Inhibition of purine phosphoribosyltransferases of Ehrlich ascites-tumour cells by 6-mercaptopurine

1. The formation of adenosine 5′-phosphate, guanosine 5′-phosphate and inosine 5′-phosphate from [8-¹⁴C]adenine, [8-¹⁴C]guanine and [8-¹⁴C]hypoxanthine respectively in the presence of 5-phosphoribosyl pyrophosphate and an extract from Ehrlich ascites-tumour cells was assayed by a method involving liquid-scintillation counting of the radioactive nucleotides on diethylaminoethylcellulose paper. The results obtained with guanine were confirmed by a spectrophotometric assay which was also used to assay the conversion of 6-mercaptopurine and 5-phosphoribosyl pyrophosphate into 6-thioinosine 5′-phosphate in the presence of 6-mercaptopurine phosphoribosyltransferase from these cells. 2. At pH 7·8 and 25° the Michaelis constants for adenine, guanine and hypoxanthine were 0·9 μm, 2·9 μm and 11·0 μm in the assay with radioactive purines; the Michaelis constant for guanine in the spectrophotometric assay was 2·6 μm. At pH 7·9 the Michaelis constant for 6-mercaptopurine was 10·9 μm. 3. 25 μm-6-Mercaptopurine did not inhibit adenine phosphoribosyltransferase. 6-Mercaptopurine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 4·7 μm) and hypoxanthine phosphoribosyltransferase (K_i 8·3 μm). Hypoxanthine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 3·4 μm). 4. Differences in kinetic parameters and in the distribution of phosphoribosyltransferase activities after electrophoresis in starch gel indicate that different enzymes are involved in the conversion of adenine, guanine and hypoxanthine into their nucleotides. 5. From the low values of K_i for 6-mercaptopurine, and from published evidence that ascites-tumour cells require supplies of purines from the host tissues, it is likely that inhibition of hypoxanthine and guanine phosphoribosyltransferases by free 6-mercaptopurine is involved in the biological activity of this drug.     

Diadenosine Homodinucleotide Products of ADP-ribosyl Cyclases Behave as Modulators of the Purinergic Receptor P2X7

ADP-ribosyl cyclases from both vertebrates and invertebrates were previously shown to produce two isomers of P1,P2 diadenosine 5′,5′"-P1, P2-diphosphate, P18 and P24, from cyclic ADP-ribose (cADPR) and adenine. P18 and P24 are characterized by an unusual N-glycosidic linkage in one of the adenylic mononucleotides (Basile, G., Taglialatela-Scafati, O., Damonte, G., Armirotti, A., Bruzzone, S., Guida, L., Franco, L., Usai, C., Fattorusso, E., De Flora, A., and Zocchi, E. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 14509–14514). P24, but not P18, proved to increase the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in HeLa cells and to negatively affect mitochondrial function. Here we show that micromolar P24, but not P18, triggers a slow and sustained influx of extracellular Ca²⁺ through the opening of the purinergic receptor/channel P2X7. On the other hand, P18 inhibits the Ca²⁺ influx induced by 0.6 mm ATP in HEK293 cells stably transfected with P2X7, with an IC₅₀ of ∼1 μm. Thus, P18 is devoid of intrinsic P2X7 stimulatory activity and behaves as an ATP antagonist. A P2X7-mediated increase of the basal [Ca²⁺]_i has been demonstrated to negatively affect Schwann cell (SC) function in rats with the inherited, peripheral neuropathy Charcot-Marie-Tooth 1A (CMT1A) (Nobbio, L., Sturla, L., Fiorese, F., Usai, C., Basile, G., Moreschi, I., Benvenuto, F., Zocchi, E., De Flora, A., Schenone, A., and Bruzzone S. (2009) J. Biol. Chem. 284, 23146–23158). Preincubation of CMT1A SC with 200 nm P18 restored the basal [Ca²⁺]_i to values similar to those recorded in wild-type SC. These results identify P18 as a new P2X7 antagonist, potentially useful in the treatment of CMT1A.     

