Inhibition Of Translocase Research Articles

The chemical structure of mumbaistatin, a novel glucose-6-phosphate translocase inhibitor produced by Streptomyces sp. DSM 11641.

The characterization of the structure of mumbaistatin (1), an effective inhibitor of the glucose-6-phosphatase system (EC 3.1.3.9), is reported. Isolation of mumbaistatin from cultures of Streptomyces sp. DSM 11641 was achieved by anion-exchange and reversed-phase chromatography. The acid-labile inhibitor was methylated for the structure determination. Single-crystal X-ray structure analysis of a triply methylated dehydration product, C31H24O11, revealed the structure of an aromatic dispirodiketal (2), a compound containing a previously undescribed ring system. Extensive 2D-NMR experiments with mumbaistatin and with the methylation products showed that mumbaistatin itself possesses the hydroxydiketodicarboxylic acid structure 1, C28H20O12, which, in the presence of acid or upon activation through methyl ester formation, undergoes self-condensation with loss of water to the dispirodiketal form (2). Mumbaistatin is an anthraquinone derivative, whose open-chain diketo form acts as a specific and powerful inhibitor of glucose-6-phosphate translocase: IC50=5 nM. The activity towards the same enzyme of the cyclized dispirodiketal derivatives is roughly one thousand times lower.

N-Bromoacetylethanolamine Phosphate as a Probe for the Identification of a Liver Microsomal Glucose-6-Phosphate Transporter Peptide in Rats and Ehrlich Ascites Tumor-Bearing Mice

Hepatic microsomal glucose-6-phosphatase is a multicomponent system composed of substrate/product translocases and a catalytic subunit. Previously we (Foster et al. (1996) Biochim. Biophys. Acta 12, 244–254) demonstrated that N-bromoacetylethanolamine phosphate (BAEP) is a time-dependent, irreversible inhibitor of glucose-6-phosphate hydrolysis in intact but not disrupted microsomes. We proposed that BAEP manifests its inhibitory effect by binding with a glucose-6-phosphate translocase protein of the glucose-6-phosphatase system. Here we provide additional evidence that BAEP inhibits glucose-6-phosphate transport in microsomal vesicles and utilize [32P]BAEP as an affinity label in the identification of a glucose-6-phosphate transport protein. In this study, we identify 51-kDa rat and mouse liver microsomal proteins involved in glucose-6-phosphate transport into and out of microsomal vesicles by utilizing (1) an Ehrlich ascites tumor-bearing mouse model, which displays a decreased sensitivity to the time-dependent inhibitory effect of BAEP, and (2) another glucose-6-phosphate translocase inhibitor, tosyl-lysine chloromethyl ketone, in conjunction with [32P]BAEP as an affinity label.

Alterations of carbohydrate and lipid intermediary metabolism during inhibition of glucose-6-phosphatase in rats

S 4048 (1-[2-(4-Chloro-phenyl)-cyclopropylmethoxy]-3,4-dihydroxy-5-(3-imidazo[4,5- b]pyridin-1-yl-3-phenyl-acryloyloxy)-cyclohexanecarboxylic acid), a derivative of chlorogenic acid, specifically inhibits the glucose-6-phosphate translocating component T1 of the glucose-6-phosphatase system. Its pharmacological effect was studied on carbohydrate and lipid parameters in rats. In starved and fed rats, S 4048 caused a dose-dependent reduction of blood glucose levels with a corresponding increase in hepatic and renal glycogen and glucose-6-phosphate. The major quantitative route of carbon flux in the liver during S 4048-induced inhibition of the glucose-6-phosphatase activity seemed to be glycogenesis. Plasma free fatty acids were increased secondarily due to the S 4048-induced hypoglycemia. Hepatic triglycerides were increased possibly due to increased re-esterification of the readily available free fatty acids. Glucose-6-phosphate translocase inhibitors may be useful for experimentally studying aspects of type 1 glycogen storage disease in laboratory animals as well as for the therapeutic modulation of inappropriately high rates of hepatic glucose production in type 2 diabetes.

