Agnes K. Nagy

Rapid Preparation of Synaptosomes from Mammalian Brain Using Nontoxic Isoosmotic Gradient Material (Percoll)

Abstract: A new procedure is described for the isolation of synaptosomes from various parts of mammalian brain. This method utilizes an isoosmotic Percoll/sucrose discontinuous gradient and has some advantages over the traditionally used synaptosomal isolation techniques: (1) it is possible to prepare suitable gradients while retaining isoosmolarity; (2) the time of the preparation is remarkably short (approximately 1 h); (3) if necessary, the gradient material can be easily removed from the samples. Intact synaptosomes were recovered from the 10%/16% (vol/vol) Percoll interphase. The fractions were identified and characterized by electron microscopy and by several biochemical markers for synaptosomes and other subcellular organelles. The homogeneity of the preparations is comparable to or better than that of synaptosomes prepared by the conventional methods. This procedure has been successfully used for the isolation of synaptosomes from very small tissue samples of various experimental animals and human brain.

Ecto-ATPase of mammalian synaptosomes: identification and enzymic characterization

Abstract: Intact synaptosomes isolated from mammalian brain tissues (rat, mouse, gerbil, and human) have an ATP hydrolyzing enzyme activity on their external surface. The synaptosomal ecto‐ATPase(s) possesses characteristics consistent with those that have been described for ecto‐ATPases of various other cell types. The enzyme has a high affinity for ATP (the apparent Km values are in the range of 2–5 × 10−5M), and is apparently stimulated equally well by either Mg2+ or Ca2+ in the absence of any other cations. The apparent activation constant for both divalent cations is approximately 4 × 10−4M in all mammalian brain tissues studied. The involvement of a nonspecific phosphatase in the hydrolysis of externally added ATP is excluded. ATP hydrolysis is maximal in the pH range 7.4–7.8 for both divalent cation‐dependent ATPase activities. Dicyclohexylcarbodiimide, 2,4‐dinitrophenol, trifluoperazine, chlorpromazine, and p‐chloromercuribenzoate (50 μM) inhibit the ecto‐ATPase, whereas ouabain (1 mM) and oligomycin (3.5 μg × mg−1 protein) show little or no inhibition of this enzyme activity. Inhibitor data suggest that the Mg2+‐ and Ca2+‐dependent ecto‐ATPase may represent two different enzymes on the surface of synaptosomes.

CHICKEN OVIDUCTAL ECTO-ATP-DIPHOSPHOHYDROLASE : PURIFICATION AND CHARACTERIZATION

An ecto-ATP diphosphohydrolase (ATPDase) was purified to homogeneity from vesiculosomes shed from chicken oviduct. First, the ecto-ATPDase-enriched vesiculosomes were concentrated by filtration, differential centrifugation, and exclusion chromatography. Next, the nonionic detergent, Nonidet P-40, was used to extract the ecto-ATPDase from vesiculosomal membranes, and the solubilized enzyme was further purified by ion exchange (DEAE-Bio-Gel) and lentil-lectin-Sepharose 4B chromatography. In the final stage, immunoaffinity chromatography was utilized to obtain purified ecto-ATPDase. More than 25,000-fold purification was achieved. Specific activity of the purified enzyme was greater than 800 μmol/min/mg of protein with MgATP as the substrate, the highest ever reported for an ATPDase. The enzyme also hydrolyzed other nucleoside triphosphates in the presence of magnesium at similar rates and CaATP and MgADP at lower rates. The molecular mass of the purified glycoprotein was 80 kDa as determined by SDS-polyacrylamide gel electrophoresis and Western blot analysis. Based on its enzymatic properties, the relationship of the chicken oviduct ecto-ATPDase with other reported ATPDases and ecto-ATPases is discussed.

Rat brain synaptosomal ATP:AMP-phosphotransferase activity.