Calcium Regulates Molecular Interactions of Otoferlin with Soluble NSF Attachment Protein Receptor (SNARE) Proteins Required for Hair Cell Exocytosis

Mutations in otoferlin, a C2 domain-containing ferlin family protein, cause non-syndromic hearing loss in humans (DFNB9 deafness). Furthermore, transmitter secretion of cochlear inner hair cells is compromised in mice lacking otoferlin. In the present study, we show that the C2F domain of otoferlin directly binds calcium (K_D = 267 μm) with diminished binding in a pachanga (D1767G) C2F mouse mutation. Calcium was found to differentially regulate binding of otoferlin C2 domains to target SNARE (t-SNARE) proteins and phospholipids. C2D–F domains interact with the syntaxin-1 t-SNARE motif with maximum binding within the range of 20–50 μm Ca²⁺. At 20 μm Ca²⁺, the dissociation rate was substantially lower, indicating increased binding (K_D = ∼10⁻⁹) compared with 0 μm Ca²⁺ (K_D = ∼10⁻⁸), suggesting a calcium-mediated stabilization of the C2 domain·t-SNARE complex. C2A and C2B interactions with t-SNAREs were insensitive to calcium. The C2F domain directly binds the t-SNARE SNAP-25 maximally at 100 μm and with reduction at 0 μm Ca²⁺, a pattern repeated for C2F domain interactions with phosphatidylinositol 4,5-bisphosphate. In contrast, C2F did not bind the vesicle SNARE protein synaptobrevin-1 (VAMP-1). Moreover, an antibody targeting otoferlin immunoprecipitated syntaxin-1 and SNAP-25 but not synaptobrevin-1. As opposed to an increase in binding with increased calcium, interactions between otoferlin C2F domain and intramolecular C2 domains occurred in the absence of calcium, consistent with intra-C2 domain interactions forming a “closed” tertiary structure at low calcium that “opens” as calcium increases. These results suggest a direct role for otoferlin in exocytosis and modulation of calcium-dependent membrane fusion.     

Organopalladium compound 7b targets mitochondrial thiols and induces caspase-dependent apoptosis in human myeloid leukemia cells

The advances in the treatment of chronic myeloid leukemia (CML) during the last years were also accompanied by the development of evading strategies by tumor cells, resulting in chemotherapy resistance in some patients. Patented organopalladium compounds derived from the reaction of N,N-dimethyl-1-phenethylamine (dmpa) with [1,2-ethanebis(diphenylphosphine)] (dppe) exhibited a potent antitumor activity in vivo and in vitro in melanoma cells. We showed here that the cyclopalladated derivative [Pd₂(R₍₊₎)C², N-dmpa)₂(μ-dppe)Cl₂], named compound 7b, was highly effective to promote cell death in the K562 human leukemia cells and its mechanisms of action were investigated. It was shown that compound 7b was able to promote exclusively apoptotic cell death in K562 cells associated to cytochrome c release and caspase 3 activation. This cytotoxic effect was not observed in normal peripheral mononuclear blood cells. The compound 7b-induced intrinsic apoptotic pathway was triggered by the protein thiol oxidation that resulted in the dissipation of the mitochondrial transmembrane potential. The preventive effect of the dithiothreitol on the compound 7b-induced cell death and all downstream events associated to apoptosis confirmed that death signal was elicited by the thiol oxidation. These findings contribute to the elucidation of the palladacycle 7b-induced cell death mechanism and present this compound as a promising drug in the CML antitumor chemotherapy.     

