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G Czerlinski

Publications and source records attributed to G Czerlinski.

16 recordsLinked to original sources
PubMed1999

Hemoglobin/O2 systems: using short-lived intermediates for mechanistic discrimination.

The kinetics of the reaction of hemoglobin with molecular oxygen, in which rapid mixing is followed by a very fast temperature jump, is numerically simulated. Four different mechanisms are considered. In two of them, oxygen reacts with the alpha-chains first, in the other two with the beta-chains first. Furthermore, either the third or the fourth measured (Adair) dissociation constant is composed of the product of a local dissociation constant and an allosteric interconversion constant. We explore whether these alternative mechanisms may be distinguished experimentally. We show that reaction steps not resolvable by rapid mixing can be resolved by chemical relaxation at appropriate points in time. Discrimination under experimental conditions is possible at higher oxygen concentrations (above 100 microM), but high resolution in time and concentration amplitude are required.

Animals↗
PubMed1998

Short-lived intermediates in hemoglobin/O2 systems.

The kinetics of the reaction of hemoglobin with molecular oxygen, in which rapid mixing is followed by a very fast temperature jump, is numerically simulated. Values for rate constants are used to the extent known, otherwise interpolated or extrapolated. It is shown that reaction steps not resolvable by rapid mixing can be resolved by subsequent chemical relaxation at appropriate points in time. Four different mechanisms are considered, all assuming no distinction between the two kinds of chains of hemoglobin. Bimolecular rate constants for oxygen binding are either the same for all four sites, or are governed by "frequency factors" (the kinetic equivalent of statistical factors for equilibrium constants in allosteric models). Furthermore, either the third or the fourth measured (Adair) dissociation constant is composed of the product of a "local" dissociation constant and an allosteric interconversion constant. These two pairs of choices give rise to four different mechanisms. Can these mechanisms be distinguished experimentally? As the final parameter values are so similar for the first two binding steps, discrimination is essentially impossible at low oxygen concentration levels (less than 100 microM with 50 microM hemoglobin). Discrimination becomes possible at higher oxygen concentrations, but high resolution in time and concentration amplitude are required. Much depends upon the differences in molar extinction coefficients of components over the accessible wave length range. Some of these values are as yet unknown or not known to a sufficient precision. Nevertheless, distinction between mechanistic alternatives is possible in principle.

Hemoglobins↗
PubMed1997

Short-lived intermediates in aspartate aminotransferase systems.

The kinetics of the reaction of aspartate aminotransferase with erythro-beta-hydroxy-aspartate, in which rapid mixing is followed (upon reaching a suitable stationary state) by a very fast temperature jump, is numerically simulated. Values for rate constants are used to the extent known, otherwise estimated. It is shown that reaction steps not resolvable by rapid mixing can be resolved by subsequent chemical relaxation. Since several absorption spectra of enzyme complexes overlap, use of a pH-indicator is investigated. When the pH-indicator is coupled to the protonic dissociation of free enzyme, the fast steps are easily detected in the chemical relaxation portion of the simulation. When the pH-indicator is coupled to the protonic dissociation of the (short-lived) quinoid intermediate, protonic dissociation is easily detectable in the stopped flow phase and in the chemical relaxation phase. Such transient protonic dissociation has not been detected experimentally, but is predicted by the simulation. When natural substrates are used, the magnitude of the rate constants makes it unlikely that transient proton dissociation can be detected by stopped flow alone, but a combination of stopped flow with very fast temperature perturbation allows detection of the transient proton through use of a suitable nonbinding pH-indicator. This is demonstrated by simulation for a specific case. Finally, an alternate mechanism is introduced and distinction of its kinetics from that of the original mechanism is demonstrated.

Aspartate Aminotransferases↗
PubMed1988

Cell sedimentation with gravity activation.

Murine monoclonal antibody T101 has been coupled to thinly polymer-coated heavy alloy particles (LaMn2Ge2). These conjugates are coupled to cultured cells of the human T-cell leukemia line RPMI 8402 (T8402). The sedimentation velocities of cells, of particles, and of cells with particles attached are measured. After determining the mean radii of cells, of particles, and of cells with particles attached, one may compute a mean number of 33 particles attached to a cell. Independently one may compute a mean number of 144 particles/cell for surface saturation. The Appendix handles the underlying theory in three parts: number of particles/cell, saturation number of particles/cell, and resolution for gravity activation. Regarding the latter, cell radii from 4 to 10 microns and particle radii from 0.01 to 1 micron are considered.

Cell Line↗
PubMed1987

Density of TEPC-15 plasmacytoma cells as function of size.

TEPC-15 plasmacytoma cells were observed under the microscope, as they sedimented at 1 G in Dulbecco's modified Eagle's medium. A mean density of 1.0422 g/cm3 (30 degrees C) was found. The density difference of smaller cells (15 micron diameter) was significantly higher than the density difference of larger cells (25 micron diameter). The ratio in density difference was close to 3.

Animals↗
PubMed1984

A stopped-flow investigation of calcium ion binding by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid.

The kinetics of calcium ion complexation by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) were investigated using the stopped-flow technique. This study was performed within the pH range 5.8 to 8.4. The reaction was found to be first order in EGTA and complex order in calcium, with an observed second-order rate constant (pH 7, T = 25 degrees C, ionic strength = 0.1 M) of about 1.5 X 10(6) M-1 s-1. The rate constant was independent of hydrogen ion concentration between pH 5.8 and 7.3; above pH 7.3 it increased in magnitude with increasing pH, and was 2.0 X 10(8) M-1 s-1 at pH 8.4. The rate constant at 16 and 38 degrees C (pH 6.8) was found to be 0.9 and 7 X 10(6) M-1 s-1, respectively. These data imply that calcium ion buffering by EGTA will require times on the order of milliseconds when EGTA is present in millimolar concentrations.

Binding Sites↗
PubMed1976

Computer-based modeling in the teaching of steady-state enzyme kinetics.

Equations are derived for the steady-state treatment of enzyme reactions with one type of inhibitor, consisting of one reaction cycle or two connected cycles. Computerized stimulation programs are described which are designed to acquaint the student thoroughly with the behavior of enzyme systems. The user has freedom in the choice of parameters for the system, including up to three product-producing rate constants for the two-cycle system.

Computer-Assisted Instruction↗
PubMed1968

Conformation and activity of chymotrypsin: the pH-dependent, substrate-induced proton uptake.

Hydrogen ion uptake by chymotrypsin during reversible binding of specific substrate is shown to be due to an ionizing group of the enzyme with a pK(apparent) approximately 9 in the free enzyme. This pK(apparent) is shifted to higher value in the enzyme-substrate complexes. Previous results indicating an equilibrium, controlled by this ionizing group, between active and inactive conformational forms of chymotrypsin are confirmed.

Amides↗