Identification of adenine binding domain peptides of the NADP+ active site within porcine heart NADP(+)-dependent isocitrate dehydrogenase

Photoaffinity labeling with [2'-32P]2N3NADP+ and [32P]2N3NAD+ was used to identify two overlapping tryptic and chymotryptic generated peptides within the adenine binding domain of NADP(+)-dependent isocitrate dehydrogenase (IDH). Photolysis was required for insertion of radiolabel, and prior photolysis of photoprobes before addition of IDH prevented insertion. Photoincorportion of 2N3NAD+ inhibited the enzymatic activity of IDH. Photolabeling of IDH with both [32P]2N3NAD+ and [2'-32P]2N3-NADP+ showed saturation effects with apparent Kds of 20 and 14 microM (+/-12%), respectively. The efficiency of photoincorporation at saturation of binding sites was determined to be about 50%. Also, photolabeling was observed with [32P]8N3ATP and [32P]2N3ATP but with saturation effects observed at lower affinity. With all radiolabeled probes reduction of photoinsertion was effected best by the addition of NADP+ followed by NAD+ and then ATP, indicating that photoinsertion with all the probes was within the NADP+ binding site. Isolation of [32P]2N3NAD+ and [2'-32P]2N3NADP+ photolabeled peptides by use of immobilized boronate and immobilized Al3+ chromatography, respectively, followed by HPLC purification resulted in the identification of overlapping peptides corresponding to Ile244-Arg249 and Leu121-Arg133 (tryptic fragments) and Lys243-His248 and Leu121-His135 (chymotryptic fragments). Trp125 and Trp245 were identified as the sites of photoinsertion based on these residues not being detectable on sequencing, the lack of chymotryptic cleavage at these residues, and the decreased rate of trypsin digestion at nearby Lys243 and Lys127. Sequence analysis of [32P]8N3ATP and [32P]2N3ATP photolabeled peptides gave essentially the same peptide regions being photolabeled but at much lower efficiency, indicating that the effects of ATP on IDH activity are dependent on competition for the same site.     

Changes in serum IGF-I and IGFBP-3 concentrations during the IGF-I generation test performed prospectively in children with short stature

The fractions of antibodies (Abs) containing only sIgA and IgG were purified from human breast milk by Protein A-Sepharose chromatography and they catalyzed phosphorylation of casein in the presence of [gamma-32P]ATP. Also, 32P-labeled low-molecular-weight non-protein products are formed which are visible as radioactive background on the polyacrylamide gel lanes during electrophoresis of Abs under denaturing conditions. Separation of sIgA from IgG using a DEAE-sorbent with subsequent gel-filtration in 0.05 M NaOH indicates that the low-molecular-weight substances partially remain tightly bound to sIgA and are separated only by gel-filtration in a buffer containing 5% dioxane (non-denaturing resolution) or by extraction of the sIgA pellet with the chloroform--methanol mixture (2:1). The 32P-labeled substances were separated by TLC in the system used for phospholipid chromatography forming two fractions (Rf 0.83 and 0.66) that were stained with iodine. The data suggest that the substances co-isolated with sIgA are phospholipids. At 25 nM ATP, casein and lipids are 32P-labeled. At 1 microM ATP, the sIgA polypeptides are also phosphorylated. Gentle removal of the lipids from Ab preparation enhanced 32P incorporation into casein and sIgA polypeptides. Considering the heterogeneity of polyclonal sIgA in protein and ATP affinity, it is suggested that phosphorylation of casein and sIgA polypeptides is catalyzed by abzymes of different clonal origin.     

