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Biomedical subjects

J M Engasser

Publications and source records attributed to J M Engasser.

10 recordsLinked to original sources
PubMed1992

Comparison of specific rates of hybridoma growth and metabolism in batch and continuous cultures.

For the mouse hybridoma cell line VO 208, kinetics of growth, consumption of glucose and glutamine, and production of lactate, ammonia and antibodies were compared in batch and continuous cultures. At a given specific growth rate, different metabolic activities were observed: a 40% lower glucose and glutamine consumption rate, but a 70% higher antibody production rate in continuous than in batch culture. Much higher metabolic rates were also measured during the initial lag phase of the batch culture. When representing the variation of the specific antibody production rate as a function of the specific growth rate, there was a positive association between growth and antibody production in the batch culture, but a negative association during the transient phase of the continuous culture. The kinetic differences between cellular metabolism in batch and continuous cultures may be the result of modifications in the physiology and metabolism of cells which, in continuous cultures, were extensively exposed to glucose limitations.

Ammonia↗
PubMed1991

Influence of inoculum age on hybridoma culture kinetics.

To determine the influence of the inoculum age on the kinetics of hybridoma growth and metabolism, spinner flasks have been inoculated with cells previously propagated in T flasks for 43, 52, 62 and 71 hr respectively. Increasing the age of the inoculum is found to result in a longer lag phase, in a lower maximum specific growth rate and in a reduced maximal cell density. During the growth phase specific rates of glucose and glutamine uptake and of ammonia and lactate production are similar. However, with the older inoculum, much higher metabolic activities are observed during the lag phase. The production of antibodies is delayed with increasing inoculum age, but the final antibody concentrations are similar, which indicates a higher specific antibody production rate when inoculating with older cells.

Ammonia↗
PubMed1990

Gamma-glutamyltransferase from human hepatoma cell lines: purification and cell culture of HepG2 on microcarriers.

After screening different human hepatoma cell lines, we observed that both HepG2 and PLC/PRF/5 naturally produced large amounts of gamma-glutamyltransferase. We optimized HepG2 cell culture conditions and observed that higher cell densities were obtained when cells were cultured on microcarriers, particularly when Cytodex 3 was used and that cell growth was optimal when DMEM, the basic medium, was supplemented with 5% fetal calf serum and 6 mmol/l glutamine. These culture conditions allowed us to produce the highest amounts of GGT after about 150 h of culture. The GGT obtained from HepG2 cells was partially purified and some of its physico-chemical properties characterized. Successive Con A gel chromatography separated the activity into two peaks, suggesting that GGT from HepG2 is not uniformly glycosylated. Papain-treated HepG2 GGT showed a Mr of about 120 kDa and migrated as a single-chain protein in SDS-PAGE. Immunological and kinetic properties of the GGT were similar to other human GGTs (liver, kidney and serum). It appears that HepG2 GGT could be a source for the preparation of a human enzyme reference material.

Blotting, Western↗
PubMed1978

A fast evaluation of diffusion effects on bound enzyme activity.

As the kinetic behavior of bound enzymes is frequently affected by substrate diffusion between the bulk solution and the catalytic sites, a fast and simple method is proposed to detect and, subsequently, to remove diffusion effects on measured enzymic activities. The procedure makes use of the effectiveness factor concept and essentially involves the direct determination on two diagrams of the magnitude of both external and internal diffusion limitations. It requires a prior estimation of the volume and external surface area of the matrix, of the substrate external transport coefficient and internal diffusivity, and of the intrinsic Michaelis constant of the bound enzyme. However, it does not necessitate the knowledge of the quantity of bound enzyme. The two basic graphs have been calculated for Michaelis-Menten kinetics. They can also be used to evaluate diffusional effects on two-substrate reactions, as illustrated with previously published data.

Diffusion↗
PubMed1977

Kinetics of soluble and collagen-bound aspartate aminotransferase: diffusional effects with a two-substrate enzymatic reaction.

The kinetic properties of aspartate aminotransferase covalently bound to collagen are compared to those of the free enzyme. In the bound state, the enzyme exhibits a greater affinity for glutamate, but a lower affinity for oxalacetate. In order to assess precisely the contribution of diffusional limitations on the heterogeneous enzyme kinetics, a simple modeling of diffusional effects on a two-substrate enzymatic reaction is developed. According to this quantitative analysis, diffusional limitations for oxalacetate alone account for the increased and decreased enzyme affinities toward its two substrates. Consequently, coupling of the enzyme to collagen does not significantly affect its intrinsic kinetic properties.

Aspartate Aminotransferases↗
PubMed1977

Comparison of intrinsic stabilities of free and bound enzymes by graphical removal of diffusional effects.

The enhanced stability usually exhibited by enzymes after immobilization may be attributed either to a stabilization effect of the solid matrix on the bound enzyme molecule or to the influences of diffusional limitations on the observed activity. To allow the comparison of the intrinsic statilities of free and bound enzymes a simple graphical procedure for the removal of external diffusional effects of stability curves is described. It is based on the determination of substrate concentration differences between the enzyme micro- and macroenvironment. Application of the method to aspartate aminotransferase bound to collagen membranes indicates that diffusional limitations for oxaloacetate are partly responsible for the observed stability enhancement. Comparison of the graphically obtained intrinsic profile with the stability curve of the soluble enzyme further demonstrate that the binding itself greatly increases the stability of aspartate aminotransferase.

Aspartate Aminotransferases↗
PubMed1975

Electrostatic effects on the kinetics of bound enzymes.

1. The effect of the interaction between the charged matrix and substrate on the kinetic behaviour of bound enzymes was investigated theoretically. 2. Simple expression is derived for the apparent Km. 3. The apparent Km can only be used for the characterization of the electrostatic effect of the ionic strength does not vary with the substrate concentration. 4. The deviations from Michaelis-Menton kinetics are graphically illustrated for cases when the ionic strength varies with the substrate concentration. 5. The inhibition of the bound enzyme by a charged inhibitor at constant ionic strength is characterized by an apparent Ki. 6. When both the inhibitor concentration and the ionic strength change there is no apparent Ki, and the inhibition profile is graphically illustrated for this case. 7. Under certain conditions the electrostatic effects manifest thenselves in a sigmoidal dependence of the enzyme activity on the concentration of the substrate or inhibitor.

Binding Sites↗