Determinants of cytochrome c pro-apoptotic activity. The role of lysine 72 trimethylation

Cytochrome c released from vertebrate mitochondria engages apoptosis by triggering caspase activation. We previously reported that, whereas cytochromes c from higher eukaryotes can activate caspases in Xenopus egg and mammalian cytosols, iso-1 and iso-2 cytochromes c from the yeast Saccharomyces cerevisiae cannot. Here we examine whether the inactivity of the yeast isoforms is related to a post-translational modification of lysine 72, N-epsilon-trimethylation. This modification was found to abrogate pro-apoptotic activity of metazoan cytochrome c expressed in yeast. However, iso-1 cytochrome c lacking the trimethylation modification also was devoid of pro-apoptotic activity. Thus, both lysine 72 trimethylation and other features of the iso-1 sequence preclude pro-apoptotic activity. Competition studies suggest that the lack of pro-apoptotic activity was associated with a low affinity for Apaf-1. As cytochromes c that lack apoptotic function still support respiration, different mechanisms appear to be involved in the two activities.     

Chromatographically distinguishable heme insertion isoforms of human hemopexin

Two spectroscopically distinct, non-interconverting forms of human hemopexin have been isolated by immobilized metal ion affinity chromatography and characterized spectroscopically. Form alpha (characterized by a bisignate Soret CD spectrum) and form beta (Soret CD characterized by a positive Cotton effect) exhibit different spectroscopic responses to addition of Zn2+ or Cu2+, yet both forms exhibit the same metal ion-induced decrease in Tm for the thermally induced release of the heme prosthetic group. Far UV-CD spectra indicate that the two isoforms possess essentially identical secondary structures, but their differential retention during metal ion affinity chromatography indicates slight differences in exposure of His residues on the protein surface. We propose that these observations result from the binding of heme in form beta with an orientation that differs from the crystallographically observed binding orientation for rabbit hemopexin by rotation of the heme prosthetic group by 180 degrees about the alpha-gamma meso-carbon axis and from interaction of metal ions at two separate binding sites.     

Self-association and chaperone activity of Hsp27 are thermally activated

The small heat shock protein 27 (Hsp27) is an oligomeric, molecular chaperone in vitro. This chaperone activity and other physiological roles attributed to Hsp27 have been reported to depend on the state of self-association. In the present work, we have used sedimentation velocity experiments to demonstrate that the self-association of Hsp27 is independent of pH and ionic strength but increases significantly as the temperature is increased from 10 to 40 degrees C. The largest oligomers formed at 10 degrees C are approximately 8-12 mer, whereas at 40 degrees C oligomers as large as 22-30 mer are observed. Similarly, the chaperone activity of Hsp27 as indicated by its ability to inhibit dithiothreitol-induced insulin aggregation also increases with increased temperature, with a particularly sharp increase in activity as temperature is increased from 34 to 43 degrees C. Similar studies of an Hsp27 triple variant that mimics the behavior of the phosphorylated protein establish that this protein has greatly diminished chaperone activity that responds minimally to increased temperature. We conclude that Hsp27 can exploit a large number of oligomerization states and that the range of oligomer size and the magnitude of chaperone activity increase significantly as temperature is increased over the range that is relevant to the physiological heat shock response.     

Preparation of lipid vesicles containing high levels of entrapped radioactive cations.

The ionophore A23187 was incorporated into the lipid bilayer of unilamellar vesicles and used to earry externally added indium ions to a chelator, nitrilotriacetic acid, which was previously entrapped in the vesicles. The extent of vesicle loading was assayed by Sephadex chromatography and γ-ray perturbed angular correlation spectroscopy. Incubation conditions were developed which allow rapid loading of more than 90% of added ¹²¹In³⁺ with specific activities of 200–300 μCi/mg lipid.     

Donepezil treatment restores the ability of estradiol to enhance cognitive performance in aged rats: Evidence for the cholinergic basis of the critical period hypothesis