Polyphosphoinositide synthesis in human neutrophils. Effects of a low metabolic energy state

Phosphatidylinositol (PtdIns) synthesis and polyphosphoinositide (PPI) formation were measured as the incorporation of [32P]orthophosphate ([32P]Pi) or [3H]inositol into non-stimulated intact human neutrophil membrane phospholipids. The rate of PtdIns "de novo" synthesis appeared to be a slow mechanism when compared to the rapid incorporation of [32P]Pi into PPIs. Of the "de novo" synthesized [3H]PtdIns, 70% was further phosphorylated to PPI. Nevertheless, this PPI pool represented less than 0.01% of the total nmols of PPIs formed evaluated as [32P]Pi labeling, indicating that PPI formation mainly involves a no "de novo" synthesized phosphatidylinositol pool. When evaluated at short incubation times, oscillations in the formation of PPIs were detected. A rapid phase was characterized after 30 s of incubation with [32P]Pi Phosphorylation levels returned to an equilibrium state within a minute, and the second phase peaked at 5 min., returning to equilibrium at 15 min. The fluctuant kinetics though not the equilibrium level of PPI formation, could be abolished by neomycin. On the other hand, a selective inhibition of the rapid phase of PPI synthesis occurred in the presence of the tyrosine kinase inhibitor genistein. When the incorporations of [gamma-32P]-adenosine triphosphate (ATP) or [32P]Pi into human neutrophil particulate fraction membranes were evaluated, PPIs synthesis showed fluctuations independently of the precursor used. Noticeably, [32P]from [32P]Pi was incorporated more efficiently into PPIs than that from [gamma-32P]ATP, when evaluated in parallel using equal specific activities for both radiolabeled precursors and under non-ATP synthesizing conditions. Moreover, the incorporation of [32P]Pi into particulate fraction PPIs was not abolished by high concentrations of non-radiolabeled ATP, and metabolically inhibited PMNs showed high rates of PPI synthesis. These data suggest that PPI formation is not necessarily a futile cycle in PMNs.     

Biosynthesis of chondroitin sulfate. Purification of glucuronosyl transferase II and use of photoaffinity labeling for characterization of the enzyme as an 80-kDa protein

A photoaffinity analogue, [beta-32P]5-azido-UDP-GlcA, was used to photolabel the enzymes that utilize UDP-GlcA in cartilage microsomes and rat liver microsomes. SDS-polyacrylamide gel electrophoresis analysis of photolabeled cartilage microsomes, which are specialized in chondroitin sulfate synthesis, showed a major radiolabeled band at 80 kDa and other minor radiolabeled bands near 40 and 60 kDa. Rat liver microsomes, which are enriched for enzymes of detoxification by glucuronidation, had a different pattern with multiple major labeled bands near 50-60 and 35 kDa. To determine that the photolabeled 80-kDa protein is the GlcA transferase II, we have purified the enzyme from cartilage microsomes. This membrane-bound enzyme, involved in the transfer of GlcA residues to non-reducing terminal GalNAc residues of the chondroitin polymer, has now been solubilized, stabilized, and then purified greater than 1350-fold by sequential chromatography on Q-Sepharose, heparin-Sepharose, and WGA-agarose. The purified enzyme exhibited a conspicuous silver-stained protein band on SDS-polyacrylamide gel electrophoresis that coincided with the major radiolabeled band of 80 kDa. SDS-polyacrylamide gel analysis of photoaffinity-labeled active fractions from the Q-Sepharose, heparin-Sepharose, and WGA-agarose also indicated only the single radiolabeled band at 80 kDa. Intensity of photolabeling in each of the fractions examined coincided with enzyme activity. The photolabeling of this 80-kDa protein was saturable with the photoprobe and could be inhibited by the addition of UDP-GlcA prior to the addition of the photoprobe. Thus, the photolabeling with [beta-32P]5-azido-UDP-GlcA has identified the GlcA transferase II as an 80-kDa protein. The purified enzyme was capable of transferring good amounts of GlcA residues to chondroitin-derived pentasaccharide with negligible transfer to pentasaccharides derived from hyaluronan or heparan.     

