The adenosine triphosphate pool and the adenyl cyclase activity inEscherichia coli strains with different UV sensitivity

Two genetically relatedEscherichia coli strains were tested for the ATP pool and for the adenyl cyclase activity in the cell membrane fractions. TheEscherichia coli strain B_S1with a high UV sensitivity showed a higher ATP level than the wild typeEscherichia coli B strain. The adenyl cyclase activity was found to be lower in the sensitive strain than in the wild typeEscherichia coli.     

Characterization of adenylate cyclase fromEscherichia coli

Adenylate cyclase activity was detected and characterized in cell-free preparations of different strains ofEscherichia coli; it was localized not only in the membrane fraction but also in the cytoplasm, the localization differing from strain to strain. The adenylate cyclase activity is highly dependent on the method used for disintegration of cells. The best results were obtained when using vortexing of the cell suspension with ballotini beads. The pH optimum of adenylate cyclase in cell-free preparations was found to be 9.0 –9.5. The enzyme has an absolute requirement for Mg²⁺ and is inhibited by sodium fluoride and inorganic diphosphate. Release of adenylate cyclase from the membrane leads to an immediate loss of the activity; it was found that adenylate cyclase is quite labile and hence it could not yet been purified. The method used to determine adenylate cyclase activity and cyclic AMP is described.     

Adenyl cyclase of oxyntic cells its association with different cellular membranes

The adenyl cyclase of the oxyntic, or acid-secreting, cells of bullfrog gastric mucosa has been found to be a membrane-bound enzyme. A method has been developed to isolate the adenyl cyclase rich membrane fractions in a hypotonic medium containing dithiothreitol, which has been found to protect the hormonal resposivenes of the adenyl cyclase.Highest specific activity of adenyl cyclase was localized in a light membrane fraction which also had abundant K⁺-stimulated ATPase and K⁺-stimulated $\text{p-}\text{nitrophenyl}$ phophatase and very low cytochrome $\text{c}$ oxidase activty. The three gastric secretagogues tested, namely histamine, pentagastrin and methylcholine, significantly stimulated the adenyl cyclase activity of the light membrane fraction.After treatment with 10 mM Mg⁺ further subfractionation of the light membrane fraction on a sucrose density gradient yielded light membrane subfraction 1, light membrane subfraction 2 and light membrane subfraction 3 in order of increasing densities. The three subfractions had different enzymatic and chemical properties. Adenyl cyclase activity has been found to be distributed in all three subfractions. However, the hormonal responsiveness of the three fractions was quite different. Light membrane subfraction 2 could be stimulated by all three secretagogues, light membrane subfraction 1 by histamine and methylcholine, while light membrane subfraction 3 was refractory to all three secretagogues. On the basis of the cholesterol to phospholipid molar ratio, RNA content, glycoprotein content and the enzymatic data it is suggested that light membrane subfraction 1 and light membrane subfraction 2 are of general plasma-membrane type, while light membrane subfraction 3 is largely of cytoplasmic origin.     

Adenosine 3',5' cyclic monophosphate in Euglena gracilis

$\text{Euglena gracilis}$ contains in high concentration the enzymes for the synthesis and degradation of cyclic AMP. The synthetic enzyme, adenyl cyclase is mainly associated with a particulate fraction which sediments at 7,000–30,000xg whereas the degradative enzyme, 3′5′ nucleotide phosphodiesterase, is soluble (does not sediment at 78,000xg). The adenyl cyclase activity is stimulated somewhat by prostaglandins and by catecholamines, agents which markedly stimulate cyclase in appropriate mammalian tissues. There is no detectable activity of guanyl cyclase, the enzyme which synthesizes cyclic GMP. $\text{Euglena}$ also contains a cyclic AMP stimulated protein kinase which is associated with a particulate fraction sedimenting at 30,000xg.     

Localization of L-asparaginase inEscherichia coli

The localization ofl-asparaginase (l-asparagine amidohydrolase, EC 3.5.1.1) EC-2 isoenzyme was studied inEscherichia coli ATCC 9637 grown under conditions of moderate aeration. The enzyme was determined in cell fractions obtained by fraction centrifugation of lysed spheroplasts. When the synthesis of the enzyme was induced byl-asparagine, its amount in the cytoplasmic fraction at the beginning of the induction exceeded as much as five times that in uninduced cells, attaining up to 20% of the total activity. In the course of growth of the culture this activity decreased gradually to zero. The membrane fraction of induced cells contained considerable amount of EC-2l-asparaginase which, at the beginning of the induction, reached up to 6% ot the total enzymic activity; in membrane fraction of control cells the activity was close to zero. The results indicate a relationship of cell structures to thel-asparagine-induced synthesis of the enzyme.     