Recent studies suggest that the ability of estradiol to enhance cognitive performance diminishes with age and/or time following loss of ovarian function. We hypothesize that this is due, in part, to a decrease in basal forebrain cholinergic function. This study tested whether donepezil, a cholinesterase inhibitor, could restore estradiol effects on cognitive performance in aged rats that had been ovariectomized as young adults. Rats were ovariectomized at 3 months of age, and then trained on a delayed matching to position (DMP) T-maze task, followed by a configural association (CA) operant condition task, beginning at 12-17 or 22-27 months of age. Three weeks prior to testing, rats started to receive either donepezil or vehicle. After one week, half of each group also began receiving estradiol. Acclimation and testing began seven days later and treatment continued throughout testing. Estradiol alone significantly enhanced DMP acquisition in middle-aged rats, but not in aged rats. Donepezil alone had no effect on DMP acquisition in either age group; however, donepezil treatment restored the ability of estradiol to enhance DMP acquisition in aged rats. This effect was due largely to a reduction in the predisposition to adopt a persistent turn strategy during acquisition. These same treatments did not affect acquisition of the CA task in middle-aged rats, but did have small but significant effects on response time in aged rats. The data are consistent with the idea that estrogen effects on cognitive performance are task specific, and that deficits in basal forebrain cholinergic function are responsible for the loss of estradiol effect on DMP acquisition in aged ovariectomized rats. In addition, the data suggest that enhancing cholinergic function pharmacologically can restore the ability of estradiol to enhance acquisition of the DMP task in very old rats following long periods of hormone deprivation. Whether donepezil has similar restorative effects on other estrogen-sensitive tasks needs to be explored.     

Synthesis of protoporphyrin IX induced by 5-aminolevulinic acid in yeast cells in the presence of 2,2;-dipyridyl

5-Aminolevulinic acid (ALA), a precursor of porphyrin synthesis, increased the production of various porphyrin compounds in Candida guilliermondii cells. Metalloporphyrins and protoporphyrin IX (PPIX) were predominantly accumulated, respectively, at ALA concentrations of 0.2-0.4 mM and at those higher than 1.5 mM. 2,2;-Dipyridyl which complexed with bivalent metals significantly increased the content of endogenous PPIX even at ALA concentrations lower than 0.5 mM. Under these conditions, the yeast sensitivity to photodynamic effect of visible light (400-600 nm) dramatically increased due to photosensitization by endogenous PPIX.     

Inactivation of the Heme Degrading Enzyme IsdI by an Active Site Substitution That Diminishes Heme Ruffling

IsdG and IsdI are paralogous heme degrading enzymes from the bacterium Staphylococcus aureus. Heme bound by these enzymes is extensively ruffled such that the meso-carbons at the sites of oxidation are distorted toward bound oxygen. In contrast, the canonical heme oxygenase family degrades heme that is bound with minimal distortion. Trp-66 is a conserved heme pocket residue in IsdI implicated in heme ruffling. IsdI variants with Trp-66 replaced with residues having less bulky aromatic and alkyl side chains were characterized with respect to catalytic activity, heme ruffling, and electrochemical properties. The heme degradation activity of the W66Y and W66F variants was approximately half that of the wild-type enzyme, whereas the W66L and W66A variants were inactive. A crystal structure and NMR spectroscopic analysis of the W66Y variant reveals that heme binds to this enzyme with less heme ruffling than observed for wild-type IsdI. The reduction potential of this variant (−96 ± 7 mV versus standard hydrogen electrode) is similar to that of wild-type IsdI (−89 ± 7 mV), so we attribute the diminished activity of this variant to the diminished heme ruffling observed for heme bound to this enzyme and conclude that Trp-66 is required for optimal catalytic activity.     

Rapid quantitative analysis of a gibberellin-sterol inhibitor using high-performance liquid chromatographic cartridge columns

A rapid, sensitive, and economical chemical analysis of the triazole, gibberellin-inhibitor, paclobutrazol (PP333, [(2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4 triazol-1-yl) pentan-3-ol]) was sought, featuring high-performance liquid chromatography (HPLC) as the final quantitation step. Three C₁₈-reverse phase columns (conventional, 250×4.6 mm; cartridge type, 125×4.6 mm; and minicolumn, 33×4.6 mm) were evaluated for their performance in HPLC separation and quantitation of PP333 applied to soil and plant foliage. The 125-mm Whatman Partisil 5 ODS-3 cartridge column was superior to the standard 250-mm DuPont Zorbax ODS unit, and provided enhanced resolution and reduced solvent consumption, analysis time, and cost. A Perkin-Elmer Pecosphere 3×3C-C₁₈ cartridge system was also superior to the 125-mm column with respect to these parameters. Although this minicolumn necessitated an additional purification step prior to HPLC analysis, its exceptionally fast analysis time and recovery period coupled with a high degree of sensitivity rendered it the most superior unit. This HPLC technology provided an efficient means of assaying for PP333 in large-scale experiments dealing with the chemical's absorption, translocation, and physiological response.