Concomitant Localization of Dopa Oxidase and Adenosine Triphosphatase in Cryostat Sections

Cryostat sections fixed 10 min in calcium formalin were incubated sequentially in 0.1% DOPA, 2 hr at 37 C, and ATPase substrate 1% hr at 37 C. The enzymatically produced calcium phosphate was visualized by 0.2% glyoxal-bis(2-hydroxyanil) (GBHA) in alkaline ethanol as a red precipitate. Nonspecific protein-bound calcium ions which obscured active sites in such formalin-fixed material were successfully removed by treatment with 0.5% ethylenediaminetetraacetic acid at pH 7.0 for 1 min before treatment with GBHA. Phosphatase sites were red; DOPA active sites, black. The method was also successfully applied to the demonstration of alkaline phosphatases. Acetone fixation inhibited both enzymes; fixation in 70% alcohol suppressed the DOPA reaction and partially inhibited ATPase.     

Localization of phosphatases in Trypanosoma rhodesiense

Phosphatase activity in Trypanosoma rhodesiense has been examined histochemically by light and electron microscopy and by enzymatic assay in homogenate fractions. Using a method with lead as capture ion, acid phosphatase was found in lysosome-like vesicles and in the flagellar pocket. No alkaline adenosine triphosphatase (ATPase) was detectable by this method. Direct assay of p-nitrophenylphosphatase activity in homogenate fractions showed that acid phosphatase activity was strongly membrane-bound, but that activity at pH 9 was minimal in both soluble and particulate fractions. "Endogenous" ATPase activity was localized specifically and reproducibly in the mitochondrial membranes and under the plasma membrane of he flagellum. This nonenzymic reaction product could not be eradicated by glycerol extraction or glucose depletion. Unlike the membrane staining, which was manifest only after lead treatment, heat-resistant electron-dense material was found in the matrix of lysosomal vesicles in trypanosomes fixed in glutaraldehyde only and not subjected to further treatment with heavy metal reagents. X-ray emission analysis showed the presence of calcium and phosphorus, indicating that the matrix might have a phosphate storage function.     

Membrane-associated thiamine triphosphatase in rat skeletal muscle.

1. Thiamine triphosphatase activity in particulate fraction, but not in soluble, of rat skeletal muscle was stimulated by several anions. 2. The stimulative effect of anions was dependent on pH of reaction medium and was reversible. 3. The activities of ATPase in rat muscle particulate preparation and thiamine triphosphatase in the brain were inhibited by the anions.     

Localization of Phosphatases in Trypanosoma rhodesiense1

ABSTRACT. Phosphatase activity in Trypanosoma rhodesiense has been examined histochemically by light and electron microscopy and by enzymatic assay in homogenate fractions. Using a method with lead as capture ion, acid phosphatase was found in lysosome-like vesicles and in the flagellar pocket. No alkaline adenosine triphosphatase (ATPase) was detectable by this method. Direct assay of p-nitrophenylphosphatase activity in homogenate fractions showed that acid phosphatase activity was strongly membrane-bound, but that activity at pH 9 was minimal in both soluble and particulate fractions. “Endogenous” ATPase activity was localized specifically and reproducibly in the mitochondrial membranes and under the plasma membrane of the flagellum. This nonenzymic reaction product could not be eradicated by glycerol extraction or glucose depletion. Unlike the membrane staining, which was manifest only after lead treatment, heat-resistant electron-dense material was found in the matrix of lysosomal vesicles in trypanosomes fixed in glutaraldehyde only and not subjected to further treatment with heavy metal reagents. X-ray emission analysis showed the presence of calcium and phosphorus, indicating that the matrix might have a phosphate storage function.     

The properties and structure of the membrane ATPase from Vibrio alginolyticus

Abstract Membrane ATPase of marine alkali-tolerant bacterium Vibrio alginolyticus has been studied. The ATPase was inhibited by diethylstilbestrol, DCCD, NBD-Cl and sodium azide, but it was insensitive to vanadate. The ATPase was solubilized by EDTA-treatment and partially purified by ion exchange chromatography. Two major polypeptides with molecular weights of 55 kDa and 58 kDa were found in this preparation. The molecular weight of the soluble ATPase was estimated to be 360 kDa. These data indicate, that the ATPase of Vibrio alginolyticus is likely to belong to F₀–F₁ type.     

