Factors Affecting the Activity and Stability of Alkaline Phosphatase in a Marine Pseudomonad

Conditions optimum for the assay of alkaline phosphatase of marine pseudomonad B-16 (ATCC 19855) and for maintaining the activity of the enzyme have been determined. The pH for optimal activity of the cell-bound enzyme was 9.0, whereas that for the enzyme after its release from the cells exceeded 9.4. Release was effected by first washing the cells in 0.5 M NaCl and then suspending them in 0.5 M sucrose. In the absence of salts, the activity of the cell-bound enzyme decreased rapidly at 25 C and less rapidly at 4 C. This loss of activity could be arrested but not restored by adding Mg(2+). In the presence of Na(+), activity of the cell-bound enzyme dropped to about 50% of that prevailing initially, but in this case adding Mg(2+) restored enzyme activity completely. The activity of the enzyme after its release from the cells into 0.5 M sucrose was approximately 50% of that of the equivalent amount of enzyme in the original cells. This activity was relatively stable at both 25 and 4 C. Adding Mg(2+) to the released enzyme restored its activity to that of the cell-bound form. The synthesis of alkaline phosphatase by the cells was not affected by adding 50 mM inorganic phosphate to the growth medium. The K(m) of the released enzyme for p-nitrophenyl phosphate was found to be 6.1 x 10(-5) M.     

Localization in the Cell and Extraction of Alkaline Phosphatase from Bacillus subtilis

Study of protoplasts, lysed protoplasts, and cells treated with lysozyme in the absence of osmotic stabilizer suggested that the alkaline phosphatase (EC 3.1.3.1.) of Bacillus subtilis is located in the protoplasmic membrane. Cytochemical evidence in support of this view is presented. The enzyme protein was strongly bound to the membrane structure and could not be solubilized by a number of treatments known to release enzymes from membranes and other lipoprotein structures. Alkaline phosphatase was, however, solubilized by treatment of intact B. subtilis cells or isolated protoplasmic membranes with strong salt solutions at pH 7.2, suggesting that electrostatic forces are responsible for the association between membrane and enzyme protein. Dialysis of alkaline phosphatase solutions against buffer of low ionic strength resulted in precipitation of the enzyme.     

Freeze-Etching and X-Ray Diffraction of the Isolated Double-Track Layer from the Cell Wall of a Gram-Negative Marine Pseudomonad

The isolated double-track layer of the cell wall of the gram-negative marine pseudomonad studied here contains a cleavage plane. This finding localizes the single cleavage plane of the cell wall and shows that the molecular architecture of this layer provides the lipid-enriched layer which cleaves preferentially in the frozen cell. The observation that the isolated double-track layer of the cell wall is sufficiently ordered at the molecular level to yield a well-defined X-ray diffraction pattern with a d-spacing of 0.44 nm shows that its molecular architecture is very similar to that of true membranes. This specific d-spacing is produced by the highly ordered packing of the hydrophobic portions of phospholipid molecules. Therefore, the double-track layer of the cell wall has been shown, by these two biophysical means, to have a molecular architecture which would allow it to function as the membrane-like “molecular sieve” layer, whose presence has been deduced from physiological data. This layer is important in the retention of cell wall-associated enzymes and in the control of the movement of large molecules through the cell wall.     

Structural and Biochemical Characterization of a Halophilic Archaeal Alkaline Phosphatase

Phosphate is an essential component of all cells that must be taken up from the environment. Prokaryotes commonly secrete alkaline phosphatases (APs) to recruit phosphate from organic compounds by hydrolysis. In this study, the AP from Halobacterium salinarum, an archaeon that lives in a saturated salt environment, has been functionally and structurally characterized. The core fold and the active-site architecture of the H. salinarum enzyme are similar to other AP structures. These generally form dimers composed of dominant β-sheet structures sandwiched by α-helices and have well-accessible active sites. The surface of the enzyme is predicted to be highly negatively charged, like other proteins of extreme halophiles. In addition to the conserved core, most APs contain a crown domain that strongly varies within species. In the H. salinarum AP, the crown domain is made of an acyl-carrier-protein-like fold. Different from other APs, it is not involved in dimer formation. We compare the archaeal AP with its bacterial and eukaryotic counterparts, and we focus on the role of crown domains in enhancing protein stability, regulating enzyme function, and guiding phosphoesters into the active-site funnel.     

