Structure of alanine dehydrogenase from Archaeoglobus: active site analysis and relation to bacterial cyclodeaminases and mammalian mu crystallin.

The hyperthermophilic archaeon Archaeoglobus fulgidus contains an L-Ala dehydrogenase (AlaDH, EC 1.4.1.1) that is not homologous to known bacterial dehydrogenases and appears to represent a previously unrecognized archaeal group of NAD-dependent dehydrogenases. The gene (Genbank; TIGR AF1665) was annotated initially as an ornithine cyclodeaminase (OCD) on the basis of strong homology with the mu crystallin/OCD protein family. We report the structure of the NAD-bound AF1665 AlaDH (AF-AlaDH) at 2.3 A in a C2 crystal form with the 70 kDa dimer in the asymmetric unit, as the first structural representative of this family. Consistent with its lack of homology to bacterial AlaDH proteins, which are mostly hexameric, the archaeal dimer has a novel structure. Although both types of AlaDH enzyme include a Rossmann-type NAD-binding domain, the arrangement of strands in the C-terminal half of this domain is novel, and the other (catalytic) domain in the archaeal protein has a new fold. The active site presents a cluster of conserved Arg and Lys side-chains over the pro-R face of the cofactor. In addition, the best ordered of the 338 water molecules in the structure is positioned well for mechanistic interaction. The overall structure and active site are compared with other dehydrogenases, including the AlaDH from Phormidium lapideum. Implications for the catalytic mechanism and for the structures of homologs are considered. The archaeal AlaDH represents an ancient and previously undescribed subclass of Rossmann-fold proteins that includes bacterial ornithine and lysine cyclodeaminases, marsupial lens proteins and, in man, a thyroid hormone-binding protein that exhibits 30% sequence identity with AF1665.

Effect of external pH on heat sensitization by local anesthetics.

The sensitization of asynchronous Chinese hamster ovary (CHO) cells to 43 degrees C by procaine, lidocaine, and tetracaine was examined, with the pH of the medium carefully controlled at approximately pH7 and 8. Thermal enhancement factors for 43 degrees C were calculated for the surviving fraction of 0.01. The thermal enhancement factors of the local anesthetics were increased at approximately pH8 by factors of 2-12, depending on the local anesthetic and its concentration. The concentration of the uncharged free base form of the local anesthetic in the culture medium correlated positively with the thermal enhancement factors of each local anesthetic and in a near linear fashion with the thermal enhancement factors for procaine. However, the concentration of the cationic form of the local anesthetics in the growth medium did not correlate with heat sensitization. We conclude that the ability of local anesthetics to sensitize cells to heat killing is dramatically influenced by the extracellular pH, with increased sensitization at the more basic pH's. Secondly, it is the extracellular concentration of the free base form of the local anesthetics that correlates with heat sensitization.

Response of the microtubular cytoskeleton following hyperthermia as a prognostic indicator of survival of Chinese hamster ovary cells.

PURPOSE: The response of the microtubular (MT) cytoskeleton to hyperthermia was assessed as a prognostic indicator of cytotoxicity. METHODS AND MATERIALS: Heat-induced collapse and subsequent recovery of the MT system were compared with survival for both nonthermotolerant (NT) and thermotolerant (TT) G1 populations of Chinese hamster ovary (CHO) cells. The response of the MT system was monitored using immunofluorescence staining. The G1 populations of NT and TT cells were heated by submersion in 45.0 and 43.0 degrees C waterbaths. RESULTS: Heat-induced perinuclear collapse of the MT system did not correlate with survival for the NT and TT populations. However, recovery of the organization of the MT cytoskeleton was correlatable with survival. The regression line of survival plotted as a function of MT recovery is fit by: y = -0.43 + 1.03x, r2 = 0.95 (p < 0.0005). CONCLUSION: Restoration of the organization of the MT cytoskeleton following hyperthermia may be used as a prognostic indicator of survival of CHO cells heated in G1.

Caffeine sensitization of Chinese hamster ovary cells to heat killing.

The effects of the methylxanthine, caffeine, on heat sensitization was investigated using Chinese hamster ovary (CHO) cells. Caffeine sensitized CHO cells to heat killing by reducing both the shoulder and the slope of the 44 degrees C survival curve. Heating was performed in suspension by addition of cells to preheated spinner flasks containing caffeine. Changes in intracellular free calcium levels, [Ca2+]i, were measured at 37 degrees C using the luminescent probe aequorin. Caffeine (1-5 mM) induced a transient increase in [Ca2+]i at 37 degrees C. The transient increase in [Ca2+]i was reduced 15-fold when 5 mM caffeine was added to aequorin-loaded cells suspended in Ca(2+)-free Hanks' balanced salt solution. However, 5 mM caffeine sensitized the cells to the same extent when they were suspended in either Ca(2+)-containing or Ca(2+)-free Hanks' balanced salt solution. The mechanism of heat sensitization by caffeine is still unknown.

Administration of bovine anti-IGF-1 immunoglobulin to dietary protein deficient rats alters dietary intake and plasma IGF-1 binding profiles, but does not affect change in body mass.

