Adenine nucleotide content and energy charge of Azotobacter vinelandii during batch growth in dialysed-soil medium

Abstract In human peripheral blood, a factor which induces gonococcal resistance to complement-mediated killing by fresh human serum is more concentrated in the white blood cells of buffy coat than in red blood cells. Futhermore, the resistance-inducing factor is present in both polymorphonuclear phagocytes and mononuclear cells (monocytes and lymphocytes) separated from the buffy coat by centrifugation on Ficoll-Hypaque gradients. These results imply that inflammatory cells mobilised from the blood to sites of infection carry a host factor which, if it is available to the gonococci, would materially increase their ability to resist a major defence mechanism and hence enhance their capacity to maintain and increase infection.     

Adenine nucleotide content and energy charge of Bacillus megaterium during batch growth in low-phosphate medium

Abstract The effect of a low phosphate concentration on intracellular adenine nucleotides, oxygen consumption and poly-β-hydroxybutyric acid synthesis, was investigated with batch cultures of Bacillus megaterium . At low phosphate concentrations the cells contained much larger amounts of poly-β-hydroxybutyric acid, but displayed lower adenylate energy charge and oxygen uptake than did control cells. The ratio of ATP to ADP was much greater in the control cells. The levels of ATP and AMP were lower in low-phosphate cells.     

Adenine nucleotide degradation during energy depletion in human lymphoblasts. Adenosine accumulation and adenylate energy charge correlation.

Adenine nucleotide breakdown to nucleosides and purine bases was measured in cultures of human lymphoblastoid cells following: 1) the inhibition of oxidative phosphorylation in the absence of glucose or 2) the addition of 2-deoxyglucose. A mutant cell line, deficient in adenosine kinase, in the presence of an adenosine deaminase inhibitor was used to measure utilization of the two pathways of AMP catabolism involving initial action of either purine 5'-nucleotidase or AMP deaminase. In such a system the appearance of adenosine induced by the oxidative phosphorylation inhibitor, rotenone, implies that approximately 70% of AMP breakdown occurs via dephosphorylation. By the same method, deamination accounts for 82% of AMP breakdown when 2-deoxyglucose is added. The occurrence of AMP dephosphorylation is not correlated with elevated concentrations of substrate or with decreased concentrations of the inhibitors of 5'-nucleotidase, ATP and ADP. Dephosphorylation occurs if, and only if, the adenylate energy charge decreases to about 0.6 in these experiments. In cultures deprived of glucose and oxygen, adenine nucleotide degradation via dephosphorylation results in recovery of normal energy charge values.     

Adenine nucleotide content and energy charge in dry cells and cysts of Azotobacter vinelandii

Abstract The levels of intracellular adenine nucleotides, energy charge, oxygen consumption and poly-β-hydroxybutyric acid stored, have been investigated in dry vegetative cells and cysts of Azotobacter vinelandii . The data show that under desiccation conditions the cysts retain viability at energy charge values of 0.20 and an ATP/ADP ratio of 0.24, whereas under the same desiccation conditions, vegetative cells die at energy charge values <0.5 and an ATP/ADP ratio of <0.6.     

The nucleotide sequence of an α-amylase gene from an alkalopsychrotrophic Micrococcus sp

An alpha-amylase gene from Micrococcus sp. 207 was cloned into Escherichia coli JM101 using the vector pHSG399. The constructed recombinant plasmid pYK63 contained a 4.8 kb chromosomal DNA fragment derived from strain 207 DNA. The cloned amylase isolated from E. coli JM101 (pYK63) produced mainly maltotetraose from starch, and exhibited temperature and pH activity profiles closely similar to those of the enzyme from the original strain. Nucleotide sequence analysis of the cloned DNA fragment revealed one open reading frame containing the gene which consisted of 3312 bp (1104 amino acids). When compared with several other alpha-amylases, three consensus sequences were identified in the region of the active site. About 300 amino acid residues were present both upstream and downstream of the active site region.     