Further observation on the lack of active uptake system for substance P in the central nervous system

Crude mitochondrial P2 fractions from bovine hypothalamus and substantia nigra, slices from rabbit spinal cord and mesencephalon and glial fractions from rabbit brain were incubated with [3H]-substance P and the uptake was measured and compared with those for 5-HT and GABA. Substance P was to some extent taken up into the fractions but this uptake was neither temperature nor time dependent and the pellet/medium ratios were less than 1. Similar results were obtained in high potassium treated slices from rabbit mesencephalon. The rate of uptake for [3H]-substance P increased linearly in proportion to the medium concentration, suggesting a non-saturable binding. These results, together with our previous observations provide strong evidence that nerve terminals and glial cells lack a temperature sensitive, active uptake system capable of terminating transmitter action of substance P at the synapse.     

The G protein alpha s subunit incorporates [3H]palmitic acid and mutation of cysteine-3 prevents this modification

We investigated whether alpha s could be acylated by palmitate by transfecting COS cells with the cDNA for the wild-type, long form of alpha s and metabolically labeling with [3H]palmitate or [35S]methionine. Cells were separated into particulate and soluble fractions and immunoprecipitated with a specific peptide antibody. [3H]Palmitate was incorporated into both endogenous and transfected alpha s. Inhibition of protein synthesis with cycloheximide did not block the radiolabeling of alpha s with [3H]palmitate. Hydroxylamine treatment caused a release of the tritium radiolabel, demonstrating that the incorporation was through a thioester bond. The tritium radiolabel was base-labile and comigrated with [3H]palmitate on thin-layer chromatography. The third residue of the wild-type alpha s was mutated from a cysteine to an alanine by site-directed mutagenesis. This mutant was expressed in COS cells and localized to the particulate fraction as determined by immunoprecipitation of the [35S]methionine-labeled cells. The cysteine-3 mutant did not undergo radiolabeling with [3H]palmitate, indicating that this residue is crucial for the modification.     

Serine 167 is the major estradiol-induced phosphorylation site on the human estrogen receptor

Serine 167 has been identified by radiolabel and amino acid sequencing as the major estrogen-induced phosphorylation site on the human estrogen receptor (hER) from human MCF-7 mammary carcinoma cells. The phosphorylation of the hER on serine 167 was estrogen-dependent, increasing 4-fold upon estradiol treatment of MCF-7 cells and accounted for almost half of the total [32P]phosphate incorporated into the recombinant hER from Sf9 insect cells and the native hER from MCF-7 cells. Casein kinase II was found to phosphorylate the purified recombinant hER on serine 167 in vitro. In addition, estradiol binding enhanced by 2-fold the phosphorylation of the purified recombinant hER by casein kinase II in vitro. Western blot analysis and [32P]phosphate incorporation confirmed the presence of casein kinase II in Sf9 cells. These results demonstrate that the hER is phosphorylated on serine 167 by casein kinase II in a hormone-dependent manner.     

Synthesis and release of ATP by soluble mitochondrial F1 in complex with its inhibitor protein during dimethylsulfoxide-water transitions

Soluble mitochondrial F1 and F1 in complex with the natural ATPase inhibitor protein (F1-IP) catalyze the spontaneous synthesis of [gamma-32P]ATP from medium [32P]phosphate and enzyme-bound ADP when incubated in media with dimethylsulfoxide (Me2SO); under these conditions, the synthesized [gamma-32P]ATP is not released into the media, it remains tightly bound to the enzymes [Gómez-Puyou, A., Tuena de Gómez-Puyou, M. & de Meis, L. (1986) Eur. J. Biochem. 159, 133-140]. Some of the characteristics of the synthesized [gamma-32P]ATP were studied in F1 and F1-IP (ATPase activities of 70 and 1-3 micromol x min(-1) x mg(-1), respectively). In Me2SO media, gamma-phosphate of synthesized ATP in F1 or F1-IP exchanges with medium phosphate. From the rates of the exchange reaction, the half-times for hydrolysis of the synthesized ATP in F1 and F1-IP were calculated: 45 min and 58 min for F1 and F1-IP, respectively. The course that synthesized [gamma-32P]ATP follows after dilution of the Me2SO synthetic mixture with aqueous buffer was determined. After dilution, the half-life of synthesized ATP in F1 was less than 1 min. In F1-IP, ATP was also hydrolyzed, but at significantly lower rates. In F1-IP, dilution also produced release of the synthesized [gamma-32P]ATP. This was assayed by the accessibility of [gamma-32P]ATP to hexokinase. About 25% of [gamma-32P]ATP synthesized in F1-IP, but not in F1, was released into the media after dilution with aqueous buffer that contained 20 mM phosphate. Release of tightly bound ATP required the binding energy of phosphate and solvation of F1-IP, however, the particular kinetics of F1-IP were also central for medium ATP synthesis in the absence of electrochemical H+ gradients.     