Phosphatidyl glycerophosphate phosphatase

An enzyme (phosphatidyl glycerophosphate phosphatase) that catalyzes the formation of phosphatidyl glycerol from phosphatidyl glycerophosphate has been rendered soluble by treatment of the particulate fraction of E. coli with Triton X-100 in the presence of EDTA, and has been partially purified. The enzyme is specific for phosphatidyl glycerophosphate and does not catalyze the hydrolysis of other simple phosphomonoesters. It requires Mg(++) for activity and is inhibited by sulfhydryl agents. Some other properties of the enzyme are also described.     

The uptake and degradation of matrix-bound lipoproteins by macrophages require an intact actin Cytoskeleton, Rho family GTPases, and myosin ATPase activity

A key cellular event in atherogenesis is the interaction of macrophages with lipoproteins in the subendothelium. In vivo, these lipoproteins are bound to matrix and often aggregated, yet most cell-culture experiments explore these events using soluble monomeric lipoproteins. We hypothesized that the internalization and degradation of matrix-retained and aggregated low density lipoprotein (LDL) by macrophages may involve the actin-myosin cytoskeleton in a manner that distinguishes this process from the endocytosis of soluble LDL. To explore these ideas, we plated macrophages on sphingomyelinase-aggregated LDL bound to smooth muscle cell-derived matrix in the presence of lipoprotein lipase. The macrophages internalized and degraded the LDL, which was mediated partially by the LDL receptor-related protein. Cytochalasin D and latrunculin A, which block actin polymerization, markedly inhibited the uptake and degradation of matrix-retained LDL but not soluble LDL. Inhibition of Rho family GTPases by Clostridium difficile toxin B blocked the degradation of matrix-retained and aggregated LDL by >90% without any inhibition of soluble LDL degradation. However, specific inhibition of Rho had no effect, suggesting the importance of Rac1 and Cdc42. Degradation of matrix-retained, but not soluble, LDL was also blocked by inhibitors of tyrosine kinase, phosphatidylinositol 3-kinase, and myosin ATPase. These findings define fundamental cytoskeletal pathways that may be involved in macrophage foam cell formation in vivo but have been missed by the use of previous cell culture models.     

Effect of Purine and Pyrimidine Analogues on Growth and RNA Metabolism in the Soybean Hypocotyl-the Selective Action of 5-Fluorouracil

The effects of several base analogues and cycloheximide on RNA synthesis, protein synthesis, and cell elongation were studied in excised soybean hypocotyl. None of the pyrimidine analogues tested affected growth or protein synthesis; only 5-fluorouracil appreciably inhibited RNA synthesis. 8-Azaguanine and 6-methylpurine markedly inhibited RNA and protein synthesis and cell elongation. Cycloheximide effectively inhibited both cell elongation and protein synthesis.The results show that 5-fluorouracil selectively inhibited ribosomal and soluble RNA synthesis without affecting the synthesis of D-RNA. These results indicate that the requirement for RNA synthesis to support continued protein synthesis and cell elongation is restricted to the synthesis of D-RNA.5-Fluorouracil was incorporated into all classes of RNA in a form believed to be 5-fluorouridylic acid.Cycloheximide markedly inhibited the accumulation of ribosomal RNA, but the results indicate that CH did not inhibit, per se, the synthesis of ribosomal RNA. The accumulation of newly synthesized D-RNA was only slightly affected by cycloheximide. These results show that the inhibition of cell elongation by cycloheximide correlates with the inhibition of protein synthesis, but not with the effect on RNA metabolism.     

ATPase and acid phosphatase activities associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.)

Phosphatase activities were measured in preparations of vacuoles isolated from storage roots of red beet (Beta vulgaris L.). The vacuoles possessed both acid phosphatase and ATPase activities which could be distinguished by their susceptibility to inhibition by low concentrations of ammonium molybdate [(NH₄)₆Mo₇O₂₄·4H₂O]. The acid phosphatase was completely inhibited by 100 M ammonium molybdate but the ATPase was unaffected. The acid phosphatase was a soluble enzyme which hydrolysed a large number of phosphate esters and had a pH optimum of 5.5. In contrast, the ATPase was partially membrane-bound, had a pH optimum of 8.0 and hydrolysed ATP preferentially, although it was also active agianst PP_i, GTP and GDP. At pH 8.0 both the ATPase and PPase activities were Mg²⁺-dependent and were further stimulated by KCl. The ATPase and PPase activities at pH 8.0 may be different enzymes. The recovery and purification of the ATPase during vacuole isolation were determined. The results indicate that the Mg²⁺-dependent, KCl-stimulated ATPase activity is not exclusively associated with vacuoles.Abbreviations BSA bovine serum albumen - MES 2-(N-Morpholino)ethanesulphonic acid - MOPS 3-(N-Morpholino)propanesulphonic acid - Na₂EDTA ethylenediaminetetra-acetic acid, disodium salt - P_i inorganic phosphate - PP_i inorganic pyrophosphate - PPase inorganic pyrophosphatase - TCA trichloroacetic acid - TES N-tris(hydroxymethyl)methyl-2-amino-ethanesulphonic acid - Tris tris(hydroxymethyl)methylamine     

Phosphoprotein phosphatase in the central nervous system of Manduca sexta.