The potential of antibodies raised against insulin-like growth factor-1 (IGF-1) as a treatment to enhance the anabolic actions of IGF-1 has been demonstrated in both rodent and ruminant models. We investigated whether treatment of genetically normal rats with anti-IGF-1 immunoglobulin (Ig, raised in cattle) would enhance growth and if anti-IGF-1 Ig treatment would ameliorate live-weight loss in genetically normal rats offered a severely protein-restricted diet. Scatchard analysis was used to characterise ammonium sulphate precipitated bovine anti-IGF-1 Ig. Anti-IGF-1 Ig binding to 125I-IGF-1 yielded an almost linear Scatchard plot, with a Hill co-efficient of 0.951 ± 0.012, indicating a single class of IGF-1 binding sites. The affinity of anti-IGF-1 Ig for IGF-1 was 2.14 ± 0.66 × 109 l/mol. The non-immune Ig preparation did not bind IGF-1. Rats were offered either a diet with a normal protein level (20%) or protein restricted (4% protein), and each dietary group was further treated with twice-daily i.p. injections of either diluent phosphate buffered saline, non-immune Ig or anti-IGF-1 Ig. Dietary protein level had a significant effect on live-weight gain, but there was no effect of non-immune Ig or anti-IGF-1 Ig on live-weight gain. Treatment with anti-IGF-1 Ig prevented the significant depression of cumulative dietary intake observed in diluent, and non-immune Ig treated groups offered the 4% protein diet. The cumulative dietary intake of the anti-IGF-1 Ig treated, 4% dietary protein group did not differ significantly from those of the groups offered the 20% protein diet. In addition, within the 4% dietary protein group, rats treated with non-immune Ig exhibited a cumulative feed intake that was intermediate between that of the diluent treated and anti-IGF-1 Ig treated groups (P < 0.05). Size exclusion chromatography was used to characterise in vitro 125I-IGF-1 binding in end-point plasma from each treatment group. In comparison to control groups, anti-IGF-1 Ig treatment resulted in substantially increased 125I-IGF-1 binding in the 30 to 40 kDa region and a concomitant reduction in elution of free 125I-IGF-1. Protein restriction markedly depressed IGF-1 binding at ∼150 kDa in the plasma of diluent and non-immune Ig treated groups. Anti-IGF-1 Ig treatment was effective in preventing this decrease in ∼150 kDa binding. Thus, anti-IGF-1 Ig appears to have a beneficial effect on dietary intake in protein-restricted rats, which is associated with induced changes in IGF-1 binding profiles in plasma.

Thermal response of synchronous CHO cells with different shapes.

This study examines the differential heat sensitivity of rounded suspension cells versus flattened monolayer cells. G1 populations of two different Chinese hamster ovary lines were used to eliminate possible cell cycle artifacts. The cell populations were heated at 43 and 45 degrees C. In all cases, cells treated in suspension were less sensitive to heat killing than cells treated as monolayers.

Protocol for freezing thermotolerant cells and maintaining thermotolerance following thawing.

Two independent laboratories have demonstrated that suspension-grown, Chinese hamster ovary (CHO) cells can be made thermotolerant, frozen and subsequently thawed such that they still express thermotolerance. Thermotolerance was determined as the ability to protect cells against hyperthermic cell killing (colony formation assay) and the ability to reduce protein aggregation within the nuclei of heated cells. Cells were frozen either following development of full or partial thermotolerance. In the former case frozen cells maintained thermotolerance upon thawing and in the latter case cells subsequently developed full thermotolerance following thawing and incubation at 37.0 degrees C. After thawing, frozen cells displayed a temporal course of thermotolerance development and decay that was similar to that for never-frozen cells. Success was obtained using either asynchronous or synchronous cell populations, and the heat sensitivity of the cells was not altered by the freezing procedure. The experimental results demonstrate the plausibility of utilizing a frozen stock of thermotolerant cells to make thermotolerance experiments more convenient.

Heat protection by deuterium oxide of heat sensitive and wild-type Chinese hamster ovary cells.

The ability of deuterium oxide (D2O) to protect a heat-sensitive and thermotolerance-impaired Chinese hamster ovary (CHO) mutant cell line, HS-36 (Harvey and Bedford 1988), from heat killing was examined and compared to the parent CHO 10B cell line (WT). Both non-thermotolerant (NT) and thermotolerant (TT) G1 populations were examined. D2O differentially protected the NT cell lines from heat killing, with thermal protection ratios (D0) of 2 x 5 and 4 x 3 for HS-36 and WT cells, respectively. D2O provided additional protection to TT cells, but now protected the TT HS-36 cells more than the TT WT cells when the thermal protection ratios of TT cells are compared with those of NT cells (1.15 versus 0.82). The differential protection from heat of the mutant and wild-type lines by D2O may be useful in studies of induced lesions in proteinaceous cellular systems (e.g. the nuclear matrix, cytoskeleton and plasma membrane) using these two paired cell lines.

Effects of verapamil and diltiazem on hyperthermic cell death in CHO cells.

Non-toxic concentrations of verapamil (0.05 and 0.075 mM) and diltiazem (0.10 and 0.25 mM) sensitize CHO cells to heat killing at 44 degrees C. These drugs sensitize by reducing both the shoulder and the slope of the exponential survival curve. Exposure to verapamil or diltiazem in conjunction with the heat sensitizer procaine HCl at 44 degrees C reduced cell survival below that observed for procaine plus heat. The mechanism by which verapamil and diltiazem sensitize CHO cells to heat killing is not understood.