Translocation of iron citrate and phosphorus in xylem exudate of soybean

Soybean plants, Glycine max (L.) Merrill, in standard solution received 2.5 μm ferric ethylenediamine di(o-hydroxyphenylacetate (FeEDDHA) and 0 to 128 μm phosphorus. Their stem exudates contained: 32 to 52 μm Fe, 120 to 5000 μm P, and 120 to 165 μm citrate. Electrophoresis of exudates with high P caused Fe trailing that precluded identification of any major form of Fe. Exudate with low P gave an anodic band of Fe citrate as the major Fe compound. Phosphate added to exudate in vitro depressed the Fe citrate peak and cause Fe trailing. EDDHA added to exudate in vitro pulled Fe from Fe citrate; citrate then migrated as a slower form and Fe migrated as FeEDDHA. A modified preculture system, involving 2-day renewals of 0.2 μm FeEDDHA with 3.2, 9.6, or 16 μm P and low levels of other ions, controlled pH depression and produced considerable change in citrate and P levels. The exudates contained: 45 to 57 μm Fe, 200 to 925 μm P, and 340 to 1025 μm citrate. The high citrate was from plants grown with low P. The major form of Fe in the exudates was Fe citrate. This is probably the form translocated in the plants.     

Identification,Purification, and Characterization of a Novel Amino Acid Racemase,Isoleucine 2-Epimerase,from Lactobacillus Species

Accumulation of d-leucine, d-allo-isoleucine, and d-valine was observed in the growth medium of a lactic acid bacterium, Lactobacillus otakiensis JCM 15040, and the racemase responsible was purified from the cells and identified. The N-terminal amino acid sequence of the purified enzyme was GKLDKASKLI, which is consistent with that of a putative γ-aminobutyrate aminotransferase from Lactobacillus buchneri. The putative γ-aminobutyrate aminotransferase gene from L. buchneri JCM 1115 was expressed in recombinant Escherichia coli and then purified to homogeneity. The enzyme catalyzed the racemization of a broad spectrum of nonpolar amino acids. In particular, it catalyzed at high rates the epimerization of l-isoleucine to d-allo-isoleucine and d-allo-isoleucine to l-isoleucine. In contrast, the enzyme showed no γ-aminobutyrate aminotransferase activity. The relative molecular masses of the subunit and native enzyme were estimated to be about 49 kDa and 200 kDa, respectively, indicating that the enzyme was composed of four subunits of equal molecular masses. The K_m and V_max values of the enzyme for l-isoleucine were 5.00 mM and 153 μmol·min⁻¹·mg⁻¹, respectively, and those for d-allo-isoleucine were 13.2 mM and 286 μmol·min⁻¹·mg⁻¹, respectively. Hydroxylamine and other inhibitors of pyridoxal 5′-phosphate-dependent enzymes completely blocked the enzyme activity, indicating the enzyme requires pyridoxal 5′-phosphate as a coenzyme. This is the first evidence of an amino acid racemase that specifically catalyzes racemization of nonpolar amino acids at the C-2 position.     

Transport of glucose and galactose in kidney-cortex cells

1. The aerobic transport of d-glucose and d-galactose in rabbit kidney tissue at 25° was studied. 2. In slices forming glucose from added substrates an accumulation of glucose against its concentration gradient was found. The apparent ratio of intracellular ([S]_i) and extracellular ([S]_o) glucose concentrations was increased by 0·4mm-phlorrhizin and 0·3mm-ouabain. 3. Slices and isolated renal tubules actively accumulated glucose from the saline; the apparent [S]_i/[S]_o fell below 1·0 only at [S]_o higher than 0·5mm. 4. The rate of glucose oxidation by slices was characterized by the following parameters: K_m 1·16mm; V_max. 4·5μmoles/g. wet wt./hr. 5. The active accumulation of glucose from the saline was decreased by 0·1mm-2,4-dinitrophenol, 0·4mm-phlorrhizin and by the absence of external Na⁺. 6. The kinetic parameters of galactose entry into the cells were: K_m 1·5mm; V_max 10μmoles/g. wet wt./hr. 7. The efflux kinetics from slices indicated two intracellular compartments for d-galactose. The galactose efflux was greatly diminished at 0°, was inhibited by 0·4mm-phlorrhizin, but was insensitive to ouabain. 8. The following mechanism of glucose and galactose transport in renal tubular cells is suggested: (a) at the tubular membrane, these sugars are actively transported into the cells by a metabolically- and Na⁺-dependent phlorrhizin-sensitive mechanism; (b) at the basal cell membrane, these sugars are transported in accordance with their concentration gradient by a phlorrhizin-sensitive Na⁺-independent facilitated diffusion. The steady-state intracellular sugar concentration is determined by the kinetic parameters of active entry, passive outflow and intracellular utilization.     

