R. Janke

Measurement of Key Metabolic Enzyme Activities in Mammalian Cells Using Rapid and Sensitive Microplate-Based Assays

Sensitive microplate‐based assays to determine low levels of key enzyme activities in mammalian cells are presented. The enzyme platform consists of four cycling assays to measure the activity of 28 enzymes involved in central carbon and glutamine metabolism. The sensitivity limit of all cycling assays was between 0.025 and 0.4 nmol product. For the detection of glutaminase activity, a new glutamate cycle system involving the enzymes glutamate dehydrogenase and aspartate transaminase was established. The relative standard deviation of the method was found to be 1.7% with a limit of detection of 8.2 pmol and a limit of quantitation of 24.8 pmol. Hence, cell extracts could be highly diluted to reduce interferences caused by other components in the extract, which in addition minimized underestimates or overestimates of actual enzyme activities. Since substrate concentrations could be maintained at a nearly constant level throughout the assay product accumulation during the reaction was low, which minimized product inhibition. As an example, the enzyme platform was used to investigate maximum enzyme activities of stationary‐phase MDCK cells grown in serum‐containing GMEM medium as typically used in influenza vaccine production. Biotechnol. Bioeng. 2010;107: 566–581.

Metabolic adaptation of MDCK cells to different growth conditions: Effects on catalytic activities of central metabolic enzymes

Lactate and ammonia are the most important waste products of central carbon metabolism in mammalian cell cultures. In particular during batch and fed‐batch cultivations these toxic by‐products are excreted into the medium in large amounts, and not only affect cell viability and productivity but often also prevent growth to high cell densities. The most promising approach to overcome such a metabolic imbalance is the replacement of one or several components in the culture medium. It has been previously shown that pyruvate can be substituted for glutamine in cultures of adherent Madin–Darby canine kidney (MDCK) cells. As a consequence, the cells not only released no ammonia but glucose consumption and lactate production were also reduced significantly. In this work, the impact of media changes on glucose and glutamine metabolism was further elucidated by using a high‐throughput platform for enzyme activity measurements of mammalian cells. Adherent MDCK cells were grown to stationary and exponential phase in six‐well plates in serum‐containing GMEM supplemented with glutamine or pyruvate. A total number of 28 key metabolic enzyme activities of cell extracts were analyzed. The overall activity of the pentose phosphate pathway was up‐regulated during exponential cell growth in pyruvate‐containing medium suggesting that more glucose‐6‐phosphate was channeled into the oxidative branch. Furthermore, the anaplerotic enzymes pyruvate carboxylase and pyruvate dehydrogenase showed higher cell specific activities with pyruvate. An increase in cell specific activity was also found for NAD+‐dependent isocitrate dehydrogenase, glutamate dehydrogenase, and glutamine synthetase in MDCK cells grown with pyruvate. It can be assumed that the increase in enzyme activities was required to compensate for the energy demand and to replenish the glutamine pool. On the other hand, the activities of glutaminolytic enzymes (e.g., alanine and aspartate transaminase) were decreased in cells grown with pyruvate, which seems to be related to a decreased glutamine metabolism. Biotechnol. Bioeng. 2011;108: 2691–2704.

Effect of influenza virus infection on key metabolic enzyme activities in MDCK cells.

BackgroundInfluenza, or “flu”, is an upper respiratory tract infectioncaused by a virus belonging to the family ofOrthomyxo-viridae. Influenza can pose a serious risk to the health ofmainly the elderly, the very young, and to people suffer-ing from medical conditions (e.g. weak immune system).For example, seasonal influenza strains are fatal to morethan 50,000 people annually in the United States alone[1]. The most effective measure for preventing influenza-related morbidity and mortality is annual vaccination.Seasonal influenza vaccines are almost exclusively pro-duced using the traditional egg-based manufacturing pro-cess. However, the main limitation of egg-basedtechnology (especially in the case of a pandemic) is thetime-consuming production process (~6 months).Furthermore, people with serious egg allergy cannot bevaccinated when trace amounts of egg protein remain inthe final formulation. Therefore, new production pro-cesses using continuous cell lines for influenza vaccinemanufacturing are currently being established [2].Influenza viruses take advantage of the host cell meta-bolism to replicate their genetic material and to synthe-size viral proteins. The influenza virus particle consistsof three major parts: the ribonucleocapsid, the matrixprotein M1, and the envelope, which is derived from theplasma membrane of the host cell. The lipid bilayer con-tains the ion channel protein M2 and the immunogenicglycoproteins hemagglutinin and neuraminidase [3]. Thereplication cycle of influenza viruses including entry,uncoating, genome transcription and replication, assem-bly and release has been studied extensively with type Astrains [4]. So far, only few studies have characterizedthe influence of influenza infection on the centralcarbon metabolism of host cells [5]. Madin-Darbycanine kidney (MDCK) cells are considered a suitablesubstrate for cell culture-based influenza vaccine manu-facturing [2,6]. In this study, key metabolic enzymeactivities were analyzed in MDCK cells infected with aninfluenza A virus strain to improve our understandingof virus-host cell interaction and cell response.Materials and methodsAll chemicals and enzymes were purchased from Sigma(Taufkirchen, Germany) or Roche (Mannheim, Ger-many). Adherent MDCK cells obtained from theECACC (No. 84121903) were routinely cultured in 6-well plates containing 4 mL of GMEM-based medium(2 mM glutamine, 30 mM glucose, 10 % (v/v) fetal calfserum, 2 g/L peptone, 48 mM NaHCO