Nucleotide sequence of the extracellular α-amylase gene in the yeast Schwanniomyces occidentalis ATCC 26077

The Schwanniomyces occidentalis (formerly castellii) ATCC 26077 (CBS 2863) alpha-amylase (AMY 26077) gene was cloned in Saccharomyces cerevisiae and sequenced. An open-reading frame encoding the AMY consists of 1536 base pairs and contains 512 amino-acid residues, which is almost the same in size as the AMY of Sch. occidentalis ATCC 26076 and CCRC 21164. The amino-acid sequence of AMY 26077 differed from that of ATCC 26076 alpha-amylase (AMY 26076) at two residues and from that of CCRC 21164 alpha-amylase (AMY 21164) at three residues. Comparison of the AMY 26077 gene with its homologues from two other strains (Sch. alluvius CBS 1153 and Sch. persoonii CBS 2169) using several restriction enzymes revealed that the AMY 26077 was very similar to AMY CBS 1153 but different from that of CBS 2169.     

Adenine nucleotide contents and energy charge of Azotobacter vinelandii grown at low phosphate concentration

The effect of a low phosphate concentration on intracellular adenine nucleotide content, oxygen consumption and poly--hydroxybutyrate deposition was investigated with N-free and NH ₄ ⁺ batch cultures of Azotobacter vinelandii. When the microorganisms were cultured under low-phosphate concentrations the cells contained much larger amounts of poly--hydroxybutyrate, but displayed lower oxygen consumption activities and energy charge values than did control cells. Also, the ratio ATP to ADP was much higher in control cells and the intracellular levels of ATP were lower in low-phosphate cells.     

In vitro colonization ability of human colon mucosa by exogenous Lactobacillus strains

Abstract A 5.4 kb Hind III DNA fragment carrying the gene encoding raw starch-digesting α-amylase (RSDA), has been previously cloned from Bacillus circulans F-2 and expressed in Escherichia coli [Kim et al. (1990) Biochim. Biophys. Acta 1048, 2233–2238]. Interestingly, when the cell extract of E. coli harboring a plasmid carrying this fragment was incubated with l M NaCl, it exhibited about 10 times higher enzyme activity than when assayed without NaCl. Differential zymograms showed two different amylase activities: one for RSDA and the other for a salt-dependent a-amylase (SDA). Even though RSDA activity was detected without NaCl, SDA activity was detected only in high concentrations of NaCl. SDA activity was fully detected at above l M NaCl. Results from subcloning of the genes, fractionation analysis of cell extracts, and immunological assays clearly suggested that the two amylases are genetically distinct and that genes for both enzymes are closely linked on the 5.4 kb DNA fragment.     

Gene cloning, nucleotide sequence and biochemical properties of a cytoplasmic cyclomaltodextrinase (neopullulanase) from Alicyclobacillus acidocaldarius, reclassification of a group of enzymes

A gene encoding a cyclomaltodextrinase (neopullulanase) was cloned from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius ATCC27009 and its nucleotide sequence was determined. The encoded CdaA protein lacked an N-terminal signal sequence and aligned well with a family of bacterial proteins described as maltogenic alpha-amylases, neopullulanases or cyclomaltodextrinases. Escherichia coli cells harboring the cloned cdaA gene produced a 66-kDa protein that degraded pullulan in a sodium dodecyl sulfate-polyacrylamide gel. A. acidocaldarius cells grown on maltose, soluble starch or pullulan synthesized the same protein. Neopullulanase activity of the protein was cytoplasmic and its pH optimum of 5.5 was close to the pH value of the cytoplasm. CdaA degraded cyclomaltodextrins rapidly and pullulan (to panose) more slowly. It is proposed that CdaA functions as a cytoplasmic cyclomaltodextrinase (EC 3.2.1.54).     