myo-Inositol homeostasis in foetal rabbit lung

In several species, lung maturation is accompanied by a decline in the phosphatidylinositol content of lung surfactant and a concomitant increase in its phosphatidylglycerol content. To examine the possibility that this developmental change is influenced by the availability of myo-inositol, potential sources of myo-inositol for the developing rabbit lung were investigated. On day 28 of gestation the myo-inositol content of foetal rabbit lung tissue (2.3±0.5μmol/g of tissue) was not significantly different from that of adult lung tissue but the activity of d-glucose 6-phosphate:1l-myo-inositol 1-phosphate cyclase (cyclase) in foetal lung tissue (81.0±9.0nmol·h⁻¹·g of tissue⁻¹) was higher than that found in adult lung tissue (23.2±1.0nmol·h⁻¹·g of tissue⁻¹). Day 28 foetal rabbit lung tissue was found also to take up myo-inositol by a specific, energy-dependent, Na⁺-requiring mechanism. Half-maximal uptake of myo-inositol by foetal rabbit lung slices was observed when the concentration of myo-inositol in the incubation medium was 85μm. When the myo-inositol concentration was 1mm (but not 100μm) the addition of glucose (5.5mm) stimulated myo-inositol uptake. myo-Inositol uptake was observed also in adult rabbit lung and was found to be sub-maximal at the concentration of myo-inositol found in adult rabbit serum. The concentration of myo-inositol in the serum of pregnant adult rabbits (47.5±5.5μm) was significantly lower than that of non-pregnant adult female rabbits (77.9±9.2μm). On day 28 of gestation the concentration of myo-inositol in foetal serum (175.1±12.0μm) was much less than on day 25, but more than that found on day 30. A transient post-partum increase in the concentration of myo-inositol in serum was followed by a rapid decline. Much of the myo-inositol in foetal rabbit serum probably originates from the placenta, where on day 28 of gestation a high cyclase activity (527±64nmol·h⁻¹·g of tissue⁻¹) was measured. The gestational decline in serum myo-inositol concentration, together with the decreasing cyclase activity of the lungs, is consistent with the view that maturation of the lungs is accompanied by decreased availability of myo-inositol to this tissue.     

Involvement of the oxidative burst in phytoalexin accumulation and the hypersensitive reaction

The role of the oxidative burst, transient production of activated oxygen species such as H₂O₂ and superoxide (O₂⁻) in elicitation of phytoalexins and the hypersensitive reaction (HR) was investigated in white clover (Trifolium repens L.) and tobacco (Nicotiana tabacum L.). H₂O₂ and O₂⁻ production was measured as chemiluminescence (CL) mediated by luminol, which was added to suspension-cultured white clover just before measurement in an out-of-coincidence mode scintillation counter. Maximum CL occurred between 10 and 20 min after addition of 0.4 × 10⁸ colony-forming units/mL of incompatible Pseudomonas corrugata or 158 μm HgCl₂. Autoclaved P. corrugata produced a slightly higher response. Elicitation of cells with 25 μm HgCl₂ did not produce CL. Preincubation of plant cells in superoxide dismutase, which converts O₂⁻ to H₂O₂, for 2 min before addition of bacteria did not significantly increase maximum CL levels (P ≥ 0.05). Preincubation of plant cells with catalase for 2 min before addition of bacteria prevented the increase in CL, confirming that H₂O₂ is the substrate for the luminol reaction. Addition of live bacteria or HgCl₂ (25 and 158 μm) to white clover increased levels of the phytoalexin medicarpin during a 24-h period, but addition of autoclaved bacteria did not elicit formation of medicarpin. Preincubation of plant cells with catalase, which quenched the bacteria-induced oxidative burst, did not decrease phytoalexin accumulation. Live bacteria infiltrated into Havana 44 tobacco leaf panels induced development of the HR, but autoclaved bacteria did not. Incubation of live bacteria with superoxide dismutase and catalase before infiltration into tobacco leaves did not interfere with development of the HR. Tobacco leaf panels infiltrated with up to 158 μm HgCl₂ did not develop an HR. These results suggest that an oxidative burst consisting of H₂O₂ and O₂⁻ does occur during these two plant defense responses, but it may not be a necessary element of the signaling system for HR and phytoalexin formation.     

