Proteins of a Polyhedral Cytoplasmic Deoxyvirus: III. Structure of Frog Virus 3 and Location of Virus-Associated Adenosine Triphosphate Phosphohydrolase

The adenosine triphosphatase associated with frog virus 3 shows high specificity for ATP or deoxyadenosine triphosphate and appears to be distinct from the corresponding activity in host cells, poxvirus, or reovirus. The enzyme activity is probably integrated into virus particles since it is firmly associated with subviral particles produced when approximately 50 to 60% of the outer viral protein is removed by detergent treatment. The occurrence of adenosine triphosphatase activity within three unrelated viruses suggests that adenosine tryphosphatase might be a necessary function for most viruses that replicate in the cell cytoplasm.     

Fractionation of membrane vesicles from coliphage M13-infected Escherichia coli.

Membrane vesicles were prepared by osmotic lysis of spheroplasts from M13-infected Escherichia coli. Reduced nicotinamide adenine dinucleotide (NADH) oxidase (reduced NAD: oxidoreductase, EC 1.6.99.3) and Mg2+-Ca2+-activated adenosine triphosphatase (ATP phosphohydrolase, EC 3.6.1.3), which are normally localized to the inner surface of the cytoplasmic membrane, were 50% acceesible to their polar substrates in these vesicles. The major coat protein of coliphage M13 is also bound to the cytoplasmic membrane (prior to phage assembly) but with its antigenic sites exposed to the exterior of the cell. Antibody to M13 coat protein was used to fractionate membrane vesicles. Neither agglutinated nor unagglutinated vesicles had altered NADH oxidase and adenosine triphosphatase specific activities. This is inconsistent with such vesicles being a mixture of correctly oriented and completely inverted membrane sacs and suggests that NADH oxidase, adenosine triphosphatase, M13 coat protein, or all three proteins rearrange during vesicle preparation.     

Biochemical studies on the origin of the ATPase of the avian myeloblastosis virus

The adenosine triphosphatase activity of the avian myeloblastosis virus obtained from the blood of the virus-infected chicken was compared with that of the host cell myeloblasts. The specific activity of the viral enzyme is unusually higher than that of the myeloblasts. A significant difference in inhibitor sensitivity was observed with quercetin. When the virus was grown in chicken embryonic fibroblasts in culture, the resulting virus showed very little adenosine triphosphatase activity, comparable to that of the fibroblasts and similar sensitivity to inhibitors. Antibody raised against the purified enzyme of avian myeloblastosis virus inhibits the enzyme activity of the myeloblasts while the activity of the fibroblasts enzyme as well as that of fibroblast-grown virus remains unaffected.     

The mode of inhibition by calcium of cell-membrane adenosine-triphosphatase activity

1. The mechanism of the inhibition of Na(+)-plus-K(+)-activated adenosine triphosphatase by calcium was investigated with an enzyme preparation from rabbit kidney cortex and with membranes of human erythrocytes. 2. CaATP, rather than ionic Ca(2+), acts as a competitive inhibitor, competing with MgATP in the Na(+)-plus-K(+)-activated adenosine-triphosphatase reaction. 3. There appears to be no competition between calcium and Na(+) for the activation of adenosine triphosphatase. 4. The inhibition of Na(+)-plus-K(+)-activated adenosine triphosphatase of cell membranes by low concentrations of CaATP and the consequent need of intact cells to keep the cytoplasmic concentration of calcium low relative to that of magnesium suggests a raison d'être for the mitochondrial calcium pump.     

Cytochemistry of Phosphatases in Myxococcus xanthus

An Mg(2+)-dependent and a K(+)-stimulated adenosine triphosphatase were localized by cytochemistry at or near both surfaces of the cytoplasmic membrane of Myxococcus xanthus. An alkaline and an acid phosphatase resided at the external surface of the membrane or in the periplasm. All enzymes could be extracted from partially fixed cells with Mg(2+)-deficient buffers. Suboptimal external phosphate elicited dissociation of adenosine triphosphatase from the membrane but not that of the unspecific phosphatases. The dissociated enzymes migrated into the cytoplasm where they were associated mainly with cytoplasmic aggregates.     

