Previously reported observations have shown that trans-trans farnesol inhibits incorporation of choline into phosphatidylcholine and reduces the growth rate of the human acute leukemia CEM-C1 cell ...line (Melnykovych, G., Haug, J.S. and Goldner, C.M. (1992) Biochem. Biophys. Res. Commun. 186, 543–548). These findings have now been followed up in order to establish a relationship between the inhibition of phosphatidylcholine synthesis and the ensuing cell shrinkage and cell death which takes place at higher concentrations of farnesol or upon long incubation. The present results show that after incubation in the presence of farnesol the cells decrease in viability. Their nuclear DNA becomes fragmented at internucleosomal linker regions, showing characteristic pattern of bands at 180 to 200 base-pair intervals. This farnesol-induced effect was also demonstrated by flow cytometry by staining the cellular DNA with propidium iodide and was partially reversible with phosphatidylcholine.
Six neoplastically-derived cell lines and three cell lines derived from normal tissues were compared for their sensitivity to isoprenoid trans-trans farnesol. Assays of cell numbers and of protein ...concentrations per culture revealed greater sensitivity of neoplastic cells than of the normal cells. Similar differences were obtained from the comparison of incorporation of methyl-3Hcholine into cellular lipids, with neoplastic cells showing greater inhibition than normal cells.
Contributions of the C-terminus toward the conformation and activity of phosphatidylinositol transfer protein (PITP) were studied by comparing properties of the 271 amino acid, full-length protein, ...PITP(1−271), and two truncated species, PITP(1−259) and PITP(1−253). Using recombinant proteins and an in vitro phospholipid transfer assay with phosphatidylcholine vesicles, the activities of PITP(1−271) and PITP(1−259) were identical, while the activity of PITP(1−253) was almost totally abolished. By most physical and chemical criteria, however, PITP(1−259) and PITP(1−253) were virtually indistinguishable and differed significantly from the full-length protein. Results of second derivative analysis of absorbance spectra were consistent with an additional two Tyr residues being exposed to the solvent in PITP(1−259) and PITP(1−253) in comparison to PITP(1−271). Only one out of four Cys residues in PITP(1−271) reacted with dithiobisnitrobenzoic acid, while two Cys residues were accessible in both truncated species. Quenching of intrinsic Trp fluorescence by acrylamide demonstrated an increase in exposure of Trp residues in both PITP(1−259) and PITP(1−253); binding of the fluorescence probe 1,8-ANS to these proteins was also significantly higher compared to PITP(1−271). These results describe a more relaxed overall tertiary structure brought about by the C-terminal truncations. This altered structure did not affect the stability of the truncated proteins, as indicated by equilibrium unfolding in guanidinium chloride. Refolding of the denatured PITP(1−259), however, was considerably slower than that of full-length PITP. Our study suggests a critical role of the C-terminal residues 254−259 in transfer activity of PITP. Residues 260−271, on the other hand, appear to be more important for the rapid folding and maintenance of a compact native conformation of the protein.
In order to prevent kidney stones and nephrolithiasis in hyperoxaluria, a new treatment that specifically reduces oxalate production and therefore urinary oxalate excretion would be extremely ...valuable. Pyridoxamine(PM) could react with the carbonyl intermediates of oxalate biosynthesis, glycolaldehyde and glyoxylate, and prevent their metabolism to oxalate. In PM treated rats, endogenous urinary oxalate levels were consistently lower and became statistically different from controls after 12 days of experiment. In ethylene glycol-induced hyperoxaluria, PM treatment resulted in significantly lower (by ~50%) levels of urinary glycolate and oxalate excretion compared to untreated hyperoxaluric animals, as well as in a significant reduction in calcium oxalate crystal formation in papillary and medullary areas of the kidney. These results, coupled with favorable toxicity profiles of PM in humans, show promise for the therapeutic use of PM in primary hyperoxaluria and other kidney stone diseases.
The mammalian mitochondrial enzyme, rhodanese, can form stable complexes with the Escherichia coli chaperonin GroEL if it is either refolded from 8 M urea in the presence of chaperonin or is simply ...added to the chaperonin as the folded conformer at 37 degreesC. In the presence of GroEL, the kinetic profile of the inactivation of native rhodanese followed a single exponential decay. Initially, the inactivation rates showed a dependence on the chaperonin concentration but reached a constant maximum value as the GroEL concentration increased. Over the same time period, in the absence of GroEL, native rhodanese showed only a small decline in activity. The addition of a non-denaturing concentration of urea accelerated the inactivation and partitioning of rhodanese onto GroEL. These results suggest that the GroEL chaperonin may facilitate protein unfolding indirectly by interacting with intermediates that exist in equilibrium with native rhodanese. The activity of GroEL-bound rhodanese can be completely recovered upon addition of GroES and ATP. The reactivation kinetics and commitment rates for GroEL-rhodanese complexes prepared from either unfolded or native rhodanese were identical. However, when rhodanese was allowed to inactivate spontaneously in the absence of GroEL, no recovery of activity was observed upon addition of GroEL, GroES, and ATP. Interestingly, the partitioning of rhodanese and its subsequent inactivation did not occur when native rhodanese and GroEL were incubated under anaerobic conditions. Thus, our results strongly suggest that the inactive intermediate that partitions onto GroEL is the reversibly oxidized form of rhodanese.