Certhrax Toxin,an Anthrax-related ADP-ribosyltransferase from Bacillus cereus

We identified Certhrax, the first anthrax-like mART toxin from the pathogenic G9241 strain of Bacillus cereus. Certhrax shares 31% sequence identity with anthrax lethal factor from Bacillus anthracis; however, we have shown that the toxicity of Certhrax resides in the mART domain, whereas anthrax uses a metalloprotease mechanism. Like anthrax lethal factor, Certhrax was found to require protective antigen for host cell entry. This two-domain enzyme was shown to be 60-fold more toxic to mammalian cells than anthrax lethal factor. Certhrax localizes to distinct regions within mouse RAW264.7 cells by 10 min postinfection and is extranuclear in its cellular location. Substitution of catalytic residues shows that the mART function is responsible for the toxicity, and it binds NAD⁺ with high affinity (K_D = 52.3 ± 12.2 μm). We report the 2.2 Å Certhrax structure, highlighting its structural similarities and differences with anthrax lethal factor. We also determined the crystal structures of two good inhibitors (P6 (K_D = 1.7 ± 0.2 μm, K_i = 1.8 ± 0.4 μm) and PJ34 (K_D = 5.8 ± 2.6 μm, K_i = 9.6 ± 0.3 μm)) in complex with Certhrax. As with other toxins in this family, the phosphate-nicotinamide loop moves toward the NAD⁺ binding site with bound inhibitor. These results indicate that Certhrax may be important in the pathogenesis of B. cereus.     

Factors Affecting Phosphate Uptake by Aerobacter aerogenes in a System Relating Cell Numbers to 32P Uptake

The uptake of phosphate, from media limited in this ion, by resting cells of Aerobacter aerogenes has been investigated and shown to be dependent upon several factors. An incubation medium composed of 10⁻²m K⁺, 5 × 10⁻³m Mg²⁺, 1 mg of glucose per ml, and 1 μCi of ³²PO₄³⁻ per ml, buffered at pH 6.55 with 0.05 mN-2-hydroxyethyl-piperazine-N′-2-ethanesulfonic acid (HEPES), was found to stimulate optimum accumulation of ³²P-orthophosphate. The temperature of incubation, incubation time, the concentration of unlabeled orthophosphate, as well as arsenate, and several metabolic inhibitors were found to affect the accumulation. The labeled cells were collected on a membrane filter, which had been previously boiled in glass-distilled water, for measurement of the radioactivity accumulated. Under optimum conditions, as few as 20,000 cells were capable of accumulating detectable amounts of ³²P-orthophosphate in 1 hr of incubation.     

Identification and Characterization of Prokaryotic Dipeptidyl-peptidase 5 from Porphyromonas gingivalis

