The subcellular distribution of triphosphoinositide phosphomonoesterase in guinea-pig brain

1. Some properties of the triphosphoinositide phosphomonoesterase from the homogenates of guinea-pig brain were studied. The enzyme has an optimum pH range 6.7-7.3, is stimulated with KCl at a concentration of 0.1m, and under these conditions has K(m)1.43x10(-4)m. 2. A factor from the ;pH5 supernatant' of guinea-pig brain stimulates the enzyme activity over and above the stimulation produced by KCl. Subcellular fractions of guinea-pig brain varied in their response to the ;pH5 supernatant'. Maximum stimulation was observed with the P(1) fraction, containing myelin and nuclei. 3. An assay system for the enzyme was developed that contained optimum concentrations of both KCl and the ;pH5 supernatant'. Acid phosphatases were inhibited by NaF, but, in contrast with previous work, no EDTA was added to the assay system to inhibit the alkaline phosphatases. This reagent inhibited the triphosphoinositide phosphomonoesterase. It was estimated that the remaining fraction of non-specific phosphatases can account for only 14% of the observed triphosphoinositide phosphomonoesterase activity. 4. Subcellular fractions of guinea-pig brain were characterized by electron microscopy and subcellular markers. The triphosphoinositide phosphomonoesterase exhibited a distribution between the fractions similar to that of 5'-nucleotidase, but different from that of alkaline phosphatase.     

Biochemical and histochemical studies on non-specific phosphomonoesterases of swine kidney worm Stephanurus dentatus (Diesing, 1839).

Biochemical and histochemical studies have been made on non-specific acid and alkaline phosphomonoesterases of S. dentatus. The two forms of acid phosphomonoesterases have been found active at pH 4.0 and 6.0. The pH optima for the two forms of aklaline phosphomonoesterases lie at 8.0 and 10.0. Studies on the distribution of acid and alkaline phosphomonoesterases in various tissues have revealed an abundance of acid phosphomonoesterase in various parts of the alimentary canal and various organs of the reproductive system. The excretory ducts show alkaline phosphomonoesterase activity only.     

Purification and Characterization of DNA Polymerase Obtained from the Membrane Fraction of E. coli

Purification studies were conducted on DNA polymerase bound to the membrane fraction of E. coli HF 4704. Purified enzyme (Fraction V) required Mg²⁺ and showed an optimun pH of 7.2. Various kinds of salt indicated a stimulative effect at concentrations lower than 0.1 m. Fraction V was unstable at an acidic condition (pH 5.0) but was rather stable at an alkaline condition (pH 9.0). The enzyme activity was lost by incubation at 45°C for 30min but was stabilized by the addition of DNA. The enzyme contained exonuclease activity but no endonuclease activity. The enzyme produced only light density DNA of various sizes. The function of this enzyme as considered to fill single stranded region of the double stranded primer DNA.     

Factors modulating alkaline phosphatase activity in the diazotrophic rice-field cyanobacterium, Anabaena oryzae

Summary Alkaline phosphatases (APase), both phosphomonoesterase (PMEase) and phosphodiesterase (PDEase) were studied in the cyanobacterium Anabaena oryzae for their specific requirements of temperature, pH, micro- and macronutrients and their activities in the presence of salinity and heavy metal stress. The alkaline phosphatases (PMEase and PDEase) are quite stable enzymes and require a narrow range of pH (pH 10–10.2) and temperature (35–40 °C) for their optimal activity.A pH of 10, 10.2 and 10.2 supported optimal activity of cellular PMEase, cellular PDEase and extracellular PMEase, respectively, whereas temperatures of 35, 38 and 40 °C were required for their optimal activity. The requirement for Ca²⁺ and Mg²⁺ as macronutrients and the significance of the micronutrients Zn²⁺, Co²⁺, Fe²⁺, Mn²⁺ and Cu²⁺ in APase activity in the cyanobacterium suggests nutritional regulation of enzyme activity in A. oryzae. The metals Pb²⁺, Cr⁶⁺ and Ni²⁺ severely inhibited APase activity, whereas the NaCl stress had a dual role, which was concentration dependent. NaCl stress at lower concentrations (≤20 mM) caused an increase in cellular PMEase activity while its higher concentration (>20 mM) favoured release of the extracellular PMEase. The decrease in cellular activity and an increase in extracellular activity suggest that the higher concentrations of salt stimulate the release of the enzyme.The data suggest that the cyanobacterium A. oryzae possess a potential application as biofertilizer in high salinity and alkaline (Ca²⁺-rich) soils because of its ability to release PO₄³⁻ enzymatically under these conditions.     