For the chaperonin substrates, rhodanese, malate dehydrogenase (MDH), and glutamine synthetase (GS), the folding efficiencies, and the lifetimes of folding intermediates were measured with either the ...nucleotide-free GroEL or the activated ATP·GroEL·GroES chaperonin complex. With both nucleotide-free and activated complex, the folding efficiency of rhodanese and MDH remained high over a large range of GroEL to substrate concentration ratios (up to 1:1). In contrast, the folding efficiency of GS began to decline at ratios lower than 8:1. At ratios where the refolding yields were initially the same, only a relatively small increase (1.6-fold) in misfolding kinetics of MDH was observed with either the nucleotide-free or activated chaperonin complex. For rhodanese, no change was detected with either chaperonin complex. In contrast, GS lost its ability to interact with the chaperonin system at an accelerated rate (8-fold increase) when the activated complex instead of the nucleotide-free complex was used to rescue the protein from misfolding. Our data demonstrate that the differences in the refolding yields are related to the intrinsic folding kinetics of the protein substrates. We suggest that the early kinetic events at the substrate level ultimately govern successful chaperonin-substrate interactions and play a crucial role in dictating polypeptide flux through the chaperonin system. Our results also indicate that an accurate assessment of the transient properties of folding intermediates that dictate the initial chaperonin-substrate interactions requires the use of the activated complex as the interacting chaperonin species.
The significance of noncovalently bound phospholipid as a structural component of phosphatidylinositol transfer protein (PITP) and its role in acquisition and maintenance of the native conformation ...of the protein have been addressed by studying the refolding of PITP after exposure to 6 M guanidinium chloride (GdnCl). Protein conformations were characterized by (1) the intrinsic tryptophan fluorescence, circular dichroism, and absorbance spectroscopy, (2) the degree of binding of the fluorescent probe 1,8-ANS, and (3) limited proteolytic digestion. When the GdnCl concentration was reduced 100-fold by rapid dilution at 25 °C, practically all of the native transfer activity was regained within 20 min. Endogenous phospholipid demonstrated a strong interaction with the native PITP. Separation of the phospholipid from the protein by chromatography on a lipophilic matrix was achieved only under denaturing conditions and resulted in spontaneous oxidation of the apo-protein, accompanied by almost complete loss of recoverable transfer activity. Under reducing conditions, however, apo-PITP recovered more than 80% of the native transfer activity and was similar to holo-PITP in the kinetics of phospholipid transfer. Renatured apo-PITP demonstrated a significant relaxation of the tertiary structure, compared to native and renatured holo-PITP. Incubation of apo-PITP with phospholipid vesicles resulted in a more compact protein conformation. We conclude that the polypeptide can spontaneously fold to a native-like conformation, sufficient for interaction with a lipid membrane and acquisition of a phospholipid ligand. Binding of a phospholipid ligand brings about the final adjustments of protein conformation to the more compact native structure.
Rat phosphatidylinositol transfer protein (PITP) is a 32 kDa protein containing 271 amino acids. It is involved in a number of cell functions including secretion and cell signaling. To further ...characterize structure/activity relationships of PITP, two C-terminal truncated derivatives, PITP(1-259) and PITP(1-253), were produced in Escherichia coli and purified to homogeneity. PITP(1-259) had transfer activity equal to 30-40% to that of native PITP in transfer of either phosphatidylcholine (PC) or phosphatidylinositol (PI) when transfer was measured using 95/5 mol% PC/PI donor and acceptor vesicles; PITP(1-253) had only slight transfer activity, even under the most favorable assay conditions. Thus, amino acids 254-258 are critical for transfer activity. The transfer activity of PITP(1-259) was strongly dependent on the composition of the donor and acceptor vesicles. With 100 mol% PC donor and acceptor vesicles, PITP(1-259) transfer activity ranged from 70 to 100% to that of PITP. The presence of 2 mol% phosphatidic acid (PA) in either donor or acceptor vesicles reduced transfer activity to between 10 and 20% that of full-length PITP under the same conditions. If both donor and acceptor contained 2% PA, PITP(1-259) was essentially inactive, though the activity of PITP was not affected significantly under these conditions. PITP(1-253) and PITP(1-259) bind much more avidly to vesicles than does PITP, and this enhanced binding reflects increased electrostatic interactions. Thus, the C-terminal residues modulate the affinity of PITP for vesicles and the efficiency of phospholipid transfer.
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