Anisakis simplex: uncoupling of oxidative phosphorylation in the muscle mitochondria of infected fish

Adenosine triphosphatase activity stimulated by Mg2+ was greater in muscle mitochondria of fish infected with larval Anisakis simplex nematodes than in uninfected fish. When muscle mitochondria were isolated in a sucrose ethylene-glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid medium from fresh uninfected fish, they were loosely coupled, and their adenosine triphosphatase activity was comparable to that of mitochondria from rat tissue. Activity in infected fish was dose dependent, increasing with the number of worms per fish. Excretory secretory products or a cytoplasmic fraction of anisakines, when incubated with coupled rat mitochondria, also caused adenosine triphosphatase activity to increase. Storage of fish flesh caused an increase in adenosine triphosphatase activity, but such aging was not significant until 5 and 10 days after death in refrigerated and frozen samples, respectively. The Mg2+ stimulated adenosine triphosphatase activity of muscle mitochondria can be used to estimate the number of nematodes per market fish. The type of medium used to isolate the mitochondria is crucial in such studies; an ionic medium with Nagarse proteinase was optimal for fish muscle mitochondria.     

Histochemical Localization of Phosphatases in Mycoplasma gallisepticum

Histochemical studies of adenosine triphosphatase and acid phosphatase activity were performed on Mycoplasma gallisepticum. The adenosine triphosphatase activity appears to be localized in the bleb and infrableb regions exclusively and is associated with the cell membrane; acid phosphatase activity is localized in the infrableb region and does not appear to be membrane-associated. These findings are consistent with data from biochemical studies of Mycoplasma cell fractions but, unlike them, reveal that adenosine triphosphatase activity is restricted to a particular part of the cell membrane.     

Cytochemical localization of transport adenosine triphosphatase in the ferret placenta

Synopsis The distribution of transport adenosine triphosphatase in the ferret placenta was examined cytochemically by light and electron microscopy. The enzyme was detected in the syncytiotrophoblast but was absent from maternal tissues. It appeared to be associated with cytoplasmic processes on syncytiotrophoblast surfaces directly related to foetal or maternal capillaries. The functional significance of transport adenosine triphosphatase is discussed with reference to the transport of solutes between the maternal and foetal circulation across the trophoblast layer.Paper given at the Royal Microscopical Society's European Histochemistry Meeting at Nottingham in September 1975.     

Assembly of the mitochondrial membrane system: isolation of nuclear and cytoplasmic mutants of Saccharomyces cerevisiae with specific defects in mitochondrial functions.

A selection procedure is described which permits a large number of Saccharomyces cerevisiae mutants to be screened for specific lesions in mitochondrial respiratory enzymes and the adenosine triphosphatase. The method has been used to isolate nuclear mutant strains with specific lesions in coenzyme QH2-cytochrome c reductase, cytochrome oxidase, and adenosine triphosphatase. In addition, two cytoplasmic mutants have been found whose primary defect is in cytochrome oxidase, and others have been found that show variable degrees of abnormalities in their mitochondrial translation products.     

Adenosine triphosphatase activity of mycoplasma membranes

Rottem, Shlomo (Hebrew University, Jerusalem, Israel), and Shmuel Razin. Adenosine triphosphatase activity of mycoplasma membranes. J. Bacteriol. 92:714-722. 1966.-Adenosine triphosphatase activity of Mycoplasma laidlawii, M. gallisepticum, and Mycoplasma sp. strain 14 was confined to the cell membrane. The enzymatic activity was dependent on magnesium, but was not activated by sodium and potassium. Ouabain did not inhibit the adenosine triphosphatase activity of the mycoplasmas, and did not interfere with the active accumulation of potassium by M. laidlawii cells. Sulfhydryl-blocking reagents and fluoride inhibited the enzymatic activity, whereas 2,4-dinitrophenol was without any effect. Membranes of M. laidlawii hydrolyzed other nucleotide triphosphates and adenosine diphosphate (ADP), but at a lower rate than adenosine triphosphate (ATP). Nucleoside-2'-(3')-phosphates, ribose-5-phosphate, glucose-6-phosphate, and pyrophosphate were not hydrolyzed by the membrane preparations. It seems that the enzyme(s) involved in ATP hydrolysis by M. laidlawii membranes is strongly bound to the membrane subunits, which would account for the failure to purify the enzyme by protein fractionation techniques. The adenosine triphosphatase activity of mycoplasma membranes resembles in its properties that of similar enzymes studied in bacteria. The mycoplasma enzyme(s) seems to differ from the adenosine triphosphatase associated with ion transport in mammalian cell membranes and from mitochondrial adenosine triphosphatase.     