Effect of carbon source and pH of the growth medium on spore germination inPhycomyces blakesleeanus

Phycomyces blakesleeanus sporangiospores responded differently to activation by physical and chemical stimuli. Spores that were physically (heat shock) activated or chemically (ammonium acetate) activated germinated and grew at pH 4.5 with the hexoses glucose, fructose, galactose, andN-acetylglucosamine, and with glycerol and amino acids. Under these conditions, physically activated spores showed a lower, although significant growth with the hexoses fructose, galactose,N-acetylglucosamine and with glycerol. On the other hand, physically activated spores incubated at alkaline pH (pH 7.3) required glucose to germinate; a requirement not observed with chemically activated spores, which showed significant growth in the other hexoses tested. Both physically and chemically activated spores incubated at pH 7.3 were unable to germinate and grow with amino acids and glycerol. These results suggest that there are different targets for activation of the spores by physical and chemical treatments. The levels of the fermentative enzymes alcohol dehydrogenase and lactate dehydrogenase and of the oxidative enzyme NAD⁺-isocitrate dehydrogenase were higher in cells grown at pH 4.5 in medium containing glucose; however, alcohol dehydrogenase and lactate dehydrogenase appear not to be affected by a change in the pH of the growth medium.     

Formation of phenol-protein complexes and their use by two stream invertebrates

Ethanol and water extracts of maple leaves and pine needles were analyzed for proteins, amino acids, sugars and phenolics. Leachates were mixed with a dissolved fungal cellulase. Within 24 h, insoluble particles formed, consisting of phenolics, proteins and amino acids. When exposed to an alkaline pH, or to a 0.04% solution of the surfactant lysolecithin, these particles released amino acids and proteins. Surface tensions of the gut fluids of Gammarus tigrinus and Tipula caloptera were considerably lower than that of distilled water, suggesting the presence of surfactants. Gut fluid of T. caloptera contained enzymes capable of digesting the proteins of particles formed with maple water extracts. The other particles did not appear susceptible to these enzymes. There was no evidence that G. tigrinus was able to digest the proteins of any of the particles examined.     

PARTIAL CHARACTERIZATION OF BASIC PROTEINS OF CHICKEN, TURTLE AND FROG CENTRAL NERVOUS SYSTEM MYELIN

Myelin basic proteins were isolated from CNS tissues of chicken, turtle and frog and compared with the corresponding protein of bovine origin. At acid pH all four proteins had comparable mobilities in polyacrylamide gels. Upon electrophoresis at alkaline pH the submammalian proteins, like the bovine protein, were separated into multiple components. The components of the chicken and frog proteins had exceptionally high and low mobilities, respectively, while those of the turtle protein had mobilities comparable to those of the bovine protein. The chicken and turtle proteins were similar to the bovine protein in amino acid composition except for containing considerably more serine and valine and having higher proportions of histidine to lysine. The frog protein differed further in having an unusually high content of tyrosine (approx 9 mol/mol protein), an unusually high arginine: glycine ratio (1.09) and practically no methylated arginine (0-0.036 mol/mol protein). Like those of mammalian origin, the submammalian proteins each contained a single tryptophan and two methionines. Arginine, serine and glycine together accounted for approximately 40 per cent of the residues in each protein. The chicken and turfle proteins each contained roughly equal amounts of N^G-monomethyl- and N^G, N^G-dimethylarginine, the two derivatives together comprising 0.5-0.6 mol/mol protein. No N^G, N^G-dimethylarginine was detected in any of the proteins examined. The microheterogeneity observed in the chicken and turtle proteins upon electrophoresis at alkaline pH was reproduced upon alkaline pH chromatography on carboxymethylcellulose. Chromatographic fractions of the chicken protein which differed electrophoretically at alkaline pH had virtualy identical amino acid compositions and apparent molecular weights and all contained comparable amounts of both N^G-monomethyl- and N^G, N^G-dimethylarginine. Treatment of the submammalian proteins with BNPS-skatole yielded two fragments comparable in size, charge and staining characteristics to those similarly produced from the bovine protein (residues 1-116 and 117-170). Fragments produced from the frog protein by treatment with BrCN were comparable in size and charge to those similarly produced from the bovine protein; those produced from the chicken and turtle proteins were much different. In immunodiffusion studies the submammalian and bovine proteins showed reactions of identity when tested against rabbit anti-chicken basic protein serum.     