Porphyromonas gingivalis, a Gram-negative asaccharolytic anaerobe, is a major causative organism of chronic periodontitis. Because the bacterium utilizes amino acids as energy and carbon sources and incorporates them mainly as dipeptides, a wide variety of dipeptide production processes mediated by dipeptidyl-peptidases (DPPs) should be beneficial for the organism. In the present study, we identified the fourth P. gingivalis enzyme, DPP5. In a dpp4-7-11-disrupted P. gingivalis ATCC 33277, a DPP7-like activity still remained. PGN_0756 possessed an activity indistinguishable from that of the mutant, and was identified as a bacterial orthologue of fungal DPP5, because of its substrate specificity and 28.5% amino acid sequence identity with an Aspergillus fumigatus entity. P. gingivalis DPP5 was composed of 684 amino acids with a molecular mass of 77,453, and existed as a dimer while migrating at 66 kDa on SDS-PAGE. It preferred Ala and hydrophobic residues, had no activity toward Pro at the P1 position, and no preference for hydrophobic P2 residues, showed an optimal pH of 6.7 in the presence of NaCl, demonstrated K_m and k_cat/K_m values for Lys-Ala-MCA of 688 μm and 11.02 μm⁻¹ s⁻¹, respectively, and was localized in the periplasm. DPP5 elaborately complemented DPP7 in liberation of dipeptides with hydrophobic P1 residues. Examinations of DPP- and gingipain gene-disrupted mutants indicated that DPP4, DPP5, DPP7, and DPP11 together with Arg- and Lys-gingipains cooperatively liberate most dipeptides from nutrient oligopeptides. This is the first study to report that DPP5 is expressed not only in eukaryotes, but also widely distributed in bacteria and archaea.     

Enzymic and substrate basis for the anaplerotic step in guard cells

From the maximum rate of malate accumulation in Vicia faba L. guard cells during stomatal opening the maximum rate of organic anion synthesis is calculated to be 200 millimoles per kilogram dry weight per hour. A minimum estimate for the phosphoenolpyruvate (PEP) carboxylase-catalyzed reaction in guard cells is 650 millimoles per kilogram dry weight per hour which is significantly higher than in any other leaf tissue. The apparent K_mpep of the guard cell enzyme is 60 μm at pH 8.7, but is probably higher at lower pH. The concentration of PEP in guard cells was 270μm (=2.2 × 10⁻¹⁵ moles/guard cell pair) during anion synthesis. These results support the possibility that the carboxylation of PEP is the anaplerotic step in guard cells.     

High-Level Heterologous Production of d-Cycloserine by Escherichia coli

Previously, we successfully cloned a d-cycloserine (d-CS) biosynthetic gene cluster consisting of 10 open reading frames (designated dcsA to dcsJ) from d-CS-producing Streptomyces lavendulae ATCC 11924. In this study, we put four d-CS biosynthetic genes (dcsC, dcsD, dcsE, and dcsG) in tandem under the control of the T7 promoter in an Escherichia coli host. SDS-PAGE analysis demonstrated that the 4 gene products were simultaneously expressed in host cells. When l-serine and hydroxyurea (HU), the precursors of d-CS, were incubated together with the E. coli resting cell suspension, the cells produced significant amounts of d-CS (350 ± 20 μM). To increase the productivity of d-CS, the dcsJ gene, which might be responsible for the d-CS excretion, was connected downstream of the four genes. The E. coli resting cells harboring the five genes produced d-CS at 660 ± 31 μM. The dcsD gene product, DcsD, forms O-ureido-l-serine from O-acetyl-l-serine (OAS) and HU, which are intermediates in d-CS biosynthesis. DcsD also catalyzes the formation of l-cysteine from OAS and H₂S. To repress the side catalytic activity of DcsD, the E. coli chromosomal cysJ and cysK genes, encoding the sulfite reductase α subunit and OAS sulfhydrylase, respectively, were disrupted. When resting cells of the double-knockout mutant harboring the four d-CS biosynthetic genes, together with dcsJ, were incubated with l-serine and HU, the d-CS production was 980 ± 57 μM, which is comparable to that of d-CS-producing S. lavendulae ATCC 11924 (930 ± 36 μM).     

Vanadate inhibits blue light-stimulated swelling of vicia guard cell protoplasts

When supplied under low chloride concentrations, vanadate inhibits the blue light-stimulated swelling of Vicia faba L. guard cell protoplasts in a dose-dependent fashion. The volume of guard cell protoplasts incubated in 10 mm K-imino-diacetic acid, 0.4 m mannitol, and 1 mm CaCl₂ remained essentially constant under 1000 μmol m⁻² s⁻¹ red light, but increased an average of 27% after 8 min of the addition of 50 μmol m⁻² s⁻¹ blue light to the background red light. At 500 μm, vanadate completely inhibits the response to blue light. Vanadate also inhibits the swelling of guard cell protoplasts stimulated by the H⁺-ATPase agonist fusicoccin. The vanadate sensitivity of the blue light-stimulated swelling implicates a proton-pumping ATPase as a component of the sensory transduction of blue light in guard cells.     