Identification of protein components of the microsomal glucose 6-phosphate transporter by photoaffinity labelling

The glucose-6-phosphatase system catalyses the terminal step of hepatic glucose production from both gluconeogenesis and glycogenolysis and is thus a key regulatory factor of blood glucose homoeostasis. To identify the glucose 6-phosphate transporter T1, we have performed photoaffinity labelling of human and rat liver microsomes by using the specific photoreactive glucose-6-phosphate translocase inhibitors S 0957 and S 1743. Membrane proteins of molecular mass 70, 55, 33 and 31 kDa were labelled in human microsomes by [3H]S 0957, whereas in rat liver microsomes bands at 95, 70, 57, 54, 50, 41, 33 and 31 kDa were detectable. The photoprobe [3H]S 1743 led to the predominant labelling of a 57 kDa and a 50 kDa protein in the rat. Stripping of microsomes with 0.3% CHAPS retains the specific binding of T1 inhibitors; photoaffinity labelling of such CHAPS-treated microsomes resulted in the labelling of membrane proteins of molecular mass 55, 33 and 31 kDa in human liver and 50, 33 and 31 kDa in rat liver. Photoaffinity labelling of human liver tissue samples from a healthy individual and from liver samples of patients with a diagnosed glycogen-storage disease type 1b (GSD type 1b; von Gierke's disease) revealed the absence of the 55 kDa protein from one of the patients with GSD type 1. These findings support the identity of the glucose 6-phosphate transporter T1, with endoplasmic reticulum protein of molecular mass 50 kDa in rat liver and 55 kDa in human liver.

Inhibition of the Ryanodine Receptor Calcium Channel in the Sarcoplasmic Reticulum of Skeletal Muscle by an ADP/ATP Translocase Inhibitor, Atractyloside

The effects of an inhibitor of ADP/ATP translocase (AAT) mainly expressed in the mitochondria inner membrane, atractyloside (ATR), on the gating property of the Ca2+channels in the sarcoplasmic reticulum (SR) vesicles from the rabbit skeletal muscle were investigated using ion flux measurement and single channel recording. At 10 μM of cytoplasmic Ca2+, ATR decreased the rate constant of choline+influx through the Ca2+channels up to about 60% and perfectly inhibited about half the population of single Ca2+channels incorporated into planar bilayers. Furthermore, the inhibition of the Ca2+channels by ATR was effective at lower Ca2+. These results support the previous results that AAT exists in the skeletal muscle SR and plays a key role in the Ca2+mobilization of the skeletal muscle cell [Yamaguchi, N., and Kasai, M. (1998)Biochem. J.335, 541–547], and the number of Ca2+channels regulated by AAT is thought to depend on the cytoplasmic Ca2+concentration.

Identification of protein components of the microsomal glucose 6-phosphate transporter by photoaffinity labelling

The glucose-6-phosphatase system catalyses the terminal step of hepatic glucose production from both gluconeogenesis and glycogenolysis and is thus a key regulatory factor of blood glucose homoeostasis. To identify the glucose 6-phosphate transporter T1, we have performed photoaffinity labelling of human and rat liver microsomes by using the specific photoreactive glucose-6-phosphate translocase inhibitors S 0957 and S 1743. Membrane proteins of molecular mass 70, 55, 33 and 31 kDa were labelled in human microsomes by [3H]S 0957, whereas in rat liver microsomes bands at 95, 70, 57, 54, 50, 41, 33 and 31 kDa were detectable. The photoprobe [3H]S 1743 led to the predominant labelling of a 57 kDa and a 50 kDa protein in the rat. Stripping of microsomes with 0.3% CHAPS retains the specific binding of T1 inhibitors; photoaffinity labelling of such CHAPS-treated microsomes resulted in the labelling of membrane proteins of molecular mass 55, 33 and 31 kDa in human liver and 50, 33 and 31 kDa in rat liver. Photoaffinity labelling of human liver tissue samples from a healthy individual and from liver samples of patients with a diagnosed glycogen-storage disease type 1b (GSD type 1b; von Gierke's disease) revealed the absence of the 55 kDa protein from one of the patients with GSD type 1. These findings support the identity of the glucose 6-phosphate transporter T1, with endoplasmic reticulum protein of molecular mass 50 kDa in rat liver and 55 kDa in human liver.