Intracellular perfusion and cell centrifugation studies on plasmalemma transport processes inChara corallina

Summary The OH^– transport system ofChara corallina was studied using the techniques of intracellular perfusion and cell centrifugation. Application of silk ligatures to internodal cells had quite a perturbative effect on the OH^– transport activity. Approximately 12 hr were required before normal pH profiles were reestablished.Tonoplast-free internodal cells developed rather weak, uniform alkalinity along the cell surface. Redevelopment of control pH patterns was never observed in these experiments. Surface pH profiles similar to those observed on tonoplast-free cells could also be obtained by subjecting cells to mild centrifugation (180×g). Organelles of the streaming cytoplasm were contained within the centrifugal cell segment by applying a ligature near the cell center. Normal pH profiles were observed along the centrifugal segment, while the centripetal segment developed weak, rather uniform alkaline profiles. Upon redistribution of the cytoplasmic organelles, normal pH profiles were established along the entire cell length.These results indicate that an organelle within the streaming cytoplasmic phase is responsible for the spatial location and control over OH^– transport. This explains the absence of control pH profiles in tonoplast-free cells, since during the disintegration of the tonoplast, most of the streaming cytoplasm coagulates at one end of the cell.Parallel pH mapping and electrophysiological studies indicated that the plasmalemma of this species contains an ATP-dependent electrogenic H⁺ transport system. Also, experiments conducted in the presence and absence of cellular ATP demonstrated that OH^– efflux can be driven passively by the membrane potential. Whether OH^– transport is strictly a passive process in normal cells remains to be resolved.     

Extracellular Alkaline Phosphatase in Multicellular Marine Algae and Their Utilization of Glycerophosphate

The production of extracellular alkaline phosphatase by multicellular marine algae in axenic culture has been investigated. The algae studied were five species of Rhodophyta: Asterocytis ramosa, Goniotrichum elegans, Nemalion helminthoides, Polysiphonia urceolata and Rhodosorus marinus; and one species of Phaeophyta: Ecrocarpus confervoides. The extent of enzyme activity varies from one species to another. It also varies with the phosphorus conditions under which the alga is grown. The pattern of glycerophosphate utilization suggests that this type of compound is not taken up directly by the alga but split by the external enzyme before uptake of the phosphate-ion only. The enzyme performs its action outside the organism and appears both associated with the cells and free in the surrounding water. Assays with culture filtrate of Asterocytis and Ectocarpus show that the enzyme is an unspecific phosphomonoesterase with optimum activity far to the alkaline side. It is activated by Zn²⁺.     

Purification and immunochemical characterization of ascitic fluid glycoproteins containing certain tumor-associated and blood group antigen markers

Ascitic fluids from patients with various types of cancer were screened for the CA 19-9 and CA 125 tumor-associated antigenic activities. Two fluids exhibiting the highest activities were tested for their binding to various lectin-Sepharose columns resulting in both being bound best to wheat germ agglutinin (WGA) Sepharose. The WGA column eluate of one fluid was further chromatographed by HPLC and three peaks were obtained with approximate molecular weights of 3.65 MDa, 664 kDa and 330 kDa, of which only the largest fraction contained the CA 19-9 activity. The fluids were also fractionated on a Sephacryl S-400 column with most of the activity being present in or near the void volume.Monoclonal antibodies were used to demonstrate that the purified glycoproteins also contained the blood group A determinant, the four Lewis determinants Le^a, Le^b, Le^x and Le^y, and the sialylated-Le^x determinant, while other antibody analyses failed to detect other blood group and/or carbohydrate sequence determinants. Some of the blood group expressions could be separated from the CA 19-9 and CA 125 active glycoproteins by adsorption with various lectins other than the WGA.Abbreviations used NeuAc N-acetyl-D-neuraminic acid - Gal galactose,D-galactopyranose - Fuc fucose,L-fucopyranose - GlcNAc N-acetyl-D-glucosamine - GalNAc N-acetyl-D-galactosamine - WGA wheat germ agglutinin - PBS phosphate buffered saline     