The existence and some enzymological properties of phosphoprotein phosphatase (EC 3.1.3.16) have been established in the larval central nervous system of the tobacco hornworm, Manduca sexta (Lepidoptera: Sphingidae). A simple, sensitive and reproducible assay employing 32-P-labeled protamine as a phosphoprotein substrate was employed to measure phosphatase activity in both soluble and particulate fractions of the insect nerve cord. The specific activity of soluble phosphatase in the Manduca sexta central nervous system is of the same order of magnitude as that in mammalian brain. Nerve cord phosphoprotamine phosphatase activity may be stimulated by a variety of monovalent salts, the optimal concentration of NaCl or KCl being 0.2 molar. Activity does not appear to be dependent on bivalent metals and is stimulated by EDTA. A reduced sulfhydryl group is obligatory for maximum activity. Phosphatase could be greatly inhibited by sodium fluoride, ATP and GTP. Cyclic AMP and cyclic GMP are without effect on enzyme activity. Although most of the phosphatase activity in the insect nerve cord appears to be of cytosolic origin, much latent activity can be unmasked by incubating membranous fractions with Triton X-100. In contrast to soluble phosphatase, the detergent-solubilized activity is moderately stimulated by Mn-2+.?     

Purification and properties of the adenosine triphosphatase released from the liver mitochondrial membrane by chloroform

1. Soluble ATPase (adenosine triphosphatase) activity is released when rat liver submitochondrial particles are shaken with chloroform, provided that ATP or glycerol is present in the suspending medium. The extraction is very rapid and appears to be complete. 2. The ATPase of the chloroform extract is about 50% pure and can be readily purified to a specific activity of 60-70mumol/min per mg of protein by (NH(4))(2)SO(4) fractionation and column chromatography on Sephadex G-200. 3. The particulate and soluble ATPases have many similar properties, including their K(m) values for ATP, activation by various metal ions, hydrolytic activity with other nucleotides and stimulation by bicarbonate ions. 4. Unlike the particulate enzyme, the soluble enzyme is cold-labile and insensitive to oligomycin. 5. The molecular weight indicated by the mobility of the soluble ATPase on Sepharose 6B is 360000. 6. The soluble ATPase combines very readily with liver submitochondrial particles depleted of ATPase by salt extraction, and oligomycin-sensitivity is restored. Very little recombination of the enzyme occurs with chloroform-extracted particles. 7. The soluble enzyme contains orcinol-reactive material, suggesting that it may be a glycoprotein. The carbohydrate content was estimated to be 1-2% by weight. 8. It is concluded that the liver ATPase obtained by the chloroform extraction method of Beechey, Hubbard, Linnett, Mitchell & Munn [(1975) Biochem. J.148, 533-537] is similar to other preparations described previously and that this method is superior in simplicity and speed.     

Interaction between brain natriuretic peptide and atrial natriuretic peptide in caecal circular smooth muscle cells

Guinea pig caecal circular smooth muscle cells were used to determine whether brain natriuretic peptide (BNP) can inhibit the contractile response produced by cholecystokinin-octapeptide (CCK-8). In addition, we examined the effect of an inhibitor of cAMP-dependent protein kinase, an inhibitor of particulate or soluble guanylate cyclase, an atrial natriuretic peptide (ANP) antagonist (ANP 1-11), and selective receptor protection on the BNP-induced relaxation of these muscle cells. The effect of BNP on cAMP formation was also examined. BNP inhibited the contractile response produced by CCK-8 in a dose-response manner, with an IC50 value of 8.5 nM, and stimulated the production of cAMP. The inhibitor of cAMP-dependent protein kinase and the inhibitor of soluble guanylate cyclase significantly inhibited the relaxation produced by BNP. In contrast, the inhibitor of particulate guanylate cyclase did not have any significant effect on the relaxation produced by BNP. ANP 1-11 significantly but partially inhibited the relaxation produced by BNP. The muscle cells where CCK-8 and ANP binding sites were protected completely preserved the inhibitory response to ANP, but partially preserved the inhibitory response to BNP. The muscle cells where CCK-8 and BNP binding sites were protected completely preserved the inhibitory response to both ANP and BNP. This study demonstrates that BNP induces relaxation of these muscle cells via both ANP binding sites coupled to soluble guanylate cyclase and distinct BNP binding sites coupled to adenylate cyclase.     