The influence of cell growth and enzyme activity changes on intracellular metabolite dynamics in AGE1.HN.AAT cells

Optimization of bioprocesses with mammalian cells mainly concentrates on cell engineering, cell screening and medium optimization to achieve enhanced cell growth and productivity. For improving cell lines by cell engineering techniques, in-depth understandings of the regulation of metabolism and product formation as well as the resulting demand for the different medium components are needed. In this work, the relationship of cell specific growth and uptake rates and of changes in maximum in vitro enzyme activities with intracellular metabolite pools of glycolysis, pentose phosphate pathway, citric acid cycle and energy metabolism were determined for batch cultivations with AGE1.HN.AAT cells. Results obtained by modeling cell growth and consumption of main substrates showed that the dynamics of intracellular metabolite pools is primarily linked to the dynamics of specific glucose and glutamine uptake rates. By analyzing maximum in vitro enzyme activities we found low activities of pyruvate dehydrogenase and pyruvate carboxylase which suggest a reduced metabolite transfer into the citric acid cycle resulting in lactate release (Warburg effect). Moreover, an increase in the volumetric lactate production rate during the transition from exponential to stationary growth together with a transient accumulation of fructose 1,6-bisphosphate, fructose 1-phosphate and ribose 5-phosphate point toward an upregulation of PK via FBP. Glutaminase activity was about 44-fold lower than activity of glutamine synthetase. This seemed to be sufficient for the supply of intermediates for biosynthesis but might lead to unnecessary dissipation of ATP. Taken together, our results elucidate regulation of metabolic networks of immortalized mammalian cells by changes of metabolite pools over the time course of batch cultivations. Eventually, it enables the use of cell engineering strategies to improve the availability of building blocks for biomass synthesis by increasing glucose as well as glutamine fluxes. An additional knockdown of the glutamine synthetase might help to prevent unnecessary dissipation of ATP, to yield a cell line with optimized growth characteristics and increased overall productivity.

Expression, purification, and characterization of a His6-tagged glycerokinase from Pichia farinosa for enzymatic cycling assays in mammalian cells

The GUT1 gene of the halotolerant yeast Pichia farinosa, encoding glycerokinase (EC 2.7.1.30), was expressed in Pichia pastoris. A purification factor of approximately 61-fold was achieved by a combination of nickel affinity and anion exchange chromatography. The specific activity of the final preparation was 201.6 units per mg protein with a yield of about 21%. A nearly homogeneous enzyme preparation was confirmed by SDS-polyacrylamide gels and mass spectrometry analysis. Glycerol stabilized the purified enzyme for long-term storage at -80°C. The pH and temperature optima were in the range of 6.5-7.0 and 45-50°C, respectively. ATP was the most effective phosphoryl group donor tested. Additionally, the enzyme phosphorylated glycerol also with ITP, UTP, GTP and CTP. The K(m) values of the enzyme for ATP and ITP were 0.428 and 0.845 mM, respectively. The kinetic properties of the enzyme with respect to UTP, GTP, and CTP suggested that glycerokinase exhibited negative cooperativity as double reciprocal plots showed a biphasic response to increasing nucleoside triphosphate concentrations. The application as a coupling enzyme in the determination of pyruvate kinase activity in cell extracts of Madin-Darby canine kidney cells showed good reproducibility when compared with a commercially available preparation of bacterial glycerokinase.