Studies on the assay and activities of guanyl and adenyl cyclase of rat liver

In applying recently developed methods for measuring adenyl and guanyl cyclase activities, we found that some modifications produced much better cyclic nucleotide recovery, lower assay backgrounds, and greater reliability than previously reported. The reliability and specificity of the assay methods were confirmed by substrate and product analysis. Kinetic analysis of rat liver guanyl and adenyl cyclase was subsequently performed to investigate regulatory properties of both enzymes. The Michaelis-Menton constant of guanyl cyclase activity of a 30,000g supernatant fraction of rat liver for guanosine 5′-triphosphate (GTP) was 0.04 mm. This enzyme was competitively inhibited by adenosine 5′-triphosphate (ATP) (K_i = 0.011 mM). Guanyl cyclase was activated in vitro by secretin but unaffected by carbamylcholine, hist-amine, methoxamirie, serotonin, glucagon, and pancreozymin. Liver homogenate adenyl cyclase had a Michaelis-Menten constant for ATP of 0.2 mm. This enzyme was activated by secretin, pancreozymin, glucagon, sodium fluoride, and isoproterenol. GTP (0.005 mm) enhanced the activation by both isoproterenol and glucagon. Methoxamine had no effect on adenyl cyclase activity in the presence or absence of GTP. These results suggest that both guanyl cyclase and adenyl cyclase may be mediators of hormone action in the liver.     

Degradation of 5′ adenosine monophosphate in a cell-free system ofEscherichia coli

Sonicated cells ofEscherichia coli contain an enzyme system degrading 5′ adenosine monophosphate (5′ AMP) to hypoxanthine. This enzyme system is located in the fraction sedimenting at 20,000 xg. It has a pH optimum at 8.0. In the fraction sedimenting at 20,000 xg the enzyme activity was inhibited by adenosine triphosphate (ATP). Adenosine and adenine are deaminated by this enzyme preparation to inosine and to hypoxanthine, these activities not being inhibited by ATP.     

Phospholipases of Escherichia coli

Phospholipase A activity was hardly detected in Escherichia coli K12 sonicate when solvent-extracted (free) ³²P-phosphatides were used as substrate. Phosphatides bound in membrane were, however, actively hydrolyzed to give the corresponding lysolipids by an endogenous enzyme. The results indicated the presence in E. coli membrane of a novel phospholipase which can be more precisely called as lipoprotein phospholipase A. Lysophospholipase was shown to be present in the cellular soluble fraction.

With free phosphatides as substrate, alcohols and some water-miscible solvents, as well as nonionic detergents, markedly stimulated phospholipase A activity of the membrane, possibly by enabling the substrate to hold physical state in someway simillar to that in the membrane. Possible role of this enzyme in membrane lipid metabolism is discussed.     

Changes in adenyl cyclase specific activity during differentiation on an established myogenic cell line

The variation of specific activity of adenyl cyclase has been studied during differentiation of an established line of myoblast, strain L₆D and of a temperature sensitive developmental variant strain, H₆, derived from it. The specific activities of both basal and NaF stimulated adenyl cyclase were found to decrease 2 to 3 folds after fusion of myoblasts into myotubes in cultures of L₆D. Cultures of strain H₆ displayed the same decrease in specific activity of adenyl cyclase when grown at temperature which allows differentiation, while no decrease was observed at the temperature which does not allow cell fusion. These results indicare that the decrease in specific activity of adenyl cyclase is associated with cell fusion and reflects membrane changes ocurring during differentiation of myogenic cells.     

Squalene-hopene cyclase (Spterp25) from Streptomyces peucetius: sequence analysis, expression and functional characterization

Squalene-hopene cyclase, which catalyzes the complex cyclization of squalene to the pentacyclic triterpene, hopene, is a key enzyme in the biosynthesis of hopanoids. The deduced amino acid sequence of the Streptomyces peucetius gene (spterp25) had significant similarity to other prokaryotic squalene-hopene cyclases. Like other triterpene cyclases, the S. peucetius squalene-hopene cyclase contains eight so-called QW-motifs with an aspartate-rich domain. The 2,025-bp squalene-hopene cyclase-encoding gene was expressed in Escherichia coli BL21(DE3)pLySs, and the in vitro activity of the recombinant cyclase was demonstrated using purified membrane protein. The cyclization product hopene was identified by gas chromatography/mass spectrometry (GC/MS).     