ACID AND ALKALINE PHOSPHATASES IN A BRAIN TUBULIN PREPARATION

The hydrolysis of p-Nitrophenylphosphate has been studied in vitro in a tubulin preparation from bovine brain. The activity at pH 6.8 was 16.4 ± 2.2 nmol/mg protein, h. At least two phosphatases were responsible for this activity. They were found to have pH-optima at 5.1 and 10.4. respectively, and their apparent K_M values were 1.23 ± 0.10 mm and 0.17 ± 0.03 mm . respectively. Mg²⁺ was found to stimulate activity at both pHs while Zn²⁺ inhibited at pH 5.1 and stimulated activity at pH 10.4. All of the alkaline and part of the acid phosphatase activity were found to be closely associated with microtubules/tubulin. Tubulin purified by phosphocellulose chromatography contained phosphatase activity, and it is suggested that such activity is an intrinsic property of tubulin itself. Phosphatase activity was also found in association with the microtubule-associated proteins that co-purify with tubulin. Two proteins of high molecular weight constituted the major part of the associated material. The results indicate an association of phosphatase activity with the larger of these two proteins.     

Production and Consumption of Dimethylsulfoniopropionate in Marine Microbial Mats

The fate of dimethylsulfoniopropionate (DMSP), a major sulfonium compound in marine ecosystems, was examined in Microcoleus chthonoplastes-dominated microbial mats. Chemical decomposition of DMSP was observed under laboratory conditions at pH values higher than 10.0. pH profiles measured in situ showed that these highly alkaline conditions occurred in microbial mats. Axenic cultures of M. chthonoplastes contained 37.3 μmol of DMSP g of protein⁻¹, which was partially liberated when the cells were subjected to an osmotic shock. DMSP-amended mat slurries showed a rapid turnover of this compound. The addition of glutaraldehyde blocked DMSP decrease, indicating biological consumption. Populations of potential dimethyl sulfide consumers were found in the top 10 mm of the mat.     

Cytoplasmic proteases of rat liver parenchymal cells

Soluble extracts of isolated rat liver parenchymal cells contained three proteases with alkaline pH optima. One protease was a high molecular weight (Mr = 500,000) enzyme which was stimulated by ATP. The other two proteases were totally dependent on calcium for activity and displayed different calcium concentration requirements. One was half-maximally activated by 150 μM Ca²⁺ while the other required only 10 μM Ca²⁺ for half-maximal activation.     

Phosphodiesterase activity of alkaline phosphatase in ATP-initiated Ca(2+) and phosphate deposition in isolated chicken matrix vesicles

Inorganic pyrophosphate is a potent inhibitor of bone mineralization by preventing the seeding of calcium-phosphate complexes. Plasma cell membrane glycoprotein-1 and tissue nonspecific alkaline phosphatase were reported to be antagonistic regulators of mineralization toward inorganic pyrophosphate formation (by plasma cell membrane glycoprotein-1) and degradation (by tissue nonspecific alkaline phosphatase) under physiological conditions. In addition, they possess broad overlapping enzymatic functions. Therefore, we examined the roles of tissue nonspecific alkaline phosphatase within matrix vesicles isolated from femurs of 17-day-old chick embryos, under conditions where these both antagonistic and overlapping functions could be evidenced. Addition of 25 microM ATP significantly increased duration of mineralization process mediated by matrix vesicles, while supplementation of mineralization medium with levamisole, an alkaline phosphatase inhibitor, reduces the ATP-induced retardation of mineral formation. Phosphodiesterase activity of tissue nonspecific alkaline phosphatase for bis-p-nitrophenyl phosphate was confirmed, the rate of this phosphodiesterase activity is in the same range as that of phosphomonoesterase activity for p-nitrophenyl phosphate under physiological pH. In addition, tissue nonspecific alkaline phosphatase at pH 7.4 can hydrolyze ADPR. On the basis of these observations, it can be concluded that tissue nonspecific alkaline phosphatase, acting as a phosphomonoesterase, could hydrolyze free phosphate esters such as pyrophosphate and ATP, while as phosphodiesterase could contribute, together with plasma cell membrane glycoprotein-1, in the production of pyrophosphate from ATP.     