Abstract: Adenylate kinase activity (ATPlAMP‐phospho‐transferase; EC 2.7.4.3) was studied in various subcellular fractions of rat brain tissues. Because of the presence of other adenosine nucleotide‐utilizing enzymes, adenylate kinase activity was assayed in both the forward and rejverse directions by using coupled enzyme systems and by bsing a specific adenylate kinase inhibitor, P1,P5‐di(adenasine‐5′) penta‐phosphate. As expected, the highest specific adenylate kinase activity (2.89 μumol/min/mg of protein) was detected in the cytosolic brain fraction. However, substantial enzyme activity (0.68 μumol/min/mg) was also found in the iintact synaptosomal fraction isolated on Percoll/sucrose gradients. The increased specific enzyme activity of purified sytfaptosomes and the differences found between the kinetic parameters of the membrane‐bound and cytosolic enzyme forms suggest that the synaptosomal adenylate kinase activity cannot be attributed to the small amount of contaminating cytosol present in our preparations. The adenylate kinase enzyme adhered to purified synaptic plasma membranes and was not released by washings with isoosmotic sucrose medium. The facts that the adenylate kinase enzyme activity could be measured in intact synaptosomal preparations and that both its substrates and its inhibitors do not cross intact plasma membranes support the possibility that the synaptosomal adenylate kinase is an ecto‐enzyme.

Molecular Cloning of the Chicken Oviduct Ecto-ATP-Diphosphohydrolase

The chicken oviduct ecto-ATP diphosphohydrolase (ATPDase), a member of the ecto-ATPase family, was purified to homogeneity previously (Strobel, R. S., Nagy, A. K., Knowles, A. F., Buegel, J., and Rosenberg, M. O. (1996) J. Biol. Chem. 271, 16323–16331). It is an 80-kDa glycoprotein with high specific activity (approximately 1,000 μmol/min/mg with MgATP as the substrate) and hydrolyzes both nucleoside triphosphates and diphosphates. Using amino acid sequence information obtained from the purified enzyme, two partial cDNA clones were obtained using reverse transcriptase-polymerase chain reaction and library screening. This is the second ecto-ATPase family member and the first ecto-ATPDase to be cloned from information derived from purified proteins. The deduced primary sequence of the chicken oviduct ecto-ATPDase indicates a protein of 493 amino acid residues with a molecular mass of 54 kDa. The predicted orientation shows it to be anchored to the membrane by two transmembranous segments near the NH2 and COOH termini with very short intracytoplasmic peptides at either end. The bulk of the protein is extracellular and contains 12 potentialN-glycosylation sites, several potential phosphorylation sites, and five sequences that are conserved in seven other related membrane proteins. Four of the conserved sequences, designated as apyrase conserved regions, are present in both ecto-ATPases and soluble E-type ATPases. The fifth conserved region, which occurs near the COOH terminus of the eight proteins, is observed only in the membrane-bound ecto-ATPases. Unexpectedly, sequence comparison revealed that the chicken oviduct ecto-ATPDase is equally distant from the two ecto-ATPases, which exhibit low activity toward ADP, and the four putative ecto-ATPDases, which are closely related to CD39.

Enzymatic Characteristics and Possible Role of Synaptosomal Ecto-Adenosine Triphosphatase from Mammalian Brain

In 1957 Engelhardt described a very active ATPase enzyme on the outer surface of avian (nucleated) red blood cells. He stated that the action of this enzyme is strictly oriented in space towards the surrounding medium: it splits rapidly any ATP that reaches the outside of the cell and leaves the ATP on the inside of the cell unaffected. He clearly distinguished this specific cell surface “ecto-enzyme” from “exo-enzymes” which are secreted into the extracellular space and also from the normal “endo-enzymes” which act within the cell. Prior to the observations of Engelhardt, a fairly active cell surface ATPase had already been reported by Acs et al. (1954) in ascites tumour cells. A detailed analysis of the Ehrlich ascites tumour cell surface ecto-ATPase was later carried out by Ronquist and Agren (1975).