A methyl-CoM methylreductase system from methanogenic bacterium strain G? 1 not requiring ATP for activity

Crude inside-out vesicles from the methanogenic strain G?1 were prepared via protoplasts. These vesicles catalyzed methane formation from methyl-CoM and H2 at a maximal rate of 35 nmol/min.mg protein. Methane formation by the vesicles did not depend on the addition of ATP. This was in contrast to conventionally prepared crude extracts from the same organism or from Methanosarcina barkeri which exhibited strict ATP dependence of methanogenesis. ATP analogues inhibited methanogenesis by extracts to a much higher extent than that by vesicles. Both, particulate and soluble components prepared from the crude vesicles by ultracentrifugation were necessary for ATP-independent methane formation from methyl-CoM and H2. Hydrogenase activity was mainly associated with the particulate fraction whereas methyl-CoM methylreductase could be assigned to the soluble fraction. The detergent sulfobetaine inhibited methane formation from methyl-CoM without affecting hydrogenase or titanium citrate-dependent methylreductase activities, indicating that an additional membraneous component is involved in methanogenesis for methyl-CoM and H2.     

The membrane ATPase of Escherichia coli. II. Release into solution,allotopic properties and reconstitution of membrane-bound ATPase

Membrane-bound ATPase of Escherichia coli was released in a soluble form by decreasing the Mg²⁺ concentration to 0.05 mM. The particulate fraction left behind was depleted by more than 90% from its initial ATPase activity.Soluble ATPase exhibits a number of different properties as compared with membrane-bound ATPase. These are a 2-fold increased K_m toward ATP, a shift of 1–1.5 pH units in the pH-dependence curve, a greatly increased resistance to inhibition by N,N′-dicyclohexylcarbodiimide (DCCD) and a stimulation by Dio 9 instead of an inhibition.Upon mixing the soluble fraction and the depleted membrane fraction, the initial properties of native membrane-bound ATPase reappear. This reconstitution requires Mg²⁺ and results in the physical binding of the activity to sedimentable material.Soluble ATPase and depleted membrane can be titrated against each other until an equivalence point is reached, beyond which the component in excess keeps its previous characteristics.During the release procedure, DCCD remains associated with the particulate fraction with conservation of the ATPase-binding sites.Such DCCD-treated depleted membranes behave as a specific inhibitor of soluble ATPase.     

Tentoxin-induced energy-independent adenine nucleotide exchange and ATPase activity with chloroplast coupling factor 1.

1. The effect of energy transfer inhibitors on energy-dependent exchange of tightly bound adenine nucleotides with washed, broken spinach thylakoids has been studied. Energy transfer inhibitors that inhibit the ATPase activity of soluble chloroplast coupling factor 1 (CF1) (e.g. phloridzin and tentoxin) do not inhibit energy-dependent adenine nucleotide exchange. Energy transfer inhibitors that block proton flux through the hydrophobic protein proton channel (CF0) (e.g. dicyclohexylcarbodiimide and triphenyltin chloride) also block light-dependent adenine nucleotide exchange. 2. Tentoxin, at relatively high concentrations, stimulates an energy-independent exchange of adenosine diphosphate. 3. High concentrations of tentoxin elicit a Ca2+-dependent ATPase activity with soluble CF1, but has no effect on the Ca2+-dependent ATPase activity of membrane-bound CF1. 4. The trypsin-activated, Ca2+-dependent, membrane-bound ATPase is not affected by high concentrations of tentoxin, whereas the dithiothreitol-activated, Mg2+-dependent ATPase is markedly inhibited. 5. The reconstitution of chloroplasts, partially depleted in CF1, with soluble CF1 is correlated with the loss of tentoxin-induced, Ca2+-dependent ATPase activity associated with soluble CF1.     