Bifunctional structure of two adenylyl cyclases from the myxobacterium Stigmatella aurantiaca

Two adenylyl cyclase genes (cyaA and cyaB) from the myxobacterium Stigmatella aurantiaca were cloned by complementation of Escherichia coli mutants defective in the cya gene. cyaA codes for a protein of 424 amino acid residues (AC1), while cyaB encodes a protein of 352 residues (AC2). Both cyclases are sensitive to adenosine: cAMP production was strongly inhibited in E coli cells and cell extracts expressing these genes. AC1 comprises a hydrophobic domain of six transmembrane helices coupled to a cytoplasmic catalytic domain endowed with adenylyl cyclase activity. A 17 amino acid residue sequence, which is a signature of G-protein coupled receptors, as well as of slime mold Dictyostelium discoideum cyclic AMP receptors, was found in the membrane domain. AC2 displays features also indicating that it is a bifunctional enzyme. The domain located upstream from the catalytic adenylyl cyclase domain shows strong similarity to receiver modules of response regulators of two-component bacterial signaling systems. In vitro mutagenesis of conserved aspartate residues in this domain was shown to interfere with cAMP synthesis.     

Membrane-associated reactions in ubiquinone biosynthesis in Escherichia coli. 3-octaprenyl-4-hydroxybenzoate carboxy-lyase

A sensitive and quantitative assay for 3-octaprenyl-4-hydroxybenzoate carboxy-lyase has been developed. This enzyme, which catalyses the third reaction in ubiquinone biosynthesis in Escherichia coli, was partially purified and some of its properties determined. It was found that a considerable proportion of the carboxylyase activity could be separated from the membrane fraction in cell extracts prepared using a French press. Gel filtration showed the molecular weight of the enzyme to be about 340 000. For optimal activity the carboxy-lyase was shown to require Mn²⁺, washed membranes or an extract of phospholipids, and an unidentified heat stable factor of molecular weight less than 10 000. The carboxy-lyase reaction was also shown to be strongly stimulated by dithiothreitol and methanol. The properties of the carboxy-lyase are compared with the three other enzymes concerned with ubiquinone biosynthesis in E. coli which have been studied in vitro. The fact that the substrate of the carboxy-lyase is membrane-bound and the enzyme is stimulated by phospholipid suggests that it normally functions in association with the cytoplasmic membrane in vivo.     

Cloning and expression of a murein hydrolase lipoprotein from Escherichia coli

On the basis of the published N-terminal amino acid sequence of the soluble lytic transglycosylase 35 (Slt35) of Escherichia coli, an open reading frame (ORF) was cloned from the 60.8 min region of the E. coli chromosome. The nucleotide sequence of the ORF, containing a putative lipoprotein-processing site, was shown by [³H]-palmitate labelling to encode a lipoprotein with an apparent molecular mass of 36 kDa. A larger protein, presumably the prolipoprotein form, accumulated in the presence of globomycin. Over-expression of the gene, designated mltB (for membrane-bound lytic transglycosylase B), caused a 55-fold increase in murein hydrolase activity in the membrane fraction and resulted in rapid cell lysis. After membrane fractionation by sucrose-density-gradient centrifugation, most of the induced enzyme activity was present in the outer and intermediate membrane fractions. Murein hydrolase activity in the soluble fraction of a homogenate of cells induced for MltB increased with time. This release of enzyme activity into the supernatant could be inhibited by the addition of the serine-protease inhibitor phenylmethyl-sulphonyl fluoride. It is concluded that the previously isolated Slt35 protein is a proteolytic degradation product of the murein hydrolase lipoprotein MltB. Surprisingly, a deletion in the mltB gene showed no obvious phenotype.     