Structure–function studies on nucleoside antibiotic mureidomycin A: synthesis of 5′-functionalised uridine models

The importance of functional groups in nucleoside antibiotic mureidomycin A (MRD A) for biological activity has been examined by derivatisation of samples of the natural product, and by synthesis of uridine-containing analogues. N-Succinyl and di- and tri-acetyl derivatives MRD A have been prepared, and were found to have reduced activity as inhibitors of E. coli translocase I. The enamide alkene of MRD A was found to be extremely resistant towards hydrogenation by a variety of reagents. Several 5′-functionalised uridine derivatives were synthesised from N 3-p-methoxybenzyl-2′,3′-isopropylideneuridine. A series of 5′-aminoacyl derivatives were prepared, and the 3-aminopropionyl (IC50 260 µM) and 7-aminoheptanoyl (IC50 1.5 mM) derivatives were found to act as reversible inhibitors. An analogue mimicking the carboxy terminus of MRD A was synthesised, and also acted as an inhibitor of translocase I (IC50 1.9 mM). A phosphonate analogue designed as a possible suicide inhibitor showed modest inhibition (IC50 3.7 mM), which was shown to be irreversible.

Role of the enamide linkage of nucleoside antibiotic mureidomycin A: synthesis and reactivity of enamide-containing analogues

The reactivity of an unusual enamide functional group in the nucleoside antibiotic mureidomycin A (MRD A) has been investigated by synthesis of enamide-containing analogues. Enamides based on 2-methoxyethylamine and tetrahydrofurfurylamine were found to be unstable and reactive, whereas a uridine-based analogue showed high stability and low reactivity. Samples of mureidomycin A and the uridine-based analogue were found to be stable towards acid-catalysed tautomerisation and nucleophilic attack. The lack of reactivity and lack of enzyme inhibition shown by the uridine-based analogue are not consistent with the involvement of the enamide group in slow-binding inhibition of translocase I.

Integrating cytosolic calcium signals into mitochondrial metabolic responses.

Stimulation of hepatocytes with vasopressin evokes increases in cytosolic free Ca2+ ([Ca2+]c) that are relayed into the mitochondria, where the resulting mitochondrial Ca2+ ([Ca2+]m) increase regulates intramitochondrial Ca2+-sensitive targets. To understand how mitochondria integrate the [Ca2+]c signals into a final metabolic response, we stimulated hepatocytes with high vasopressin doses that generate a sustained increase in [Ca2+]c. This elicited a synchronous, single spike of [Ca2+]m and consequent NAD(P)H formation, which could be related to changes in the activity state of pyruvate dehydrogenase (PDH) measured in parallel. The vasopressin-induced [Ca2+]m spike evoked a transient increase in NAD(P)H that persisted longer than the [Ca2+]m increase. In contrast, PDH activity increased biphasically, with an initial rapid phase accompanying the rise in [Ca2+]m, followed by a sustained secondary activation phase associated with a decline in cellular ATP. The decline of NAD(P)H in the face of elevated PDH activity occurred as a result of respiratory chain activation, which was also manifest in a calcium-dependent increase in the membrane potential and pH gradient components of the proton motive force (PMF). This is the first direct demonstration that Ca2+-mobilizing hormones increase the PMF in intact cells. Thus, Ca2+ plays an important role in signal transduction from cytosol to mitochondria, with a single [Ca2+]m spike evoking a complex series of changes to activate mitochondrial oxidative metabolism.