Biochemical properties and evaluation of washing performance in commercial detergent compatibility of two collagenolytic serine peptidases secreted by Aspergillus fischeri and Penicillium citrinum

Filamentous fungi secrete diverse peptidases with different biochemical properties, which is of considerable importance for application in various commercial sectors. In this study, we describe the isolation of two fungal species collected from the soil of decayed organic matter: Aspergillus fischeri and Penicillium citrinum. In a submerged bioprocess, we observed better peptidase production with the fungus P. citrinum, which reached a peak production at 168?h with 760?U/mL, in comparison with the fungus A. fischeri, which reached a peak production at 72?h with 460?U/mL. In both situations, the fermentative medium contained 0.5% crushed feathers as a source of nitrogen. On performing biochemical characterization, we detected two alkaline serine peptidases: The one secreted by P. citrinum had optimal activity at pH 7.0 and at 45°C, while the one secreted by A. fischeri had optimal activity in pH 6.5–8 and at 55–60°C. Metallic ions were effective in modulating these peptidases; in particular, Cu²⁺ promoted negative modulation of both peptidases. The peptidases were stable and functional under conditions of nonionic surfactants, temperatures up to 45°C for 1?h, and incubation over a wide pH range. In addition, it was observed that both peptidases had the capacity to hydrolyze collagen and performed well in removing an egg protein stain when supplemented into a commercial powder detergent; this was especially true for the peptidase from P. citrinum.     

Genome sequencing and heterologous expression of antiporters reveal alkaline response mechanisms of Halomonas alkalicola

Halomonas alkalicola CICC 11012s is an alkaliphilic and halotolerant bacterium isolated from a soap-making tank (pH > 10) from a household-product plant. This strain can propagate at pH 12.5, which is fatal to most bacteria. Genomic analysis revealed that the genome size was 3,511,738 bp and contained 3295 protein-coding genes, including a complete cell wall and plasma membrane lipid biosynthesis pathway. Furthermore, four putative Na⁺/H⁺ and K⁺/H⁺ antiporter genes, or gene clusters, designated as HaNhaD, HaNhaP, HaMrp and HaPha, were identified within the genome. Heterologous expression of these genes in antiporter-deficient Escherichia coli indicated that HaNhaD, an Na⁺/H⁺ antiporter, played a dominant role in Na⁺ tolerance and pH homeostasis in acidic, neutral and alkaline environments. In addition, HaMrp exhibited Na⁺ tolerance; however, it functioned mainly in alkaline conditions. Both HaNhaP and HaPha were identified as K⁺/H⁺ antiporters that played an important role in high alkalinity and salinity. In summary, genome analysis and heterologous expression experiments demonstrated that a complete set of adaptive strategies have been developed by the double extremophilic strain CICC 11012s in response to alkalinity and salinity. Specifically, four antiporters exhibiting different physiological roles for different situations worked together to support the strain in harsh surroundings.

     

Relationship of K+-Uptaking System with H+-Translocating ATPase in Enterococcus hirae, Grown at a High or Low Alkaline pH