Characterization ofZymomonas mobilis alkaline phosphatase activity inEscherichia coli

Zymomonas mobilis phoA gene encoding alkaline phosphatase was expressed inEscherichia coli CC118 carrying the recombinant plasmid pZAP1. The pH optimum for this enzyme was 9.0 and showed a peak activity at 42°C. This enzyme required Zn²⁺ for its catalytic activity; however, Mg²⁺ or Ca²⁺ significantly affected the activity. This enzyme was found to be ethanolabile, and ethanol inhibition was reversed by addition of Zn²⁺. Kinetics ofZ. mobilis alkaline phosphatase production inE. coli CC118 (pZAP1) showed that the enzyme activity was growth associated and localized in the cellular fraction, and the maximum activity was found in the stationary phase.     

Location of anaerobic respiratory enzyme trimethylamine N-oxide reductase in the cytoplasmic membrane ofEscherichia coli strain K10

Trimethylamine N-oxide (TMAO) reductase, which is anaerobically induced by TMAO, is a terminal enzyme in anaerobic electron transport inEscherichia coli. When the organism was anaerobically grown with TMAO, a marked increase in the specific activity of TMAO reductase was observed mainly in a cell membrane fraction and stopped after exhausting TMAO. On the other hand, activity was moderately increased in a soluble fraction of the cell even after exhaustion of TMAO. Immunoblot analysis with an antiserum against the TMAO reductase purified from the soluble fractions showed that the cells growing with TMAO contained only a membrane-bound enzyme, which has a molecular mass of 94 kDa, while a soluble enzyme with 92 kDa appeared in the stationary growth phase lacking TMAO. Experiments with right-side-out and inside-out vesicles of cytoplasmic membrane indicated that the membrane-bound enzyme faces the cytoplasm. The soluble enzyme was mainly found in the cytoplasm of the cell, but also at a negligible amount in the periplasm. The membrane-bound form of TMAO reductase functioning in anaerobic electron transport seems to be cleaved and released into the cytoplasm as soluble enzyme after exhaustion of TMAO.     

Effect of guanyl nucleotides on the stimulation of adenyl cyclase activity in human thyroid plasma membranes by thyroid-stimulating hormone and prostaglandin E2

Thyroid homogenates and thyroid plasma membranes were prepared from human thyroid and the effects of thyroid-stimulating hormone (thyrotropin), NaF, and prostaglandins E₁ and E₂ on adenyl cyclase activity in these preparations were studied. The basal level of adenyl cyclase activity in plasma membranes was 5–8 times greater than that of the original homogenates. Adenyl cyclase activity in plasma membranes was stimulated 4.7-fold by 100 munits/ml of thyrotropin and 5-fold by 10 mM of NaF, but the activity in the homogenates was only stimulated 2-fold by either thyrotropin or NaF. Prostaglandin E₁ (10^?6?10^?3 M) and prostaglandin E₂ (10^?7?10^?4 M) failed to stimulate adenyl cyclase activity in plasma membranes, but they did stimulate adenyl cyclase activity in the homogenates. A marked stimulatory effect of prostaglandin E₂ (10^?5 M) on adenyl cyclase activity in plasma membranes resumed in the presence of GTP (10^?7?10^?4 M), although GTP itself only slightly stimulated enzyme activity. GDP and GMP were also effective in this respect, although their potencies varied from compound to compound. GTP potentiated slightly the action of thyrotropin on adenyl cyclase in plasma membranes, but it significantly depressed an increase of enzyme activity produced by NaF. Since GTP did not affect the ATP-regenerating system, it seems that GTP, GDP or GMP was required for the manifestation of prostaglandin E₂ action on adenyl cyclases of human thyroid plasma membranes.     

Characterization of Substances that Restore Impaired Cell Division of UV-Irradiated E. coli B

Substances which restore impaired cell division in UV-irradiated E. coli B were surveyed among various bacteria. The active substance was found only in several genera of Gram-negative bacteria, i.e., Escherichia, Enterobacter, Salmonella and some species of Pseudomonas. The activity in the dialyzed cell extract of E. coli B/r was observed in the presence of β-NAD and was enhanced by Mg^{2 +} and Mn²⁺. The active substance was very labile, but the activity was protected by 1 mM dithiothreitol in the process of purification. The activity of a fraction recovered through DEAE-cellulose column chromatography was stimulated by the presence of membrane fraction. Upon treatment with lipid-degrading enzymes and proteases, the division-stimulating activity was lost or reduced. It appears that the inactivation by lipase and phospholipase A₂ was due to the formation of lysophospholipids and that a proteinous substance participated in the recovery of impaired cell division of UV-irradiated E. coli B.     