Amphiphile-induced phosphatidylserine exposure in human erythrocytes.

Nonionic and anionic water-soluble amphiphiles were shown to increase strongly the binding of fluorescein isothiocyanate-conjugated annexin V (FITC-annexin V) in human erythrocytes pretreated with the aminophospholipid translocase (APLT) inhibitor n-ethylmaleimide (NEM). At high sublytic amphiphile-concentrations the binding of FITC-annexin V, monitored in a flow cytometer, was time- and temperature-dependent and occurred heterogeneously in the cell population, with 43-81% of cells being stained above background following incubation for 60 minutes at 37 degrees C. The increased FITC-annexin V binding apparently indicates an increased flop rate of phosphatidylserine (PS) to the outer membrane leaflet. When the NEM-pretreatment was omitted, the FITC-annexin V binding was markedly, but not completely, reduced. In erythrocytes incubated with a zwitter-ionic amphiphile, a small increase in FITC-annexin V binding was detected, while cationic amphiphiles did not induce an increased FITC-annexin V binding. The potency of amphiphiles to induce PS exposure was not related to the type of shape alteration or vesiculation induced. Our results indicate a significant role of the charge status of a membrane intercalated amphiphile for its capability to induce PS exposure.

Chlorogenic acid and synthetic chlorogenic acid derivatives: novel inhibitors of hepatic glucose-6-phosphate translocase.

The enzyme system glucose-6-phosphatase (EC 3.1.3.9) plays a major role in the homeostatic regulation of blood glucose. It is responsible for the formation of endogenous glucose originating from gluconeogenesis and glycogenolysis. Recently, chlorogenic acid was identified as a specific inhibitor of the glucose-6-phosphate translocase component (Gl-6-P translocase) of this enzyme system in microsomes of rat liver. Glucose 6-phosphate hydrolysis was determined in the presence of chlorogenic acid or of new synthesized derivatives in intact rat liver microsomes in order to assess the inhibitory potency of the compounds on the translocase component. Variation in the 3-position of chlorogenic acid had only poor effects on inhibitory potency. Introduction of lipophilic side chain in the 1-position led to 100-fold more potent inhibitors. Functional assays on isolated perfused rat liver with compound 29i, a representative of the more potent derivatives, showed a dose-dependent inhibition of gluconeogenesis and glycogenolyosis, suggesting glucose-6-phosphatase as the locus of interference of the compound for inhibition of hepatic glucose production also in the isolated organ model. Gl-6-P translocase inhibitors may be useful for the reduction of inappropriately high rates of hepatic glucose output often found in non-insulin-dependent diabetes.

Tauroursodeoxycholic acid for the treatment of chronic hepatitis: A dose-response study

Background: Tauroursodeoxycholic acid (TUDCA) is of potential benefit in cholestatic disorders. However, the effects of TUDCA on cytosolic free calcium [(Ca2+)i], which regulates hepatocyte secretion, are unknown. Methods: The effect of TUDCA on (Ca2+)i was investigated in groups of isolated rat hepatocytes by microspectrofluorometry and in single cells by confocal line scanning microscopy. Results: Administration of TUDCA (5–50 μmol/L) induced a nearly fourfold increase of basal levels of (Ca2+)i. After a 15 minute treatment period, the TUDCA (10 μmol/L)-induced change in (Ca2+)i was higher than that of other monodi-, and trihydroxy bile acids at equimolar concentrations. Pretreatment with TUDCA (10 μmol/L) markedly reduced or abolished increases in (Ca2+)i induced by phenylephrine (1 μmol/L), the microsomal Ca2+ translocase inhibitor 2,5-di-(tert-butyl)-1,4-benzohydroquinone (25 μmol/L), or taurolithocholic acid (10–25 μmol/L). In Ca2+-free medium, TUDCA caused only a reduced and transient increase in (Ca2+)i. TUDCA (10 μmol/L) induced Ca2+ oscillations in all single cells that responded. However, levels of inositol-1,4,5-trisphosphate (IP3) in hepatocytes were not increased by treatment with TUDCA (10 μmol/L). Conclusions: TUDCA at physiological concentrations potently modulates (Ca2+)i signals in hepatocytes by (1) mobilizing microsomal IP3-sensitive Ca2+ stores by an IP3-independent mechanism, (2) initiating Ca2+ oscillations, and (3) inducing influx of extracellular Ca2+.

