour of the GPI-AP fractions, (ii) the interference of detergents with GPI-AP binding to octyl-Sepharose, and (iii) the elution of GPI-AP bound to octyl-Sepharose. It was shown that polyoxyethylene-type detergents surprisingly interact much stronger than n-octylglucoside with GPI-AP, which is in contrast to the known behaviour of GPI-proteins in natural membranes. Gel filtration chromatography of Triton X-100 at concentrations above the critical micellar concentration yields three different micelle species with apparent molecular weights of about 166, 54, and 16 kDa. GPI-AP fraction II, which is shown to bear only one anchor per dimer, does not bind to any of these micelles. We demonstrate that a complex is formed containing about 150 Triton X-100 molecules and about 4700 molecules of water per molecule of GPI-AP dimer. The experimental findings are in accordance with a simple geometrical model based on the physical data of fatty acids and the arrangement, mean size, and shape of Triton X-100 molecules.

1-Propanol↗
PubMed1996

Microheterogeneity of the hydrophobic and hydrophilic part of the glycosylphosphatidylinositol anchor of alkaline phosphatase from calf intestine.

Digestion of calf intestine alkaline phosphatase with pronase and subsequent dephosphorylation of the released peptidyl-(Etn-P)2-glycosyl-PtdIns with HF generated 8 glycosyl-Ins species the largest of which (G1 and G2) have the following proposed structures: [sequence: see text] G3 and G5 are lower homologues of G1 and G2, respectively, being one alpha 1-2 linked mannopyranosyl residue shorter. G4 is analogous to G2 lacking the N-acetylgalactosaminyl residue and G6 is the next lower homologue of G4. Most of G4 and G6 occur substituted with a palmitoyl (G4, G6) or a myristoyl residue (G6) probably attached to the inositol moiety. Thus, the basic ManxGlc-Ins species are either substituted with an N-acetylgalactosaminyl residue or a fatty acid ester. The structures were deduced from compositional analysis, molecular-mass determination by matrix-assisted laser desorption MS, sequential hydrolysis with appropriate exoglycosidases and treatment with CrO3. Purification of the glycosylinositol species was achieved by a novel reverse-phase HPLC technique using fluorescent fluoren-9-yl-methoxy-carbonyl (Fmoc) derivatives. These stable derivatives were susceptible to hydrolysis with exoglycosidases which allowed sequential cleavages to be carried out and kinetics to be followed at the picomole level. We observed recently that native alkaline phosphatase separates on octyl-Sepharose into four distinct fractions of increasing hydrophobicity (F1-F4). Here we show that all four fractions contain G1-G6. The acylated species G4 and G6 were restricted to F2 and F4 which had been shown earlier to contain, on average, 2.5 and 3 fatty acid residues/subunit, respectively. In all four fractions the diradylglycerol moiety was predominantly diacylglycerol, alkylacylglycerol being less than 10% which is in contrast to most glycosyl-PtdIns--anchored proteins of mammalian origin.

Acetylgalactosamine↗
PubMed1996

Dependence on intracellular pH and Mg2+ of metabolic compartmentation of phosphoinositides in human erythrocytes.

Effects of intracellular pH and Mg2+ on turnover and extent of metabolic compartmentation of phosphomonoester groups of phosphoinositides and phosphatidate were investigated in human erythrocytes by short-term and equilibrium labeling with [32P]Pi under steady-state conditions. At pH 6.7, the specific radio-activities of phosphoinositides reached apparent equilibrium values, in the range of 70% of that ATP-gamma-P after long-term labelling. At pH 7.2, these values were in the range of 40-50% of that ATP-gamma-P. This demonstrates a decreased accessibility of phosphoinositides to enzymatic phosphorylation and dephosphorylation at the transition from acidic to normal incubation conditions. These changes were more pronounced at pH 7.8. High intracellular [Mg2+] initially activated the turnover of phosphoinositides at pH 7.2. The activation changed into an inhibition and an increase of metabolic compartmentation after three hours preincubation of erythrocytes at high [Mg2+]. The long-term Mg2+ effects are reversible to a great extent by a subsequent re-reduction of [Mg2+]. In conclusion, deprotonation of phosphoinositides by low [H+] or high intracellular [Mg2+] at normal pH induces a decreased accessibility for their specific lipid kinases and phosphatases. This effect may be the result of lateral phase separation of acidic phospholipids as a consequence of divalent cation complexation under both experimental conditions, high pH and high [Mg2+] at normal pH.