Chromosomes with two intact axial cores are induced by G2 checkpoint override: evidence that DNA decatenation is not required to template the chromosome structure

Trypanosoma cruzi has a plasma membrane ATP transport system that may consist of an exterior receptor domain (ATP-R) and an interior domain regulated by tyrosine and serine/threonine kinases. The addition of exogenous ATP to freely swimming trypomastigotes resulted in a receptor-mediated inward movement of the nucleotide, and the system obeyed mass action law (Km, 9.42 microM and Vmax, 77.7 nmol x min(-1) x 4 x 10(6) trypomastigotes(-1)). Preloaded [3H]ADPi was not exchanged for ATP following the addition of increasing concentrations of exogenous ATPo to swimming trypomastigotes. Trypomastigote [ATP]o <--> ATP-R --> [ATP]i transport was [ATP]o-dependent and saturable at 100 microM. [ATP]o <--> ATP-R --> [ATP]i transport was abrogated by the tyrosine kinase inhibitors, genistein and lavendustin A. [ATP]o <--> ATP-R --> [ATP]i transport was also inhibited by the serine/threonine kinase inhibitor, staurosporin. Suramin, the antagonist of P2x and P2y purinergic receptors and the inhibitor of tyrosine kinase growth factor receptors, was also a very effective competitive inhibitor of the trypomastigote ATP transport system. The action of exogenous [gamma32P]ATPo resulted in the initial and simultaneous phosphorylation of a 63-kDa polypeptide (p63) and of a 92.4-kDa polypeptide (p92.4), which was followed by the abrupt phosphorylation of many other substrate proteins. The trypomastigote p63/p92.4 polypeptides may represent substrate proteins of a putative ATP-R-related tyrosine phosphokinase, and ATP receptors may transmit their signals by phosphorylation of specific substrate proteins.     

Stimulation of P2Y receptors activates c-fos gene expression and inhibits DNA synthesis in cultured cardiac fibroblasts

OBJECTIVES: The aims of this study were to determine (1) whether neonatal rat cardiac fibroblasts (CAFB) express P2Y receptors; (2) whether CAFB respond to extracellular ATP by inducing expression of c-fos mRNA; and (3) whether extracellular ATP modulates norepinephrine (NE)-stimulated cell growth in CAFB. METHODS: Expression of P2Y1 and P2Y2 receptors and induction of c-fos were examined by Northern blot analysis. CAFB growth was assessed by measuring [3H]thymidine incorporation and DNA content. P2Y receptor pharmacology was studied using various ATP analogues. RESULTS: Northern blot analysis of polyA enriched RNA confirmed that at least 2 subtypes of P2Y receptors (P2Y1 and P2Y2) are expressed in cultured CAFB. Extracellular ATP induced the expression of c-fos mRNA through a pathway that was sensitive to inhibitors of protein kinase C (PKC), but not to inhibitors of intracellular Ca2+ signaling. Extracellular ATP inhibited the NE-stimulated increases in DNA content and in [3H]thymidine incorporation into DNA. Whereas the potency order for stimulation of c-fos expression was ATP = UTP > ADP > adenosine, the potency order to inhibit the NE-induced increase of [3H]thymidine incorporation into DNA was ATP > ADP > UTP > adenosine. CONCLUSIONS: These data demonstrate that CAFB express both P2Y1 and P2Y2 receptor mRNA and that CAFB respond to P2Y receptor stimulation by induction of c-fos and inhibition of DNA synthesis. These findings suggest that the effects of ATP on [3H]thymidine incorporation into DNA and on expression of c-fos mRNA are exerted via distinct P2Y receptor subtypes.     