Calcium Stimulation of Procoagulant Activity in Human Erythrocytes: ATP DEPENDENCE AND THE EFFECTS OF MODIFIERS OF STIMULATION AND RECOVERY

The human erythrocyte membrane is generally considered to have no procoagulant activity. The normal membrane is characterized as having an asymmetric distribution of phospholipid species such that negatively charged and aminophospholipids are predominantly located on the inner leaflet of the membrane bilayer. Elevation of cytoplasmic Ca2+ in erythrocytes produces an assortment of biochemical and structural responses that include diminished phospholipid asymmetry and an elevation in procoagulant activity. Maintenance of the normal asymmetric distribution of phospholipid species is believed to be largely mediated by a phospholipid translocase mechanism. We have utilized a recently developed single-step kinetic assay of procoagulant activity to investigate the mechanisms of Ca2+ stimulation of procoagulant activity and recovery from the procoagulant state upon removal of Ca2+. This study demonstrated that stimulation of procoagulant activity by elevated cytoplasmic Ca2+ is greatly diminished in ATP-depleted erythrocytes. Phospholipid translocase inhibitors failed to fully inhibit recovery from the procoagulant state after removal of Ca2+. The data indicate that recovery of endogenous lipid from a procoagulant cofiguration may not be entirely mediated by the phospholipid translocase. Additionally, the data are inconsistent with the phospholipid translocase mediating the Ca(2+)-induced elevation of procoagulant activity, although the involvement of other protein(s) is indicated.

Involvement of mitochondria in the assimilatory metabolism of anaerobic Saccharomyces cerevisiae cultures

The possible physiological role of mitochondria in anaerobically grown Saccharomyces cerevisiae was investigated via enzyme localization and inhibitor studies. Almost all of the activity of citrate synthase (EC 4.1.3.7) was recovered in the mitochondrial fraction after differential centrifugation of spheroplast lysates. The enzyme exhibited a high degree of latency which was demonstrated by sonication of the mitochondrial fractions. Since citrate synthase is an important enzyme in anabolic reactions, a consequence of this localization is the requirement for transport of metabolites across the mitochondrial membranes. Such transport is likely to require energy which, as a result of anaerobiosis, cannot be supplied by respiration. It was therefore investigated whether ATP translocation into the mitochondria by an ADP/ATP translocase might be involved in anaerobic mitochondrial energy metabolism. It was shown that addition of the ADP/ATP translocase inhibitor bongkrekic acid to anaerobic cultures indeed inhibited growth, although only partially. It is concluded that mitochondria of S. cerevisiae fulfil a vital role in anaerobic sugar metabolism.