Erythrocytes↗
PubMed1995

Glycosylphosphatidylinositol-alkaline phosphatase from calf intestine as substrate for glycosylphosphatidylinositol-specific phospholipases--microassay using hydrophobic chromatography in pipet tips.

An electrophoretically homogeneous glycosylphosphatidylinositol- alkaline phosphatase fraction from calf intestine, obtained by hydrophobic chromatography, was used as "enzyme-labeled" substrate for testing phospholipase activity. The reaction products were separated by (i) hydrophobic chromatography in pipet tips and (ii) Triton X-114 phase partitioning. The chromatographic method presented permits high test frequencies, does not need temperature-controlled sample handling, and is only slightly disturbed by detergents, organic solvents, and proteins. The method was used to characterize phosphatidylinositol- specific phospholipase C from Bacillus cereus and phospholipase D from calf serum. Measurement of substrate hydrolysis by phospholipases is apparently linear to enzyme concentration and time. Relative activity of both enzymes is maximum at pH 6.5, corresponding to the optimal pH range found with other glycosylphosphatidylinositol substrates and phosphatidylinositol-specific phospholipases of other sources. Maximum activity of phospholipase C was found at 0.03% Triton X-100, 0.01% Brij 35, and 0.2% n-octylglucoside. The activity is not affected by Ca(2+), NaHCO(3), o-phenanthroline, or EDTA, increasingly inhibited by MgCl(2), MnCl(2), and ZnCl(2), and slightly activated by Na+ and K+. Calf serum phospholipase D shows maximum activity at 0.05% Triton X-100, 0.02% Brij 35, and 0.4% n-octylglucoside. The apparent Km values for phospholipase C (12.25 micron) and phospholipase D (4.94 micron) found with glycosylphosphatidylinositol-alkaline phosphatase are compared with values published for other glycosylphosphatidylinositol substrates.

Alkaline Phosphatase↗
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↗
PubMed1993

Determination, purification, and characterization of alpha-NADH.

alpha-NADH can be determined in the presence of high concentrations of beta-NADH using anion exchange HPLC combined with oxidation of beta-NADH by lactate dehydrogenase. The method is suited for the detection of a large number of impurities. Residual absorption is a poor measure of alpha-NADH. By combination of preparative anion exchange chromatography and reversed-phase HPLC, alpha-NADH can be prepared with 95-98% purity starting from beta-NADH. Commercially available preparations contain only 80-90% alpha-NADH. The anomerization kinetics of alpha- and beta-NADH has been investigated at pH 7.0 and 7.3 taking into account the irreversible conversion reactions of alpha- and beta-NADH. The H+ ion activity related anomerization rate constants kappa 1 (alpha to beta) and kappa 2 (beta to alpha) have been found to be 170 and 29 M-1 s-1, respectively, at pH 7.0 and 192 and 36 M-1 s-1, respectively, at pH 7.3. The equilibrium constants kappa 1 /kappa 2 are 5.8 and 5.3 at pH 7.0 and pH 7.3, respectively. Absorption and fluorescence properties of alpha-NADH preparations have been determined newly. At 25 degrees C, maximum absorption was found at 260.1 and 346.6 nm, whereas at 37 degrees C the wavelengths were 260.1 and 345.3 nm. Maximum fluorescence excitation was found at 348 nm, and maximum emission was at 453 nm.

Anions↗
PubMed1993

Multiwavelength photometry of thermochromic indicator solutions for temperature determination in multicuvettes.

If buffer/indicator systems are used as optical thermometers in multicuvettes, the temperature resolution is limited by errors of optical measurements produced mainly by variations of pathlength and blank transmittance of the wells. Theoretical and practical approaches show that, in multicuvettes, a between-well temperature resolution of < 0.05 degrees C can be achieved by multiwavelength photometry with use of the Tris/cresol red indicator system. Using up to three absorbances (A0, A1, A2) measured in the same well at different wavelengths for calculation of differences, quotients, and quotients of differences, we found the optimum temperature signal to be In(A1/A2), with equal-ranking absorbances changing with temperature in opposite directions. We have used the method successfully to measure the temperature profiles and temporal dynamics of temperatures at all positions in two types of multicuvettes under controlled conditions, or not controlled thermostatically.

Indicators and Reagents↗
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↗