ELECTRON MICROSCOPE OBSERVATIONS ON THE SURFACE ADENOSINE TRIPHOSPHATASE-LIKE ENZYMES OF HELA CELLS INFECTED WITH HERPES VIRUS

HeLa cells infected with herpes simplex virus have been examined in thin sections by electron microscopy after cytochemical staining for the presence of surface enzymes splitting adenosine triphosphate. As with uninfected HeLa cultures (18), the opaque enzyme reaction product was localized at the plasma membranes of about half the cells, tending to be present where there were microvilli and absent on smooth surfaces. Where mature extracellular herpes particles were found in association with cell membranes showing the enzyme activity, they were invariably likewise stained, and conversely, those mature particles which lay close against cells without reaction product at the surface were themselves free of it. Particles found budding into cytoplasmic vacuoles were also always without opaque deposit since this was never seen at vacuolar membranes, even in cells having the activity at the surface. The enzyme reaction product thus provided a marker indicating the manner in which the particles escape from cells and mature by budding out through cellular membranes, carrying, in the process, a portion of the latter on to themselves to form the outer viral limiting membrane. In some instances, virus particles were observed with more opaque material covering them than was present at the cell membrane with which they were associated. This finding has been taken as evidence for a physiological waxing and waning of surface enzyme activity of adenosine triphosphatase type. The fine structure of the mature extracellular virus as prepared here, using glutaraldehyde fixation, is also recorded. The observations and interpretations are discussed in full.     

The transport of alpha-aminoisobutyrate into Crithidia fasciculata.

The transport of alpha-aminoisobutyrate into Crithidia fasciculata was characterized under aerobic and anaerobic conditions. Kinetic data for alpha-aminoisobutyrate transport were consistent with the operation of a single system of broad specificity that showed no marked dependence on Na+. Under anaerobic conditions alpha-aminoisobutyrate transport was inhibited by uncouplers such as 2,4-dinitrophenol, lipophilic cations such as methyltriphenylphosphonium ion and adenosine triphosphatase inhibitors such as dicyclohexylcarbodi-imide and NaN3. A working model in which alpha-aminoisobutyrate enters this organism by an H+-symport mechanism, the electrochemical gradient of protons being maintained by an H+-translocating adenosine triphosphatase on the cytoplasmic membrane, is proposed.     

Protection of Vaccinia from Heat Inactivation by Nucleotide Triphosphates

The presence of adenosine triphosphate, guanosine triphosphate, cytosine triphosphate, or uridine triphosphate reduced the rate of inactivation of vaccinia when heated at 50 C. The virus-associated nucleoside triphosphate phosphohydrolases (adenosine triphosphatase, guanosine triphosphatase, cytosine triphosphatase, and uridine triphosphatase) and ribonucleic acid polymerase were also protected from heat inactivation by these compounds. These obervations are best explained by postulating that ribonucleoside triphosphates bind to enzymes in the virus particle, and that these enzyme-substrate complexes are more resistant to thermal denaturation than are the enzymes without their substrates. The kinetics of heat inactivation of the vaccinia ATP phosphohydrolase activity is biphasic, suggesting that there are two proteins in the vaccinia particle that have this enzyme activity but they have different kinetics of heat inactivation. Any of the vaccinia-associated nucleotide phosphohydrolase activities are protected from heat inactivation by the presence of any one of the respective nucleoside triphosphates. This observation suggests that there is a single enzymatic site in vaccinia that is able to react with any ribonucleoside triphosphate.     

Comparison of Effects of Sublethal Microwave Radiation and Conventional Heating on the Metabolic Activity of Staphylococcus aureus