Effect of Charged Residues in the N-domain of Sup35 Protein on Prion [PSI+] Stability and Propagation

Recent studies have shown that Sup35p prion fibrils probably have a parallel in-register β-structure. However, the part(s) of the N-domain critical for fibril formation and maintenance of the [PSI⁺] phenotype remains unclear. Here we designed a set of five SUP35 mutant alleles (sup35^KK) with lysine substitutions in each of five N-domain repeats, and investigated their effect on infectivity and ability of corresponding proteins to aggregate and coaggregate with wild type Sup35p in the [PSI⁺] strain. Alleles sup35-M1 (Y46K/Q47K) and sup35-M2 (Q61K/Q62K) led to prion loss, whereas sup35-M3 (Q70K/Q71K), sup35-M4 (Q80K/Q81K), and sup35-M5 (Q89K/Q90K) were able to maintain the [PSI⁺] prion. This suggests that the critical part of the parallel in-register β-structure for the studied [PSI⁺] prion variant lies in the first 63–69 residues. Our study also reveals an unexpected interplay between the wild type Sup35p and proteins expressed from the sup35^KK alleles during prionization. Both Sup35-M1p and Sup35-M2p coaggregated with Sup35p, but only sup35-M2 led to prion loss in a dominant manner. We suggest that in the fibrils, Sup35p can bind to Sup35-M1p in the same conformation, whereas Sup35-M2p only allowed the Sup35p conformation that leads to the non-heritable fold. Mutations sup35-M4 and sup35-M5 influence the structure of the prion forming region to a lesser extent, and can lead to the formation of new prion variants.     

Kinesin-2 KIF3AB Exhibits Novel ATPase Characteristics

KIF3AB is an N-terminal processive kinesin-2 family member best known for its role in intraflagellar transport. There has been significant interest in KIF3AB in defining the key principles that underlie the processivity of KIF3AB in comparison with homodimeric processive kinesins. To define the ATPase mechanism and coordination of KIF3A and KIF3B stepping, a presteady-state kinetic analysis was pursued. For these studies, a truncated murine KIF3AB was generated. The results presented show that microtubule association was fast at 5.7 μm⁻¹ s⁻¹, followed by rate-limiting ADP release at 12.8 s⁻¹. ATP binding at 7.5 μm⁻¹ s⁻¹ was followed by an ATP-promoted isomerization at 84 s⁻¹ to form the intermediate poised for ATP hydrolysis, which then occurred at 33 s⁻¹. ATP hydrolysis was required for dissociation of the microtubule·KIF3AB complex, which was observed at 22 s⁻¹. The dissociation step showed an apparent affinity for ATP that was very weak (K_½,ATP at 133 μm). Moreover, the linear fit of the initial ATP concentration dependence of the dissociation kinetics revealed an apparent second-order rate constant at 0.09 μm⁻¹ s⁻¹, which is inconsistent with fast ATP binding at 7.5 μm⁻¹ s⁻¹ and a K_d,ATP at 6.1 μm. These results suggest that ATP binding per se cannot account for the apparent weak K_½,ATP at 133 μm. The steady-state ATPase K_m,ATP, as well as the dissociation kinetics, reveal an unusual property of KIF3AB that is not yet well understood and also suggests that the mechanochemistry of KIF3AB is tuned somewhat differently from homodimeric processive kinesins.     