Potassium ion pool was studied in glycolyzing Enterococcus hirae, grown at high or low alkaline pH (pH 9.5 and 8.0, respectively). Energy-dependent increase of K⁺ pool was lower for the wild-type cells, grown at pH 9.5, than that for the cells grown at pH 8.0. It was inhibited by N,N′-dicyclohexylcarbodiimide (DCCD). The stoichiometry of DCCD-inhibited K⁺ influx to DCCD-inhibited H⁺ efflux for the wild-type cells, grown at pH 9.5 or 8.0, was fixed for different K⁺ external activity. DCCD-inhibited ATPase activity of membrane vesicles was significantly stimulated by K⁺ for the wild-type cells grown at pH 9.5, and required K⁺ for the wild-type cells grown at pH 8.0, while the levels of α and β subunits of the F₁ and b subunit of the F₀ were lower for the cells grown at pH 9.5 than that for the cells grown at pH 8.0. Such an ATPase activity was residual in membrane vesicles from the atpD mutant with a nonfunctional F₀F₁. ATPase activity of membrane vesicles from the mutant with defect in Na⁺-ATPase was higher for the cells grown at pH 9.5 than that for the cells grown at pH 8.0, and was inhibited by DCCD. An energy-dependent increase of K⁺ pool in this bacterium, grown at a high or low alkaline pH, is assumed to occur through a K⁺ uptaking system, most probably the Trk. The latter functions in a closed relationship with the H⁺-translocating ATPase F₀F₁. Received: 30 June 1997 / Accepted: 4 August 1997     

Cloning of an alkaline phosphatase gene from the moderately thermophilic bacterium<Emphasis Type="Italic"> Meiothermus ruber</Emphasis> and characterization of the recombinant enzyme

A gene that codes for an alkaline phosphatase was cloned from the thermophilic bacterium Meiothermus ruber, and its nucleotide sequence was determined. The deduced amino acid sequence indicates that the enzyme precursor including the putative signal sequence is composed of 503 amino acid residues and has an estimated molecular mass of 54,229 Da. Comparison of the peptide sequence with that of the prototype alkaline phosphatase from Escherichia coli revealed conservation of the regions in the vicinity of the corresponding phosphorylation site and metal binding sites. The protein was expressed in E. coli and its enzymatic properties were characterized. In the absence of exogenously added metal ions, activity was negligible; to obtain maximal activity, addition of free Mg²⁺ ions was required. Zn²⁺ ions had an inhibitory effect on the activity of the M. ruber enzyme. The pH and temperature optima for activity were found to be 11.0 and 62°C, respectively. The enzyme was moderately thermostable: it retained about 50% activity after incubation for 6 h at 60°C, whereas at 80°C it was completely inactivated within 2 h. The Michaelis constant for cleavage of 4-nitrophenylphosphate was 0.055 mM. While having much in common with other alkaline phosphatases, the M. ruber enzyme presents some unique features, such as a very narrow pH range for activity and an absolute requirement for magnesium for activity.Communicated by G. P. Georgiev     

Development of resistance in Cronobacter sakazakii ATCC 29544 to thermal and nonthermal processes after exposure to stressing environmental conditions

Aims: The objective was to study the response of Cronobacter sakazakii ATCC 29544 cells to heat, pulsed electric fields (PEF), ultrasound under pressure (Manosonication, MS) and ultraviolet light (UV‐C) treatments after exposure to different sublethal stresses that may be encountered in food‐processing environments. Methods and Results: Cronobacter sakazakii stationary growth‐phase cells (30°C, 24 h) were exposed to acid (pH 4·5, 1 h), alkaline (pH 9·0, 1 h), osmotic (5% NaCl, 1 h), oxidative (0·5 mmol l^?1 H₂O₂, 1 h), heat (47·5°C, 1 h) and cold (4°C, 4 h) stress conditions and subjected to the subsequent challenges: heat (60°C), PEF (25 kV cm^?1, 35°C), MS (117 μm, 200 kPa, 35°C) and UV‐C light (88·55 mW cm^?2, 25°C) treatments. The inactivation kinetics of C. sakazakii by the different technologies did not change after exposure to any of the stresses. The combinations of sublethal stress and lethal treatment that were protective were: heat shock–heat, heat shock–PEF and acid pH–PEF. Conversely, the alkaline shock sensitized the cells to heat and UV‐C treatments, the osmotic shock to heat treatments and the oxidative shock to UV‐C treatments. The maximum adaptive response was observed when heat‐shocked cells were subjected to a heat treatment, increasing the time to inactivate 99·9% of the population by 1·6 times. Conclusions: Cronobacter sakazakii resistance to thermal and nonthermal preservation technologies can increase or decrease as a consequence of previous exposure to stressing conditions. Significance and Impact of the Study: The results help in understanding the physiology of the resistance of this emerging pathogen to traditional and novel preservation technologies.