Adenylate cyclase system of human skeletal muscle: Characteristics of catecholamine stimulation and nucleotide regulation

The subcellular localization of adenylate cyclase was examined in human skeletal muscle. Three major subcellular membrane fractions, plasmalemma, sarcoplasmic reticulum and mitochondria, were characterized by membrane-marker biochemical studies, by dodecyl sulfate polycrylamide gel electrophoresis and by electron microscopy. About 60% of the adenylate cyclase of the homogenate was found in the plasmalemmal fraction and 10–14% in the sarcoplasmic reticulum and mitochondria. When the plasmalemmal preparation was subjected to discontinuous sucrose gradients, the distribution of adenylate cyclase in different subfractions closely paralleled that of (Na⁺ + K⁺)-ATPase. The highest specific activity was found in a fraction which setteled at the 0.6–0.8 M sucrose interface. The electron microscopic study of this fraction revealed the presence of flattened sacs of variable sizes and was devoid of mitochondrial and myofibrillar material. The electron microscopy of each fraction supported the biochemical studies with enzyme markers. The three major membrane fractions also contained a low K_m phosphodiesterase activity, the highest specific activity being associated with sarcoplasmic reticulum.The plasmalemmal adenylate cyclase was more sensitive to catecholamine stimulation than that associated with sarcoplasmic reticulum or mitochondria. The catecholamine-sensitive, but not the basal, enzyme was further stimulated by GTP. The plasmalemmal adenylate cyclase had typical Michaelis-Menten kinetics with respect to ATP and the apparent K_m for ATP was approx. 0.3. mM. The pH optimum for that enzyme was 7.5. The enzyme required Mg²⁺, and the concentration to achieve half-maximal stimulation was approx. 3 mM. Higher concentrations of Mg²⁺ (about 10 mM) were inhibitory. Solubilization of the plasmalemmal membrane fraction with Lubrol-PX resulted in preferential extraction of 106 000- and 40 000-dalton protein components. The solubilized adenylate cyclase lost its sensitivity for catecholamine stimulation, and the extent of fluoride stimulation was reduced to one-sixth of that of the intact membranes. It is concluded that the catalytically active and hormone-sensitive adenylate cyclase is predominantly localized in the surface membranes of the cells within skeletal muscle. (That “plasmalemmal” fraction is considered likely to contain, in addition to plasmalemma of muscle cells, plasmalemma of bloodvessel cells (endothelium, and perhaps smooth muscle) which may be responsible for a certain amount of the adenylate cyclase activity and other propertiesobserved in that fraction.)The method of preparation used in this study provides a convenient material for evaluating the catecholamine-adenylate cyclase interactions in human skeletal muscle.     

Permeabilization of Escherichia coli cells for enhanced penicillin acylase activity

Summary Escherichia coli cells with penicillin acylase activity were permeabilized with aqueous solutions of the cationic detergent N-cetyl-N,N,N-trimethylammonium bromide (CTAB), at pH 8.0 and the activity was found to have almost doubled. The concentration of CTAB, the time and temperature of treatment were optimised for maximum enzyme activity and were found to be 0.2%, 20 min and 5°C respectively. Subsequently, the cell bound activity was retained for a longer period by chemical cross-linking with 0.1% glutaraldehyde.     

Characterization of Fish Cu/Zn–Superoxide Dismutase and Its Protection from Oxidative Stress

Copper/zinc superoxide dismutase was cloned from the zebrafish (Danio rerio). The full coding region of the zebrafish superoxide dismutase (ZSOD) complementary DNA was ligated with pET-20b(+) and successfully expressed in Escherichia coli strain AD494(DE3)pLysS. The active enzyme was purified by His tagging. The ZSOD yield was 6 mg from 0.2 L of E. coli culture, and the specific activity was 2000 U/mg as assayed using a RANSOD kit. The enzyme stability was characterized by reaction to temperature, pH, and detergent treatment. The results showed enzyme activity was still active after heat treatment at 70°C for 10 minutes, resistant to pH treatment from 2.3 to 12, and resistant to treatment with sodium dodecyl sulfate (SDS) under 4%. In addition, the recombinant ZSOD was used to protect fish from 100 ppm of paraquat-induced oxidative injury by soaking fish larva in 55 µg/ml SOD enzyme. The results were significant.