This study was conducted in an attempt to characterize some of the effects of sublethal microwave radiation on cells of Staphylococcus aureus. Cultures were exposed to microwave radiation for 10, 20, 30, and 40 s. The effects of a conventional heat treatment were also compared by placing flasks containing cultures in a boiling water bath for the amount of time required to reach temperatures equivalent to those found in cultures exposed to microwave radiation. Control, microwave-treated, and conventionally heat-treated cultures were centrifuged, pellets were resuspended in distilled water, and the resulting suspensions were passed through a French pressure cell. Cell lysates and walls were then isolated and assayed for enzymatic activity. Thermonuclease production was also determined at various levels of exposure of cells to microwave radiation. Activities of malate and α-ketoglutarate dehydrogenases, cytochrome oxidase, and cytoplasmic adenosine triphosphatase were higher in microwave-treated cells than in control cells. Membrane adenosine triphosphatase, alkaline phosphatase, and lactate dehydrogenase activities were unaffected when cells were exposed to microwave radiation. The activity of glucose-6-phosphate dehydrogenase was decreased by exposure of cells to microwave radiation. In conventionally heated cells, activities of glucose-6-phosphate and malate dehydrogenases and cytoplasmic adenosine triphosphatase increased activities of α-ketoglutarate and lactate dehydrogenases decreased, and alkaline phosphatase activity remained unaffected. Increased levels of thermonuclease activity were observed when cells were exposed to microwave radiation for 10 or 20 s. Data indicate that microwave radiation affects S. aureus in a manner which cannot be explained solely by thermal effects.     

A Fifth Gene (uncE) in the Operon Concerned with Oxidative Phosphorylation in Escherichia coli

Three mutant unc alleles (unc-408, unc-410, and unc-429) affecting the coupling of electron transport to oxidative phosphorylation in Escherichia coli K-12 have been characterized. Genetic complementation analyses using previously defined mutant unc alleles indicated that the new mutant unc alleles affect a previously undescribed gene designated uncE. The phenotype of strains carrying the uncE408 or uncE429 allele is similar in that Mg(2+)-adenosine triphosphatase activity is only found in the cytoplasmic fraction, and membranes do not bind the F(1) portion of adenosine triphosphatase purified from a normal strain. In contrast, adenosine triphosphatase activity is present both in the cytoplasm and on the membranes from a strain carrying the unc-410 allele, and normal F(1) binds to F(1)-depleted membranes from this strain. The adenosine triphosphatase solubilized from membranes of a strain carrying the unc-410 allele reconstituted ATP-dependent membrane energization in F(1)-depleted membranes from a normal strain. Genetic complementation tests using various Mu-induced unc alleles in partial diploid strains show that the uncE gene is in the unc operon and that the order of genes is uncB E A D C. The unc-410 allele differs from the uncE408 and uncE429 alleles in that complementation tests with the Mu-induced unc alleles indicate that more than one gene is affected. It is concluded that this is due to a deletion which includes part of the uncE gene and another gene, or genes, between the uncE and uncA genes.     

Membrane adenosine triphosphatase in synchronous cultures of Rhodobacter sphaeroides

Studies of intracytoplasmic membrane biogenesis utilizing synchronized cultures of Rhodobacter sphaeroides have revealed that most intracytoplasmic membrane proteins accumulate continuously throughout the cell cycle while new phospholipid appears discontinuously within the intracytoplasmic membrane. The resulting changes in the structure of the membrane lipids was proposed to influence the activities of enzymes associated with the intracytoplasmic membranes (Wraight, C.A., Leuking, D.R., Fraley, R.T. and Kaplan, S. (1978) J. Biol. Chem. 253, 465-471). We have extended the study of intracytoplasmic membrane biogenesis in R. sphaeroides to include the membrane adenosine triphosphatase. The membrane bound Mg2+-dependent, oligomycin-sensitive adenosine triphosphatase activity was measured throughout the cell cycle for steady-state synchronized cells of R. sphaeroides and found to accumulate discontinuously. Following treatment with an uncoupling reagent (2,4-dinitrophenol) the intracytoplasmic membrane associated adenosine triphosphatase activity was stimulated uniformly in membranes isolated at different stages of the cell cycle. The adenosine triphosphatase was also measured by quantitative immunoblots utilizing specific antibody to compare the enzyme activity and enzyme protein mass. Immunologic measurement of the adenosine triphosphatase in isolated membranes indicated a constant ratio of enzyme to chromatophore protein exists during the cell cycle in contrast to the discontinuous accumulation of adenosine triphosphatase activity. These results are discussed in light of the cell-cycle specific synthesis of the intracytoplasmic membrane.     

Relative deficiency of Ca2 plus-dependent adenosine triphosphatase activity of red cell membranes in hereditary spherocytosis

The Ca²⁺-dependent adenosine triphosphatase activity associated with the plasma membrane of normal human erythrocytes is similar to that of erythrocytes from patients with hereditary spherocytosis. When spherocytic ghosts are compared to age-matched controls, however, they show a significantly decreased Ca²⁺-dependent adenosine triphosphatase activity. The role of the relative deficiency of Ca²⁺-dependent adenosine triphosphatase in spherocytic ghosts is discussed in the light of the effects of intracellular [Ca²⁺] on the deformability and the rigidity of the cell membrane. This enzyme may be involved in the molecular mechanism of hereditary spherocytosis.     