Effect of adrenaline on 32P incorporation into rat fat-cell phospholipids

1. The phospholipid composition of fat-cells prepared from rat epididymal fat-pad was determined. 2. The incorporation of [³²P]P_i into the phospholipids of fat-cells incubated in glucose-free medium and the effect of adrenaline and of α- and β-adrenergic blocking agents, were studied. 3. Incorporation of [³²P]P_i into fat-cell phospholipid increased with time; incubation with adrenaline resulted in increased incorporation that was related to the concentration of adrenaline. 4. The pattern of incorporation of [³²P]P_i into the individual phospholipids of fat-cells after incubation for 1h was determined; adrenaline (5.4μm) resulted in increased incorporation into phosphatidylcholine. 5. Incubation of fat-cells with propranolol (34μm) and adrenaline (5.4μm) resulted in abolition of adrenaline-stimulated lipolysis; there was a decrease in the specific radioactivity of phosphatidylcholine and an increase in the specific radioactivity of phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol and cardiolipin compared with cells incubated with adrenaline alone. 6. Incubation of fat-cells with phenoxybenzamine (0.1mm) and adrenaline (5.4μm) resulted in stimulation of lipolysis, and in diminished specific radioactivities of phosphatidylcholine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol and choline plasmalogen compared with cells stimulated with adrenaline alone.     

Photoreduction and Photophosphorylation with Tris-Washed Chloroplasts

The artificial electron donor compounds p-phenylenediamine (PD), N, N, N′, N′-tetramethyl-p-phenylenediamine (TMPD), and 2,6-dichlorophenol-indophenol (DCPIP) restored the Hill reaction and photophosphorylation in chloroplasts that had been inhibited by washing with 0.8 m tris (hydroxymethyl) aminomethane (tris) buffer, pH 8.0. The tris-wash treatment inhibited the electron transport chain between water and photosystem II and electron donation occurred between the site of inhibition and photosystem II. Photoreduction of nicotinamide adenine dinucleotide phosphate (NADP) supported by 33 μm PD plus 330 μm ascorbate was largely inhibited by 1 μm 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) while that supported by 33 μm TMPD or DCPIP plus ascorbate was relatively insensitive to DCMU. Experiments with the tris-washed chloroplasts indicated that electron donors preferentially donate electrons to photosystem II but in the presence of DCMU the donors (with the exception of PD at low concentrations) could also supply electrons after the DCMU block. The PD-supported photoreduction of NADP showed the relative inefficiency in far-red light characteristic of chloroplast reactions requiring photosystem II. With phosphorylating systems involving electron donors at low concentrations (33 μm donor plus 330 μm ascorbate) photophosphorylation, which occurred with P/e₂ ratios approaching unity, was completely inhibited by DCMU but with higher concentrations of the donor systems, photophosphorylation was only partially inhibited.     

Plasma Membrane Cholesterol as a Regulator of Human and Rodent P2X7 Receptor Activation and Sensitization

P2X7 receptors are nonselective cation channels gated by high extracellular ATP, but with sustained activation, receptor sensitization occurs, whereby the intrinsic pore dilates, making the cell permeable to large organic cations, which eventually leads to cell death. P2X7 receptors associate with cholesterol-rich lipid rafts, but it is unclear how this affects the properties of the receptor channel. Here we show that pore-forming properties of human and rodent P2X7 receptors are sensitive to perturbations of cholesterol levels. Acute depletion of cholesterol with 5 mm methyl-β-cyclodextrin (MCD) caused a substantial increase in the rate of agonist-evoked pore formation, as measured by the uptake of ethidium dye, whereas cholesterol loading inhibited this process. Patch clamp analysis of P2X7 receptor currents carried by Na⁺ and N-methyl-d-glucamine (NMDG⁺) showed enhanced activation and current facilitation following cholesterol depletion. This contrasts with the inhibitory effect of methyl-β-cyclodextrin reported for other P2X subtypes. Mutational analysis suggests the involvement of an N-terminal region and a proximal C-terminal region that comprises multiple cholesterol recognition amino acid consensus (CRAC) motifs, in the cholesterol sensitivity of channel gating. These results reveal cholesterol as a negative regulator of P2X7 receptor pore formation, protecting cells from P2X7-mediated cell death.