Brain regional correspondence between Alzheimer's disease histopathology and biomarkers of protein oxidation

Four biomarkers of neuronal protein oxidation [W/S ratio of MAL-6 spin-labeled synaptosomes, phenylhydrazine-reactive protein carbonyl content, glutamine synthetase (GS) activity, creatine kinase (CK) activity] in three brain regions [cerebellum, inferior parietal lobule (IPL), and hippocampus (HIP)] of Alzheimer's disease (AD)-demented and age-matched control subjects were assessed. These endpoints indicate that AD brain protein may be more oxidized than that of control subjects. The W/S ratios of AD hippocampal and inferior parietal synaptosomes are 30 and 46% lower, respectively, than corresponding values of tissue isolated from control brain; however, the difference between the W/S ratios of AD and control cerebellar synaptosomes is not significant. Protein carbonyl content is increased 42 and 37% in the Alzheimer's HIP and IPL regions, respectively, relative to AD cerebellum, whereas carbonyl content in control HIP and IPL is similar to that of control cerebellum. GS activity decreases an average of 27% in the AD brain; CK activity declines by 80%. The brain regional variation of these oxidation-sensitive biomarkers corresponds to established histopathological features of AD (senile plaque and neurofibrillary tangle densities) and is paralleled by an increase in immunoreactive microglia. These data indicate that senile plaque-dense regions of the AD brain may represent environments of elevated oxidative stress.     

Phosphoinositide 3-kinase in rat liver nuclei

Biochemical and immunochemical data from the present investigation reveal the existence of a p85/p110 phosphoinositide 3-kinase (PI 3-kinase) in rat liver nuclei. 32P-Labeling of membrane phosphoinositides by incubating intact nuclei with [gamma-32P]ATP results in the formation of [32P]phosphatidyl-inositol 3,4, 5-trisphosphate [PtdIns(3,4,5)P3], accompanied by small quantities of [32P]phosphatidylinositol 3-phosphate [PtdIns(3)P]. Studies with subnuclear fractions indicate that the PI 3-kinase is not confined to nuclear membranes. The nuclear soluble fraction also contains PI 3-kinase and an array of inositide-metabolizing enzymes, including phospholipase C (PLC), phosphoinositide phosphatase, and diacylglycerol (DAG) kinase. As a result, exposure of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to the nuclear extract in the presence of [gamma-32P]ATP generates a series of 32P-labeled D-3 phosphoinositides and phosphatidic acid (PA) in an interdependent manner. On the basis of the immunological reactivity and kinetic behavior, the nuclear PI 3-kinase is analogous, if not identical, to PI 3-kinase alpha, and constitutes about 5% of the total PI 3-kinase in the cell. Moreover, we test the premise that nuclear PI 3-kinase may, in part, be regulated through the control of substrate availability by PtdIns(4,5)P2-binding proteins. Effect of CapG, a nuclear actin-regulatory protein, on PI 3-kinase activity is examined in view of its unique Ca2+-dependent PtdIns(4, 5)P2-binding capability. In vitro data show that the CapG-mediated inhibition of nuclear PI 3-kinase is prompted by PKC phosphorylation of CapG and elevated [Ca2+]. This CapG-dependent regulation provides a plausible link between nuclear PLC and PI 3-kinase pathways for cross-communications. Taken together, these findings provide definite data concerning the presence of an autonomous PI 3-kinase cycle in rat liver nuclei. The nuclear location of PI 3-kinase may lead to a better understanding regarding its functional role in transducing signals from the plasma membrane to the nucleus in response to diverse physiological stimuli.     