Adenosine 5''-triphosphate-linked transhydrogenase in cytoplasmic membranes of colicin-treated and untreated Escherichia coli.

The adenosine 5'-triphosphate (ATP)-linked transhydrogenase reaction, present in the particulate fractions of Escherichia coli, was previously shown to be inhibited in these fractions when the bacteria were treated with colicins K or El. The purpose of this study was to characterized the ATP-linked transhydrogenase reaction and the colicin-caused inhibition of the reaction in purified cytoplasmic membranes. Particulate fractions from bacteria treated or untreated with colicins were separated on sucrose gradients into cell wall membrane and cytoplasmic membrane fractions. The ATP-linked transhydrogenase reaction was found to be exclusively associated with the cytoplasmic membrane fractions. The reaction was inhibited by carbonylcyanide m-chlorophenlhdrazone, dinitrophenol, N,N'-dicyclohexylcarbodiimide, and trypsin. Although the cytoplasmic membrane fractions were purified from the majoriy of the cell wall membrane and its bound colicins, they showed the inhibitory effects of colicins K and El on the ATP-linked transhydrogenase reaction. The inhibition of ATP-linked transhydrogenase reaction induced by the colicin could not be reversed by subjection the isolated membranes to a variety of physical and chemical treatments. Cytoplasmic membranes depleted of energy-transducing adenosine triphosphatase ATPase) complex (coupling factor) lost the ATP-linked transhydrogenase activity. The ATPase complexes isolated from membranes of bacteria treated or untreated with colicins El or K reconstituted high levels of ATP-linded transhydrogenase activity to depleted membranes of untreated bacteria. The same ATPase complexes reconstituted low levels of activity to depleted membranes of the treated bacteria.     

Enzymes and Nucleotides in Virions of Rous Sarcoma Virus

In addition to the previously described deoxyribonucleic acid (DNA) polymerase, DNA ligase, DNA exonuclease, and DNA endonuclease activities, purified virions of Schmidt-Ruppin strain of Rous sarcoma virus (SRV) have nucleotides and nucleotide kinase, phosphatase, hexokinase, and lactate dehydrogenase activities. The SRV virions have no glucose-6-phosphate dehydrogenase activity. All enzyme activities, but glucose-6-phosphate dehydrogenase and adenosine triphosphatase, were increased by disruption of the virions. The DNA polymerase, DNA ligase, and hexokinase activities had a higher specific activity in purified virion cores. It is suggested that during assembly virions of SRV may pick up cytoplasmic components which bind to virion proteins. The role of these components in viral replication is not known at present.     

Control of expression of the herpes simplex virus-induced deoxypyrimidine triphosphatase in cells infected with mutants of herpes simplex virus types 1 and 2 and intertypic recombinants.

Infection of cells with herpes simplex virus type 1 (HSV-1) induces high levels of deoxypyrimidine triphosphatase. The majority of the enzyme activity is found in infected cell nuclei. A similar activity is induced by HSV type 2 (HSV-2) which, in contrast to the HSV-1 enzyme, fractionates to more than 99% in the soluble cytoplasmic extract. Of a series of temperature-sensitive mutants of HSV-1 studied, only the immediate-early mutants in complementation group 1-2 (strain 17 mutants tsD and tsK and strain KOS mutant tsB2) induced reduced levels of triphosphatase at nonpermissive temperature. Of a series of temperature-sensitive mutants of HSV-2 strain HG52, ts9 and ts13 failed to induce wild-type levels of the enzyme at nonpermissive temperature; ts9 was the most defective mutant with regard to triphosphatase expression of both herpes simplex virus serotypes. After shift-up from permissive to nonpermissive temperature, triphosphatase activity in cells infected with ts9 decreased rapidly, whereas all other mutants continued to exhibit enzyme levels comparable with controls kept at the permissive temperature. The type 1-specific nuclear expression of the triphosphatase was mapped physically by the use of HSV-1 x HSV-2 intertypic recombinants, based on enzyme levels different by more than two orders of magnitude found in nuclei of HSV-1- and HSV-2-infected cells. The locus for the type-specific expression maps between 0.67 and 0.68 fractional length on the HSV genome.