A Highly Active Alkaline Phosphatase from the Marine Bacterium Cobetia

An alkaline phosphatase with unusually high specific activity has been found to be produced by the marine bacterium Cobetia marina (strain KMM MC-296) isolated from coelomic liquid of the mussel Crenomytilus grayanus. The properties of enzyme, such as a very high specific activity (15000 DE U/1 mg of protein), no activation with divalent cations, resistance to high concentrations of inorganic phosphorus, as well as substrate specificity toward 5′ nucleotides suggest that the enzyme falls in an intermediate position between unspecific alkaline phosphatases (EC 3.1.3.1) and 5′ nucleotidases (EC 3.1.3.5).     

Active Transport of Choline by a Marine Pseudomonad

A marine pseudomonad, BAL-31, accumulates the phospholipid nitrogen base, choline, although no detectable amount of choline is incorporated into polar lipids. Metabolic inhibitors such as cyanide and azide block the uptake process as does starving for oxygen by using nitrogen gas. Only very close structural analogues show any inhibition of transport, indicating that the uptake process has great structural specificity. The export of choline out of the cells is also an energy-dependent process and is markedly reduced during oxygen depletion. The constitutive level of choline transport is increased by approximately a factor of three after a brief induction period. Two other gram-negative bacteria also accumulate choline, whereas a gram-positive bacterium, Bacillus subtilis, and a yeast, Saccharomyces cerevisiae, fail to show any detectable accumulation.     

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⁺.     

Expression and Localization of Escherichia coli Alkaline Phosphatase Synthesized in Salmonella typhimurium Cytoplasm

The Escherichia coli structural gene for alkaline phosphatase was inserted into Salmonella typhimurium by episomal transfer in order to determine whether this enzyme would continue to be localized to the periplasmic space of the bacterium even though it was formed in a cell that does not synthesize alkaline phosphatase. The S. typhimurium heterogenote synthesized alkaline phosphatase under conditions identical to that observed with E. coli. This enzyme appeared to be identical to that synthesized by E. coli, and was quantitatively released from the bacterial cell by spheroplast formation with lysozyme. These results showed that localization is not a property unique to the E. coli cell and suggested that, in E. coli, enzyme location is related to the structure of the protein. Formation of alkaline phosphatase in the S. typhimurium heterogenote was repressed in cells growing in a medium with excess inorganic phosphate, even though only one of the three regulatory genes for this enzyme is on the episome. Thus, S. typhimurium can supply the products of the other two regulatory genes essential for repression even though this bacterium seems to lack the structural gene for alkaline phosphatase.     

Impact of a Genetically Engineered Bacterium with Enhanced Alkaline Phosphatase Activity on Marine Phytoplankton Communities

An indigenous marine Achromobacter sp. was isolated from coastal Georgia seawater and modified in the laboratory by introduction of a plasmid with a phoA hybrid gene that directed constitutive overproduction of alkaline phosphatase. The effects of this "indigenous" genetically engineered microorganism (GEM) on phosphorus cycling were determined in seawater microcosms following the addition of a model dissolved organic phosphorus compound, glycerol 3-phosphate, at a concentration of 1 or 10 (mu)M. Within 48 h, a 2- to 10-fold increase in the concentration of inorganic phosphate occurred in microcosms containing the GEM (added at an initial density equivalent to 8% of the total bacterial population) relative to controls containing only natural microbial populations, natural populations with the unmodified Achromobacter sp., or natural populations with the Achromobacter sp. containing the plasmid but not the phoA gene. Secondary effects of the GEM on the phytoplankton community were observed after several days, evident as sustained increases in phytoplankton biomass (up to 14-fold) over that in controls. Even in the absence of added glycerol 3-phosphate, a numerically stable GEM population (averaging 3 to 5% of culturable bacteria) was established within 2 to 3 weeks of introduction into seawater. Moreover, alkaline phosphatase activity in microcosms with the GEM was substantially higher than that in controls for up to 25 days, and microcosms containing the GEM maintained the potential for net phosphate accumulation above control levels for longer than 1 month.     