Probes of initial phosphorylation events in ATP synthesis by chloroplasts

Rapid mixing and quenching techniques have been used with chloroplasts activated by an acid-base transition or by light to assess the nature and characteristics of the substances initially labeled by inorganic [32P]phosphate during ATP synthesis. With light-activated chloroplast fragments, but not with acid-base-activated preparations, an initial rapid labeling of a small amount of ADP is observed. With the acid-base activated preparations a slower continued labeling of ADP occurs that is uncoupler-sensitive, that does not proceed via [gamma-32]ATP of the medium and for which medium ADP furnishes the AMP moiety. The results point to ADP as the initial acceptor of phosphate for ATP synthesis, with a slow side reaction in which bound ATP phosphorylates bound AMP to give a bound ADP. The phosphorylation of bound ADP by medium [32P]phosphate in the absence of added ADP is confirmed, but the reaction is too slow to serve as an intermediate in photophosphorylation. The appearance of label from [32P]phosphate in ATP in the acid-base transition at 25 degrees shows a lag of only about 3 to 7 ms, consistent with the absence of any phosphorylated intermediate. The lag is followed by a linear rate of [gamma-32]ATP formation that is about as fast as that observed in steady photophosphorylation, consistent with a proton gradient serving for transmission of energy from electron transfer reactions to the ATP-synthesizing complex.     

Characterization of the intracellular and the plasma membrane Ca2+-ATPases in fractionated pig brain membranes using calcium pump inhibitors

The Ca2+-ATPase activity of isolated membranes and purified plasma membrane ATPase from pig brain was measured in the presence of specific inhibitors. The inhibition of the enzymatic activity by vanadate presents a lower affinity in microsomes than in the synaptic plasma membrane vesicles, showing K0.5 of 0.4 and 0.2 microM, respectively. The purified enzyme showed a higher sensitivity to vanadate with a K0.5 of 0.10 microM. Thapsigargin (Tg) and 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ) were stronger inhibitors of the Ca2+-ATPase activity in microsomes than in the synaptic membrane vesicles. The activity of the purified enzyme was not affected by Tg and only partially by BHQ. Cyclopiazonic acid inhibited the enzymatic activity in all fractions, being more sensitive in microsomes. The microsome preparation incorporated 32P from [gamma-32P]ATP into two main proteins that appear at approx 110,000 and 140,000. According to the inhibition pattern, the lower phosphorylated band was identified as the sarco(endo)plasmic reticulum Ca2+-ATPase, being in a higher percentage than the upper band. Synaptic membrane vesicles also incorporated radioactive 32P into two protein bands. The 140,000 protein (upper band) shows the typical behavior of the purified plasma membrane Ca2+-ATPase, being more abundant in this preparation than the organellar Ca2+-pump (lower band). This study highlights the heterogeneous nature of the Ca2+-ATPase activity measured in brain membrane fractions.     

Protein phosphorylation in normal and neoplastic development. Phosphorylation of proteins endogenous to foetal tissues and tumours

The abilities of proteins endogenous to normal and neoplastic tissues to serve as substrates in a protein-phosphorylation reaction in vitro were compared. After the tissue extracts were incubated with [gamma-32P]ATP, the phosphorylated proteins were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and the dried gels were subjected to radioautography. Considerable incorporation of 32P into a protein of mol.wt. 135000 was observed with extracts from foetal tissues and tumours, but only minimal incorporation into this protein occurred when extracts from adult tissues were used. The ability of this protein to become phosphorylated in vitro may be related to cell proliferation. When ascites cells were incubated with [32P]Pi, one of the major phosphoproteins migrated on sodium dodecyl suphate/polyacrylamide gels at mol.wt. 135000, suggesting that this protein can be phosphorylated both in intact cells and broken-cell preparations. A protein of mol.wt. 87000 was highly phosphorylatable in extracts from solid tumours, but was not phosphorylated in extracts from ascites tumours, foetal or adult tissues. The phosphorylation pattern of these two proteins can thus distinguish solid neoplasms and normal adult tissues from ascites tumours and from foetal tissues. A protein of mol.wt. 49000, which was the most labelled protein in adult tissues, was also one of the major phosphoproteins in foetal and neoplastic tissues. Numerous mechanisms are postulated to explain how the extent of 32P incorporation into a protein could vary as a function of biological state.     

