Dooha Kim

Effect of glycerol on protein aggregation: Quantitation of thermal aggregation of proteins from CHO cells and analysis of aggregated proteins

Abstract 1. 1.|In an effort to elucidate the mechanism by which glycerol protects cells against heat killing, the effects of glycerol on heat-induced protein aggregation were studied by employing a cell free system containing cytosolic proteins from CHO cells. 2. 2.|Heat-induced unfolding and subsequent aggregation of cytosolic proteins were quantified by a novel turbidimetric assay system utilizing rhodanese (RDN) in 6 M guanidinium chloride. The turbidity caused by protein-RDN aggregation showed a correlation to the amount of aggregated proteins. 3. 3.|Aggregation of the proteins was suppressed by glycerol present during the heating in concentration up to 15% (w/v). Glycerol suppressed protein aggregation at various heat doses (0–30 min at 45.5°C) and temperatures (42–45°C), implying that glycerol stabilizes proteins against thermal denaturation. 4. 4.|The aggregated proteins were analyzed by an SDS-PAGE and individual proteins were quantified. 5. 5.|An unknown 28 kDa protein and a 70 kDa protein showed the most pronounced change in aggregation after 30 min heating at 45.5°C. The 28 kDa protein showed a 5.7-fold increase in control, but only 1.9-fold increase in 10% glycerol. The 70 kDa protein showed a 6.8-fold increase in control, while a 3.5-fold increase in 10% glycerol. 6. 6.|Other proteins such as 24, 33, 43, 46, 50, and 55 kDa protein showed 28, 38, 55, 39, 30, and 1% respective reduction in aggregation by 10% glycerol. 7. 7.|These results demonstrate that glycerol affords a differential heat protection for each protein.

Electroporation of extraneous proteins into CHO cells: Increased efficacy by utilizing centrifugal force and microsecond electrical pulses☆

A novel electroporation system employing an oscillating electric pulse and centrifugal force was used to introduce extraneous proteins into CHO cells. Following the electrical pulse, the compression and subsequent rebound induced by the centrifugal acceleration and deceleration, respectively, enhanced protein uptake, presumably by a hydrodynamic pumping of extracellular solutions through the permeabilized membrane. Protein uptake was quantitated by measuring the amount of radiolabeled, extraneous, CHO proteins introduced into unlabeled CHO cells. The amount of protein introduced into electroporated CHO cells was enhanced up to four-fold by a combination of electric pulse and centrifugal force compared to that introduced by electric pulse only. The optimum gradient of centrifugal force (GCF, temporal change of centrifugal force) was 590 and -470 g/s during acceleration and deceleration, respectively. The optimum electric field was 5 kV/cm with a 30-microsecond pulse length. At this optimum electroporation condition, approximately 5 pg of proteins (up to 200 kDa molecular weight) were introduced per CHO cell. These same settings also permitted electroporation of other membrane impermeable substances including propidium iodide and ethidium bromide. Introduction of extraneous materials into the cytoplasm during electroporation was confirmed by the ability of anti alpha-tubulin to stain the microtubules and propidium iodide and ethidium bromide to stain the nuclei. Cells electroporated with optimum device settings exhibited no significant decrease in clonogenic survival.

Employment of a turbidimetric assay system to study the biochemical role of HSP70 in heat-induced protein aggregation

Abstract 1. 1.|The biochemical role of the 70 kDa constitutive heat shock protein (cHSP70) in heat-induced protein aggregation was studied by a turbidimetric assay system. 2. 2.|When rhodanese (2.5 mg/ml) was diluted 100-fold into heated cytoplasmic proteins (500 μg/ml), turbidity caused by protein-rhodanese aggregation rapidly increased and reached maximum within 5 min. 3. 3.|The amount of protein aggregates was reduced 2-fold by adding cHSP70 (20 μg/ml), but not bovine serum albumin, during heating at 4.5°C. Similar results were observed when cHSP70 was added to cytoplasmic proteins from cells heated at 4.5°C for 10 min. 4. 4.|The reduction of protein aggregation by treatment with HSP70 was dependent on ATP content. The aggregation was also significantly reduced when cytoplasmic proteins from thermotolerant cells that contain high level of HSP70 were used. The reduction effects were markedly decreased by adding anti-SHp70 antibody (0.1 mg/ml) to cytoplasms before heating at 45.5°C for 20 min. 5. 5.|Thus, data from the turbidimetric assay system indicate that HSP70 plays an important role in reduction of protein aggregation, and perhaps dissociation of protein aggregates in the presence of ATP.