Localization of Polyribosomes Containing Alkaline Phosphatase Nascent Polypeptides on Membranes of Escherichia coli

A procedure has been developed for extracting membranes from bacterial cells under conditions that keep a large fraction of bacterial polyribosomes intact. Freeze-thawing spheroplasts in the presence of deoxyribonuclease, followed by differential centrifugation, permits a separation of free and membrane-associated polyribosomes. The latter fraction contains as much as 40% of cell ribosomal ribonucleic acid (RNA) and 55% of cell messenger RNA (mRNA). Nascent polypeptides were divided almost equally between the two fractions, but 70 to 80% of alkaline phosphatase nascent chains, detected both chemically and immunologically, were derived from polyribosomes associated with the bacterial membrane. Analysis of the fractions for mRNA specific for the lac and trp operons by RNA-deoxyribonucleic acid hydridization showed somewhat larger amounts on membrane than on free polyribosomes, but enrichment for nascent alkaline phosphatase (a secreted protein) on membranes was consistently greater, suggesting that polyribosomes making secreted proteins are more tightly bound to membranes. Electron micrographs of the membrane preparations show relatively intact membranes with clusters of polyribosomes on their inner surfaces.     

Biochemical Characterization and Subcellular Localization of the Red Kidney Bean Purple Acid Phosphatase

Phosphatases are known to play a crucial role in phosphate turnover in plants. However, the exact role of acid phosphatases in plants has been elusive because of insufficient knowledge of their in vivo substrate and subcellular localization. We investigated the biochemical properties of a purple acid phosphatase isolated from red kidney bean (Phaseolus vulgaris) (KBPAP) with respect to its substrate and inhibitor profiles. The kinetic parameters were estimated for five substrates. We used 31P nuclear magnetic resonance to investigate the in vivo substrate of KBPAP. Chemical and enzymological estimation of polyphosphates and ATP, respectively, indicated the absence of polyphosphates and the presence of ATP in trace amounts in the seed extracts. Immunolocalization using antibodies raised against KBPAP was unsuccessful because of the non-specificity of the antiserum toward glycoproteins. Using histoenzymological methods with ATP as a substrate, we could localize KBPAP exclusively in the cell walls of the peripheral two to three rows of cells in the cotyledons. KBPAP activity was not detected in the embryo. In vitro experiments indicated that pectin, a major component of the cell wall, significantly altered the kinetic properties of KBPAP. The substrate profile and localization suggest that KBPAP may have a role in mobilizing organic phosphates in the soil during germination.     

Regulatory Influences on the Production of Gamma-Aminobutyric Acid by a Marine Pseudomonad