Types of purinoceptors and phospholipase A2 involved in the activation of the platelet-activating factor-dependent transacetylase activity and arachidonate release by ATP in endothelial cells

Acyl analogs of PAF are the major products synthesized during agonist stimulation of endothelial cells. We have previously shown that PAF: 1-acyl-2-lyso-sn-glycero-3-phosphocholine transacetylase in calf pulmonary artery endothelial cells is activated by ATP through protein phosphorylation, and the increase in transacetylase activity by ATP contributes to the biosynthesis of acyl analogs of PAF (J. Biol. Chem. 272, 17431-17437, 1997). To understand the mechanisms(s) by which ATP stimulates acyl analogs of PAF production, we have identified the subtypes of the purinergic receptor that are linked to the activation of two enzymes involved in the generation of acyl analogs of PAF, namely, transacetylase and phospholipase A2. Experiments with transient transfection of the cells with antisense and sense thio-oligonucleotide to cytosolic phospholipase A2 (cPLA2) were also performed to evaluate whether downstream activation of cPLA2 is involved in ATP-receptor mediated induction of arachidonate release and synthesis of radylacetyl-GPC. We found that the P2u/P2Y2 receptor, which recognizes a pyrimidine nucleotide, UTP, as well as purine nucleotides, shows a potency profile of UTP > ATP = ATP gamma S > 2-methylthio-ATP in mediating the activation of PAF: lysophospholipid transacetylase. On the other hand, ADP beta S and 2-methylthio-ATP have similar potencies as ATP but have lower potencies than UTP and ATP gamma S in stimulating the release of arachidonate. These results suggest that both P2u/P2Y2 and P2y/P2Y1 receptor subtypes promote arachidonate release. In addition, transient transfection of endothelial cells with cPLA2 antisense but not the sense thio-oligonucleotide inhibited the stimulation of arachidonate release and [3H]acetate incorporation into radyl[3H]acetyl-GPC. Thus, our data suggest that a receptor-mediated process is involved in the activation of transacetylase for the induced synthesis of acyl analogs of PAF in endothelial cells. Furthermore, it is likely that cPLA2 supplies the lysophospholipids as substrates for the transacetylation reaction.     

Preferential utilization of ketone bodies for the synthesis of myelin cholesterol in vivo

1. The distribution of radioactivity among lipid classes of myelin and other subcellular brain fractions of young rats (18-21 days) was determined after in vivo injection of (3-(14)C-labelled ketone bodies, [U-(14)C] glucose or [2-(14)C] glucose. 2. The incorporation ratios (sterol/fatty acids) were 0.67, 1.48, 0.25, 0.62 and 0.54 for whole brain, myelin, mitochondria, microsomes and synaptosomes, respectively, with (3-(14)C)-labelled ketone bodies as substrate and 0.37, 0.89, 0.19, 0.34 and 0.29 with [U-(14)C] glucose as substrate. These data show that, both in whole brain and in subcellular brain fractions, acetyl groups derived from ketone bodies are used for sterol synthesis to a large extent than acetyl groups originating from glucose. 3. The specific radioactivity of cholesterol is much higher in myelin than in whole brain or in the other brain fractions, particularly after administration of labelled ketone bodies as substrate. 4. The incorporation patterns of acetoacetate and D-3-hydroxybutyrate were very similar, indicating that both ketone bodies contribute acetyl groups for lipid synthesis via the same metabolic route. 5. Our data suggest that a direct metabolic path from ketone bodies towards cholesterol exists - possibly via acetoacetyl-CoA formation in the cytosol of brain cells - and that this process is most active in oligodendrocytes.     