Affinity separation of an acid phosphatase from rat tissues and Gaucher spleen with immobilized Cibacron Blue

An acid phosphatase species which was activated by Fe2+ was determined to be partially soluble but mainly particulate in rat spleen. The particulate enzyme could be extracted into 1 M KCl. This enzyme bound to Cibacron Blue-immobilized Sepharose (Blue-Sepharose) and was desorbed by 2 M KCl with a good yield, while the other acid phosphatases in rat spleen did not adsorb on Blue-Sepharose. The enzymes eluted on Blue-Sepharose chromatography of both the soluble and particulate fractions were electrophoretically identical. The enzyme hydrolyzed aryl monophosphates, phosphoproteins, and nucleoside di- and triphosphates. The activity for the three kinds of substrate was similarly activated by Fe2+, ascorbic acid and cysteine, and inhibited by molybdate, Cu2+ and F-. Cibacron Blue inhibited the enzyme competitively with respect to a substrate, p-nitrophenyl phosphate, but kinetic analysis suggested that more than one dye molecule binds to the enzyme. The Blue-Sepharose technique could be applied not only to quantitative separation of acid phosphatases similar to the spleen enzyme from bone and epidermis of rat, but also to that of a tartrate-resistant acid phosphatase from human spleen with Gaucher's disease.     

Particulate glucan synthetase activity: generation and inactivation after treatments with indoleacetic acid and cycloheximide

Growing regions from epicotyls of Pisum sativum L. var Alaska contain a particulate enzyme which transfers glucose from guanosine diphosphate glucose to alkali-soluble and -insoluble products (glucan synthetase activity). When the epicotyl is decapitated to remove the source of natural hormone, the tissue below ceases growth and loses synthetase activity as well as the capacity to continue forming cellulose in vivo. If indoleacetic acid (IAA) is added to the cut apex, massive amounts of cellulose are deposited in the next few days. Particulate glucan synthetase activity is either maintained or greatly increased depending on whether endogenous activity levels are relatively high or low at the time of hormone addition. These effects appear to be due in part to IAA-dependent generation of a protein essential for synthetase activity since they are severely inhibited by concentrations of cycloheximide which are effective at preventing protein synthesis. Nevertheless, the addition of cycloheximide alone to the epicotyl reduces the rate of disappearance of synthetase activity, i.e., a protective effect. Also, a soluble thermolabile component is present in the aging epicotyl which promotes loss of synthetase activity when added to the particulate enzyme in vitro. Accordingly, turnover of pea glucan synthetase activity may be controlled in part by an inactivating protein which is itself subject to turnover.     

Loss of supercoiled viral DNA after infection of permissive cells by simian virus 40.

During infection of BS-C-1 cells (a permissive line of monkey kidney cells) with simian virus 40, there was a loss of infecting viral supercoiled (component I) DNA molecules which was partially inhibited by cycloheximide. The kinetics of loss of component I in the presence of cycloheximide suggested that the loss was the result of at least two activities acting on the DNA, one cycloheximide sensitive and the second cycloheximide insensitive. Nucleotides from degraded infecting viral DNA were reincorporated into cell DNA that was synthesized during the infection.     

Possible involvement of calmodulin in the regulation of ATPase activity in guard cells

Calcium ions play an important role in the regulation of stomatal movement and the mechanism underlying this action is yet to be determined. It is suggested that guard cell plasma membrane ATPase is a target for calcium action and that this effect is mediated by calmodulin. In this study, the effects of calcium and two calmodulin antagonists on ATPase activity in a crude homogenate of Commelina communis L. guard cell protoplasts were examined. The homogenate contained Mg²⁺-dependent, K⁺-simulated ATPase activity, which was inhibited by CaCl² while stimulated by the calmodulin antagonists, compound 48/80 and chlorpromazine. The calmodulin antagonists partially reversed the inhibitory effect of calcium ions. The results support the possibility of calmodulin involvement in the regulation of guard cell ATPase activity by calcium ions.     

Activation-induced cell death in T cell hybridomas is due to apoptosis. Morphologic aspects and DNA fragmentation.

Some T cell hybridomas, upon activation via the TCR, rapidly undergo cell death. In this paper, we demonstrate that this activation-induced cell death (AICD) is accompanied by morphologic changes seen at the electron and light microscopy levels. The most striking changes are an extensive condensation of the chromatin and formation of membrane blebs. In addition to the morphologic changes, a significant portion of genomic DNA is broken at an interval of approximately 200 bp, producing a ladder of oligonucleosome-sized fragments after gel electrophoresis. Taken together, these observations indicate that AICD proceeds via apoptosis, or programmed cell death. This is additionally supported by the observation that AICD-associated phenomena are at least partially inhibited by cycloheximide or actinomycin D. Curiously, AICD and its associated DNA fragmentation are completely inhibited by aurintricarboxylic acid, a known nuclease inhibitor. The possible relationship between AICD in vitro, and the negative selection process (wherein selection may proceed via AICD of developing, autoreactive thymocytes) is discussed.