A pseudomonad capable of producing γ-aminobutyric acid (GABA) was isolated from seawater via an enrichment in which glutamate was the sole carbon and nitrogen source. The organism grew optimally at pH 7.3 and at 25°C. Putrescine, alanine, and glucose-nitrate also served as effective growth substrates. The isolate grew poorly on GABA. Cell suspensions of the organism in 0.02 M phosphate buffer (pH 7.6) containing NaCl (19.4 g liter^-1) and MgCl₂. 6H₂O(3 g liter^-1) produced GABA from succinic semialdehyde in combination with glutamate or alanine but not from any substrate alone. Little or no GABA was produced with putrescine or glucose-nitrate as substrates. GABA production in the amino acid cosubstrate systems was transitory with optimum levels occurring in the suspension fluid after 3 h of incubation (0.3 and 0.03 mM for glutamate and alanine cosubstrates, respectively). However, yields of GABA in the cell suspension fluid were low, and quantities near that predicted from stoichiometry could be obtained only by extracting cell suspensions with methanol. GABA release in the suspension fluid was increased with higher pH or by decreasing NaCl. Substitution of the salt by the equivalent Tris-HCl or KCl likewise resulted in increased GABA release. When nigericin (10 μg ml^-1) was added to cell suspensions in which NaCl was not decreased, GABA release increased in a way similar to that observed in suspensions with decreased NaCl. The ionophore also decreased GABA uptake by cell suspensions of GABA-grown cells, and the effect was duplicated by lowering NaCl in cell suspensions. The results indicate a role for an Na⁺-dependent transport system in GABA release.     

Immunocytochemical Localization of Enzymes Involved in Lignification of the Cell Wall

O -methyltransferase, and cinnnamyl alcohol dehydrogenase were localized to differentiating xylem. These enzymes are particularly abundant during secondary wall formation. Immunolabeling was observed on polysomes and in the cytosol of the cells during secondary wall formation, indicating that these enzymes are synthesized in the polysomes and released in the cytosol. The synthesis of monolignols might occur in the cytosol. Immunolabeling of anionic peroxidase was also localized to the differentiating xylem, particularly during secondary wall formation. The labeling, however, was observed in the rough endoplasmic reticulum (r-ER), the Golgi apparatus, and the plasma membrane, indicating that peroxidase is synthesized in the r-ER, transported to the Golgi apparatus, and localized on the plasma membrane by fusion of the Golgi vesicles to the membrane. Received 3 September 2001/ Accepted in revised form 16 October 2001     

Heterogeneity and Distribution of Lipopolysaccharide in the Cell Wall of a Gram-Negative Marine Bacterium

Lipopolysaccharide (LPS) extracted from Alteromonas haloplanktis 214, variants 1 and 3, separated into three fractions when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fractions appeared in the gels as bands which stained for carbohydrate with the periodate-Schiff reagent. Variant 1, a smooth variant of the organism, and variant 3, a rough colonial variant, produced identical banding patterns. Under similar conditions, LPS from Neisseria meningitidis SDIC, Escherichia coli O111:B4, and Salmonella typhimurium LT2 gave rise to one, two, and three bands, respectively. LPS from Pseudomonas aeruginosa (ATCC 9027) failed to stain clearly with the reagent used. The banding pattern obtained with A. haloplanktis LPS was found not to be due to artifacts produced by the extraction or solubilization procedures employed or to the amount of protein associated with the LPS. When Triton X-100 replaced sodium dodecyl sulfate in the electrophoresis system, LPS failed to migrate into the gel. The lipid A but not the degraded polysaccharide fraction obtained by mild acid hydrolysis of the LPS migrated into the gel on electrophoresis. The three carbohydrate-staining bands obtained with A. haloplanktis LPS and referred to as LPS I, II, and III, in order of increasing electrophoretic mobility, were detected in each of the three outer layers of the cell wall of the organism. Estimations from densitometer scans indicated that 17% of the total LPS in the cell was present in the outer membrane, with the remainder divided almost equally between the loosely bound outer layer and the periplasmic space. Of the three fractions, LPS II was present in each of the layers in greatest amounts. Less LPS I and more LPS III were present in the outer membrane than in the periplasmic space. Pulse-labeling studies indicated that LPS I and II may be synthesized independently, whereas LPS III, which appeared only in cells in the stationary phase of growth, may be a degradation product of LPS I.     