Effect of L-Carnitine on Mitochondrial Acyl CoA Esters in the Ischemic Dog Heart

Many studies have shown that L-carnitine has a positive effect on ischemic myocardium, probably by reducing accumulation of long-chain acyl coenzyme A (CoA) esters. Previous studies have involved whole-heart extracts and have not assessed changes of CoA ester levels in mitochondria, the site of translocase inhibition. To more precisely assess L-carnitine effects, we measured long-chain acyl CoA ester levels in cytosol and in mitochondria in the ischemic canine heart. Dogs were divided into four groups: a sham-operated control group; an untreated group; and high- and low-dose L-carnitine-treated groups (30 mg/kg and 100 mg/kg). After 60 min of ischemia, the heart was excised, and the cytosolic and mitochondrial fractions were isolated. CoA esters and the activity of carnitine palmitoylcarnitine transferase (CPT) I and II were measured in both compartments. Approximately 89% of cellular free CoA, 90% of cellular acetyl CoA, 97% of cellular shot-chain acyl CoA, and 92% of cellular long-chain acyl CoA were located in the mitochondrial space under the normal condition. Under the ischemic condition, mitochondrial free CoA was significantly decreased. Conversely, mitochondrial acetyl CoA and long-chain acyl CoA were significantly increased. Treatment with L-carnitine significantly decreased acetyl CoA and long-chain acyl CoA in the ischemic mitochondrial space in a dose-dependent manner. These results support the hypothesis that L-carnitine reduces accumulation of long-chain acyl CoA within the ischemic mitochondrial space and thereby improves mitochondrial function and adenine nucleotide translocation.

Selective inhibition of the bacterial translocase reaction in peptidoglycan synthesis by mureidomycins.

Mureidomycins (MRDs) A and C inhibited strongly the formation of undecaprenyl pyrophosphoryl N-acetylmuramyl-pentapeptide (lipid intermediate I), which is an intermediate in bacterial peptidoglycan synthesis (50% inhibitory concentration [IC50] of MRD A, 0.05 microgram/ml). However, they did not inhibit the formation of dolichyl pyrophosphoryl N-acetylglucosamine (Dol-p-p-GlcNAc), dolichyl phosphoryl glucose, or dolichyl phosphoryl mannose, the precursors for mammalian glycoprotein synthesis, or the formation in Bacillus subtilis of lipid-linked N-acetylglucosamine for teichoic acid synthesis (IC50s, > 100 micrograms/ml). In contrast, tunicamycin (TCM) inhibited strongly the formation of Dol-p-p-GlcNAc (IC50, 0.03 microgram/ml) but inhibited weakly the formation of bacterial lipid intermediate I (IC50, 44 micrograms/ml). When the effects of MRDs A and C and TCM on the growth of mammalian cells were compared, MRDs did not show any toxicity, even at 1,000 micrograms/ml, whereas TCM inhibited the growth of BALB/3T3 cells at 10 micrograms/ml. On the basis of these results, it was concluded that MRDs are the first specific and potent inhibitors of the translocase reaction in bacterial peptidoglycan synthesis, showing a high level of toxicity against bacteria and a low level of toxicity against mammalian cells. A specific inhibitor of translocase could be a potent antibiotic with highly selective toxicity.

Investigation of cardiac metabolism using stable isotopes and mass spectrometry.

The technique described in this communication enables detailed investigations of cardiac metabolism using 13C-labeled substrates and mass spectrometric measurements of 13CO2 in the coronary effluent. To validate this technique for further studies isolated working rat hearts were perfused with 13C-labeled substrates in a bicarbonate-free perfusion fluid. The fraction of CO2 produced by oxidation of labeled substrate was calculated by the 13CO2/CO2 ratio in the coronary perfusate. The oxidation of 13C-acetate showed a linear correlation with 13C-acetate concentrations between 0.015 and 0.16 mmol/l. An inhibitor of acylcarnitine translocase, 2-(3-methylcinnamylhydrazono)-propionate (BM42.304) decreased CO2 production from 13C-palmitate from 48% +/- 4% to 31% +/- 3% (n = 11, SEM). Taking into account considerations of tracer kinetic theory rapidly accessible intracellular palmitate stores were estimated to be less than 900 nmol/g ww. This technique allows specific investigations of the oxidation of labeled substrates in the heart and may be useful for basic research and/or clinical diagnosis, thus avoiding the hazards of radiolabeled substrates.