Identification of GIYWHHY as a novel peptide substrate for human p60c-src protein tyrosine kinase

We have recently determined that -Ile-Tyr- were the two critical residues as a peptide substrate for p60c-src protein tyrosine kinase (Lou, Q. et al., Lett. Peptide Sci., 1995, 2, 289). Here, we report on the design and synthesis of a secondary 'one-bead, one-compound' combinatorial peptide library based on this dipeptide motif (XIYXXXX, where X = all 19 eukaryotic amino acids except for cysteine). This secondary library was screened for its ability to be phosphorylated by p60c-src PTK using [gamma 32P]ATP as a tracer. Five of the strongest [32P]-labeled peptide-beads were identified and microsequenced: GIYWHHY, KIYDDYE, EIYEENG, EIYEEYE, and YIYEEED. A solid-phase phosphorylation assay was used to evaluate the structure-activity relationship of GIYWHHY. It was determined that Ile2, Tyr3, His5, and His6 were crucial for its activity as a substrate.     

The vesicle transport protein Vps33p is an ATP-binding protein that localizes to the cytosol in an energy-dependent manner

Molecular mechanisms of vesicle transport between the prevacuolar compartment and the vacuole in yeast or the lysosome in mammalian cells are poorly understood. To learn more about the specificity of this intercompartmental step, we have examined the subcellular localization of a SEC1 homologue, Vps33p, a protein implicated to function in transport between the prevacuolar compartment and the vacuole. Following short pulses, 80-90% of newly synthesized Vps33p cofractionated with a cytosolic enzyme marker after making permeabilized yeast cells. However, during a chase, 20-40% of Vps33p fractionated with permeabilized cell membranes in a time-dependent fashion with a half-time of approximately 40 min. Depletion of cellular ATP increased the association rate to a half-time of approximately 4 min and caused 80-90% of newly synthesized Vps33p to be associated with permeabilized cell membranes. The association of Vps33p with permeabilized cell membranes was reversible after restoring cells with glucose before permeabilization. The N-ethylmaleimide-sensitive fusion protein homologue, Sec18p, a protein with known ATP binding and hydrolysis activity, displayed the same reversible energy-dependent sedimentation characteristics as Vps33p. We determined that the photosensitive analog, 8-azido-[alpha-32P]ATP, could bind directly to Vps33p with low affinity. Interestingly, excess unlabeled ATP could enhance photoaffinity labeling of 8-azido-[alpha-32P]ATP to Vps33p, suggesting cooperative binding, which was not observed with excess GTP. Importantly, we did not detect significant photolabeling after deleting amino acid regions in Vps33p that show similarity to ATP interaction motifs. We visualized these events in living yeast cells after fusing the jellyfish green fluorescent protein (GFP) to the C terminus of full-length Vps33p. In metabolically active cells, the fully functional Vps33p-GFP fusion protein appeared to stain throughout the cytoplasm with one or two very bright fluorescent spots near the vacuole. After depleting cellular ATP, Vps33p-GFP appeared to localize with a punctate morphology, which was also reversible upon restoring cells with glucose. Overall, these data support a model where Vps33p cycles between soluble and particulate forms in an ATP-dependent manner, which may facilitate the specificity of transport vesicle docking or targeting to the yeast lysosome/vacuole.     

Frontiers of digestive surgery using laparoscopy

AIM: To investigate the effects of tyrphostins, (AG213, AG1394, AG114, AG1109, AG555) on the activity of casein kinase (CK) II. METHODS: CK II was partially purified from rat livers by sequential DE52 and heparin-Sepharose chromatography. CK II activity was assayed by incubating CK II with dephosphorylated casein and [gamma-32 P]ATP. RESULTS: AG213 inhibited the activity of CK II with IC50 44.7 mumol.L-1 (41.5-47.9 mumol.L-1), and AG1394 (144 mumol.L-1) strongly inhibited the activity of CK II with an inhibitory ratio of 89%. AG114 (174 mumol.L-1) and AG1109 (126 mumol.L-1) had inhibitory effects on the activity of CK II (p < 0.01). AG555 (136 mumol.L-1) had little effect on CK II activity. CONCLUSION: Some tyrphostins are potent inhibitors of CK II.