Increase in Cell Wall-Associated Phosphatase Activity in Cucumber Roots during Calcium Starvation: Binding Nature and Properties of the Phosphatase and Cell Wall Analysis

In Ca^2$-starved cucumber roots, about 23% of phosphatase assayedat pH 9.0 (ALPase) in the crude cell walls was solubilized witheither 2 M NaCl or purified endo type polygalacturonase (endo-PG)from yeast culture broth. Coexistence of NaCl and endo-PG hadlittle effect on further release of ALPase, and a small amountof the activity was solubilized from the NaCl-pretreated cellwalls by incubation with endo-PG. Ionically bound ALPase, therefore,seemed to be localized in the fraction which was hydrolyzedby endo-PG in the crude cell walls of Ca^2$-starved cucumberroots. In the control roots, however, ALPase was not effectivelysolubilized by the treatment with endoPG. Ca^2$ starvation reducedthe contents of rhamnose, uronic acids and galactose among non-cellulosicsugars in the cell walls, suggesting that the structure of pecticsubstances, possibly rhamnogalacturonan, is altered during thestarvation. Activities of both ionically and covalently bound ALPases greatlyincreased during Ca^2$ starvation. The increased ALPase in theNaCl-solubilized fraction hydrolyzed most phosphate esters tested,whereas the enzyme from control roots only cleaved nucleoside2'(3')-monophosphates and p-nitrophenylphosphate. Differencesin the properties between both types of roots were also foundwhen the effects of various inhibitors were tested. Profilesof ALPase-isozymes after polyacrylamide gel electrophoresiswere also altered by Ca^2$ starvation. (Received June 2, 1982; Accepted July 20, 1982)     

Variation in the Fine Structure of a Marine Achromobacter and a Marine Pseudomonad Grown Under Selected Nutritional and Temperature Regimes

Certain features of the fine structure of a marine achromobacter and a marine pseudomonad were dependent upon the conditions of growth. Cells of achromobacter grown at 10 C in a low peptone-seawater (SW) medium displayed the characteristic morphology of the achromobacter: a regularly undulant outer element of the cell wall and a planar inner element, tightly packed ribonucleoprotein (RNP) particles in the cytoplasm, deoxyribonucleic acid (DNA) disposed in a lobate manner, and dense inclusion bodies. Few mesosomes, however, were seen. Cells of achromobacter grown at 10 C in a high peptone-SW medium had larger and more highly organized mesosomes. At 22 C, in a low peptone-SW medium, no mesosomes were seen, but the inclusions were more frequently seen and were larger in the achromobacter cells. At 22 C, in a high peptone-SW medium, these cells revealed the greatest variation in cellular morphology. They contained both small and large mesosomes, or no mesosomes, and both small and large inclusions, or no inclusions. Pseudomonad cells at 10 C in a low peptone-SW medium revealed a typical gram-negative morphology: double-layered, irregularly undulant cell wall; more nearly planar cytoplasmic membrane; densely stained, lightly packed RNP particles; finely fibrillar, axially disposed DNA; simple mesosomes. At 10 C, in a high peptone-SW medium, pseudomonad cells revealed associated strands of material and intracytoplasmic ringlike structures. At 22 C, in a low peptone-SW medium, pseudomonad cells had a more undulant cell-wall and a more nearly planar cytoplasmic membrane. At 22 C, in a high peptone-SW medium, these cells revealed prominent blebs of the cell wall.     

一个耐热碱性磷酸酯酶突变型的研究

为了进一步揭示蛋白质的耐热机制,对含有耐热碱性磷酸酯酶（ＦＤ－ＴＡＰ）的表达质粒ｐＴＡＰ５０３Ｆ进行了随机诱变,用菌落原位显色法从约５０００个转化子中筛选到４个耐热性下降的突变型克隆,并对其中１克隆（ＴＡＰＭ３）进行了部分酶学性质、ＤＮＡ和氨基酸序列的研究。酶学性质研究表明,与野生型相比,该突变型酶的耐热性有较大幅度的下降,而热激活性无明显改变。ＤＮＡ序列分析表明在１２３９位ＴＡＰＭ３发生Ｇ→Ａ转