Effects of tauroursodeoxycholic acid on cytosolic Ca2+ signals in isolated rat hepatocytes

Background: Tauroursodeoxycholic acid (TUDCA) is of potential benefit in cholestatic disorders. However, the effects of TUDCA on cytosolic free calcium [(Ca2+)i], which regulates hepatocyte secretion, are unknown. Methods: The effect of TUDCA on (Ca2+)i was investigated in groups of isolated rat hepatocytes by microspectrofluorometry and in single cells by confocal line scanning microscopy. Results: Administration of TUDCA (5–50 μmol/L) induced a nearly fourfold increase of basal levels of (Ca2+)i. After a 15 minute treatment period, the TUDCA (10 μmol/L)-induced change in (Ca2+)i was higher than that of other monodi-, and trihydroxy bile acids at equimolar concentrations. Pretreatment with TUDCA (10 μmol/L) markedly reduced or abolished increases in (Ca2+)i induced by phenylephrine (1 μmol/L), the microsomal Ca2+ translocase inhibitor 2,5-di-(tert-butyl)-1,4-benzohydroquinone (25 μmol/L), or taurolithocholic acid (10–25 μmol/L). In Ca2+-free medium, TUDCA caused only a reduced and transient increase in (Ca2+)i. TUDCA (10 μmol/L) induced Ca2+ oscillations in all single cells that responded. However, levels of inositol-1,4,5-trisphosphate (IP3) in hepatocytes were not increased by treatment with TUDCA (10 μmol/L). Conclusions: TUDCA at physiological concentrations potently modulates (Ca2+)i signals in hepatocytes by (1) mobilizing microsomal IP3-sensitive Ca2+ stores by an IP3-independent mechanism, (2) initiating Ca2+ oscillations, and (3) inducing influx of extracellular Ca2+.

Domain V of Giardia lamblia large-subunit rRNA: structure of the peptidyl transferase loop from an early-branching eukaryote and correlation with antibiotic sensitivity

Large subunit rRNA (LSR) sequences that have been implicated in peptide bond formation form a specific secondary structure called the peptidyl transferase loop (PTL). Although well conserved, the PTLs of eubacteria, archaebacteria, and eukaryotes have several distinct differences. These differences correlate with different sensitivities to peptidyl transferase and translocase inhibitors. To shed light on the basis for these kingdom-specific differences in PTL structure and function, we have analyzed the sequence and secondary structure of LSR domain V, which contains the PTL, from Giardia lamblia. This parasitic protozoan derives from a very early branch in eukaryotic evolution, and its rRNA was previously shown to have bacteria-like features. In vitro and cell-free systems were also used to test the sensitivity of G. lamblia protein synthesis to specific PTL-targeted inhibitors. Our results indicate that the PTL structure and inhibitor sensitivity typical of higher eukaryotes is conserved in G. lamblia. However, several adjacent domain V sequences more closely resemble archaebacterial rRNA, confirming the ‘primitive’ nature of G. lamblia rRNA. Thus, the eukaryotic PTL has been conserved over a vast evolutionary period. We speculate that the eukaryotic PTL is primordial and employs specific RNA-RNA interactions to catalyze protein synthesis. Three potential interactions were identified.

Phospholipid transverse mobility modifications in plasma membranes of activated platelets: An ESR study

Spin labeled phospholipid analogs were used to directly study changes in aminophospholipid translocase activity in activated platelets. In thrombin-activated platelets, the translocase activity was slightly stimulated, whereas no vesicle formation or proteolysis of cytoskeletal protein occurred. Ca 2+ ionophore A23187-mediated activation produced vesiculation and proteolysis. Additionally, the translocase activity was completely inhibited, probably due to a sharp rise the intracellular Ca 2+ concentration, as shown when platelets were activated in the presence of various A23187 and Ca 2+ concentrations and by the recovery of the translocase activity when Ca 2+ was complexed with EGTA. No translocase activity was found in vesicles. Whereas vesiculation and translocase inhibition can occur independently of proteolysis, this later accentuated the shedding phenomenon.