Employment of a turbidimetric assay system to measure heat-induced protein aggregation

1. n1.|We developed a turbidimetric assay system for quantitation of heat-induced protein aggregation which is presumably caused by protein denaturation. n n2. n2.|Rhodanese in 6 M guanidinium chloride was employed in the assay system, because this protein recognizes hydrophobic sites on denatured proteins and aggregates. n n3. n3.|Turbidity caused by protein-rhodanase aggregation was recorded at 320 nm by using a u.v./VIS spectrophotometer. n n4. n4.|When heated, alcohol dehydrogenase (ADH) aggregates with rhodanese. The increase of ADH-rhodanese aggregation was correlated with the loss of enzymatic activity. n n5. n5.|These results indicated that the aggregation was proportional to the extent of ADH denaturation which assumingly caused the loss of ADH activity during heating at 45.5°C. n n6. n6.|Similar results were observed when cytosolic proteins from CHO cells were heated at 45.5°C. Heated cytosolic proteins promoted aggregation by complex formation with rhodanese. The aggregation increased with increasing heat dose. n n7. n7.|Therefore, the rhodanese assay system can be employed usefully to quantitate the protein aggregation after heat stress.

Differences in Thermotolerance Induced by Heat or Sodium Arsenite: Correlation between Redistribution of a 26-kDa Protein and Development of Protein Synthesis-Independent Thermotolerance in CHO Cells

In previous studies, we have demonstrated the differences in thermotolerance induced by heat and sodium arsenite (Lee et al., Radiat. Res. 121, 295-303, 1990). In this study, we investigated whether a 26-kDa protein might play an important role in evincing these differences. Chinese hamster ovary (CHO) cells treated for either 1 h with 100 microM sodium arsenite (ARS) or 10 min at 45.5 degrees C became thermotolerant to a test heat treatment at 43 degrees C administered 6 or 12 h later, respectively. After the test heating at 43 degrees C for 1.5 h, the level of 26-kDa protein in the nucleus was decreased by 92% in nonthermotolerant cells, 78% in ARS-induced thermotolerant cells, and 3% in heat-induced thermotolerant cells. Inhibiting protein synthesis with cycloheximide (CHM, 10 micrograms/ml) after ARS treatment eliminated thermotolerance to 43 degrees C and delayed restoration of the 26-kDa protein in the nucleus. In contrast, CHM neither prevented the development of thermotolerance nor inhibited the restoration of the 26-kDa protein in heat-induced thermotolerant cells. However, when cells were exposed to cold (4 degrees C), immediately after initial heating, restoration of the 26-kDa protein and development of thermotolerance did not occur. These results demonstrate a good correlation between the restoration and/or the presence of this 26-kDa protein and the development of protein synthesis-independent thermotolerance.

Alteration of heat sensitivity by introduction of HSP70 or anti-HSP70 antibody in cho cells

Abstract 1. 1.An electroporation system employing an oscillating electric pulse and centrifugal force was used to introduce HSP70 or anti-HSP70 antibody into Chinese hamster ovary cells. 2. 2.There was a 1.5- or 1.9-fold increase in the amount of intracellular HSP70 by electroporation in the presence of 0.75 or 1.5 mg/ml, respectively. 3. 3.Cells electroporated with HSP70 became resistant to hyperthermic killing; i.e. the survival increased 6–7-fold from 1.2 × 10 −2 to 7–8 × 10 −2 heating at 45.5°C for 20 min. 4. 4.In contrast, introduction of 1 mg/ml anti-HSP70 antibody sensitized cells to hyperthermic killing; i.e. the reciprocal of the survival slope was decreased from 1.68 to 1.25 min. 5. 5.Thus, our results support the hypothesis that HSP70 plays an important role in heat resistance.

Purification and characterization of a 26 kDa protein that shows heat-induced reduction and restoration in the nuclei of Chinese hamster ovary cells

1. n1. We previously reported that heat-induced preferential reduction and restoration of a 26 kDa protein in the nucleus of CHO cells might be correlated to thermotolerance development [Lee et al. (1989) J. Cell. Physiol.141, 510–516; Lee et al. (1990) J. Cell. Physiol.145, 324–332; Lee et al. (1991) Radiat. Res.127, 325–334]. n n2. n2. To understand further possible biological function of the 26 kDa protein in the cell, we purified and characterized the 26kDa protein to homogeneity in two-dimensional gel electrophoresis. DEAE-ion exchange, Sephadex G-50, hydroxylapatite and Reactive Yellow 3 affinity chromatography were employed in the purification. n n3. n3. The 26 kDa protein bound tightly to dye-ligand affinity matrix such as Reactive yellow 3, Cibacron Blue 3GA and Reactive Red 120-agarose, and it could be eluted by higher than 1 M NaCl concentration or by either NAD or NADP. n n4. n4. The protein probably binds some nucleotides, and has a hydrophobic nature with an extremely basis PI value of about 10. The protein was precipitated at the concentration of at least 1 mg per ml in 10 mM Tris-HCl, pH 7.5.