Reduced cortical ecto-ATPase activity in rat brains during prolonged status epilepticus induced by sequential administration of lithium and pilocarpine

Considerable evidence indicates that ATP, acting intracellularly of as a neurotransmitter, can influence nerve cell physiology in a variety of ways. Defects in the functioning of ATP-metabolizing enzymes could therefore lead to disturbances in neurotransmission and creation of sustained neuronal discharges characteristic of status epilepticus. In this study we investigated synaptosomal ATPase changes in rat brains during lithium/pilocarpine-induced status epilepticus. After 2 h of continuous electroencephalographic spiking, both Mg(2+)- and Ca(2+)-dependent ecto-ATPases were significantly decreased in freshly prepared synaptosomal preparations from the status rats. The intracellularly acting Ca2+Mg(2+)-ATPase (Ca-pump) was also decreased, but no changes occurred in synaptosomal Na+K(+)-ATPase activity. The difference between ecto-ATPase activities of the control and status rat brains was not affected by repeated freezing-thawing and lengthy storage. Possible involvement of reduced synaptosomal divalent cation-dependent ATPases in the pathophysiology of status epilepticus is discussed.

Diabodies Targeting Epithelial Membrane Protein 2 Reduce Tumorigenicity of Human Endometrial Cancer Cell Lines

Purpose: Endometrial cancer is the most common gynecologic malignancy. One promising biomarker is epithelial membrane protein 2 (EMP2), and its expression is an independent prognostic indicator for tumors with poor clinical outcome expression. The present study assesses the suitability of EMP2 as a therapeutic target. Experimental Design: Human monovalent anti-EMP2 antibody fragments were isolated from a human phage display library and engineered as bivalent antibody fragments (diabodies) with specificity and avidity to both EMP2 peptides and native cell-surface EMP2 protein. Diabodies were assessed using cell death and apoptosis assays. In addition, the efficacy of EMP2 diabodies on endometrial cancer tumors was determined using mouse xenograft models. Results: Treatment of human endometrial adenocarcinoma cell lines with anti-EMP2 diabodies induced significant cell death and caspase-3 cleavage in vitro. These responses correlated with cellular EMP2 expression and were augmented by progesterone, which physiologically induces EMP2 expression. In vivo, treatment of subcutaneous human xenografts of HEC-1A cell lines with anti-EMP2 diabodies suppressed tumor growth and induced cell death in the xenograft. Conclusions: These findings suggest that EMP2 may be a potential pharmacologic target for human endometrial cancer.

Blockade of epithelial membrane protein 2 (EMP2) abrogates infection of Chlamydia muridarum murine genital infection model

New methods are needed to eradicate or prevent Chlamydia trachomatis infections. Blockade of epithelial membrane protein 2 (EMP2) by genetic silencing or neutralizing polyclonal antibody reduced chlamydial infectivity in vitro. This study tests the prediction that recombinant anti-EMP2 diabody could reduce early chlamydial infection of the genital tract in vivo. In a murine infection model, pretreatment with anti-EMP2 diabody, as compared with control diabody, significantly reduced bacterial load, tissue production of inflammatory cytokines, recruitment of polymorphonuclear leukocytes, and local tissue inflammation. These findings support EMP2 as a potential preventative and therapeutic target for genital chlamydial infection.

ECTO-Atpases of the Nervous System

During the past several years, convincing evidence has accumulated that ATP is stored within vesicles and co-released with various neurotransmitters upon depolarization of the nerve endings1–5. When released into neural junctions, ATP can fulfill multiple roles. As a co-transmitter, ATP modulates the release and effectivity of other neurotransmitters1, 2, 6–8 either by acting through its own, postjunctionally localized receptors9, or by altering the sensitivity of cholinoreceptors10 and adrenoreceptors 11. ATP has been well- established as a neurotransmitter in the peripheral nervous system4,9. The same role has also been suggested for ATP in the central nervous system12, which was supported by evidence showing that ATP could act as a fast excitatory transmitter in the brain13–15. In this role, ATP is assumed to act through a P2x-like purinoreceptor16–17 which is directly coupled to a non-voltage-dependent, high Ca2+-permeability ion channel18,19. On the other hand, ATP represents a major source of adenosine, a powerful neurosupressant4,23, which was shown to inhibit the release of excitatory neurotransmitters, such as acetylcholine24,25, dynorphin and glutamate8. Extracellular ATP can also serve as a substrate for ecto-protein kinases 20 and is thus involved in phosphorylation of various proteins on the neuronal cell surface21. In addition to its action in neurons, extracellular ATP is also assumed to be a signal molecule in neuronal-astrocytic interactions22.