Adenine nucleotides and energy charge evolution during the induction of flowering in spinach leaves

Changes in adenine nucleotides pool size levels have been investigated in spinach leaves (Spinacia oleracea. L. cv. Nobel) in order to characterize the transition from the vegetative to the reproductive development. The transient changes reported in this study are the earliest responses observed to date in leaves during photoperiodic induction. These results are discussed in relation to Prigogine's theory of systems far from equilibrium.Abbreviations AN adenine nucleotide(s) - MIT mimicked inductive treatment (inductive treatment on already induced plants)     

Biochemical and immunological characterization of α-amylase isoenzymes of Araucaria araucana

Seven α-amylase isoenzymes present in quiescent seeds of the South American conifer Araucaria araucana were purified by affinity chromatography and partially characterized. The molecular masses of these isoenzymes were 45.7, 47.0, 50.2, 51.2, 52.0, 53.5 and 55.2 kDa. The two main isoforms were separated from each other and from the rest of the isoenzymes by anion-exchange chromatography using a linear gradient of 0 to 0.6 M NaCl and slightly different CaCl₂ concentrations. All isoenzyme bands stained with periodic acid/dansylhydrazine, suggesting that they are glycoproteins. Electroblotting of the isoenzymes onto polyvinylidene difluoride membranes allowed determination of the amino acid composition and NH₂-terminal sequence of the 53.5-, 50.2-and 47.0-kDa isoenzymes. Amino acid compositional analysis demonstrated that these enzymes are rich in glycine, aspartic acid/asparagine, alanine, serine, proline and glutamic acid/glutamine. The NH₂-terminal sequences of the three isoenzymes are identical. Comparison of the amino acid compositions and the NH₂-terminal sequence of these isoenzymes with the cereal and Vigna radiata α-amylases demonstrated that there is no relation between them. However, polyclonal antibodies generated against barley α-amylase cross-reacted with all the A . araucana α-amylases. Peptide mapping analysis of the isoenzymes using cyanogen bromide suggests that there are genetic differences between them.     

Cloning and expression of the thermostable α-amylase gene from Clostridium thermosulfurogenes (DSM 3896) in Escherichia coli

Abstract The gene coding for a thermostable α-amylase from Clostridium thermosulfurogenes (DSM 3896) was cloned in Escherichia coli using pUC18 as a vector. The recombinant plasmid pCT2 of an amylolytic positive transformant of E. coli contained a 2.9 kbp fragment of chromosomal DNA of C. thermosulforogenes carrying the α-amylase gene. In E. coli the gene was apparently transcribed by its own promoter. Comparative studies showed no difference between the original and the heterologously in E. coli expressed enzyme. The latter was not secreted into the medium.     

The cloning and nucleotide sequence of a Corynebacterium glutamicum 3-deoxy-d- arabinoheptulosonate-7-phosphate synthase gene

Abstract The aro gene of Corynebacterium glutamucum CCRC 18310 encoding 3-deoxy- d -arabinoheptulosonate-7-phosphate (DAHP) synthase was isolated by complementation of a DAHP synthase-deficient mutant of Escherichia coli AB3257. The specific activity of DAHP synthase was increased four-fold in a C. glutamicum strain harboring the cloned aro gene. The complete nucleotide sequence of the aro gene and 5' and 3' flanking regions has been determined. The sequence contained an open reading frame of 368 codons, from which a protein with a molecular mass of 39 340 Da could be predicted. The deduced amino acid sequence shows high identity with the aro gene products of E. coli and Salmonella typhimurium .     

Coupling of fermentation and microfiltration for α-amylase production from Bacillus amyloliquefaciens

Abstract: A strain of Bacillus amyloliquefaciens secreting α-amylase was cultivated continuously in a fermentor coupled to a filtration unit. Under the tested operating conditions, the maximum flux was 91 m^-2 h^-1 during the first day and 61 m^-2 h^-1 during the 2nd day. The α-amylase retention was around 30%. Compared to a batch process, continuous cultivation with cell recycling led to lower α-amylase concentrations but to a doubling of volumetric productivities.     

Adenylate nucleotide levels and energy charge in Arthrobacter crystallopoietes during growth and starvation

The adenylate nucleotide concentrations, based on internal water space, were determined in cells of Arthrobacter crystallopoietes during growth and starvation and the energy charge of the cells was calculated. The energy charge of spherical cells rose during the first 10 h of growth, then remained nearly constant for as long as 20 h into the stationary phase. The energy charge of rod-shaped cells rose during the first 4 h of growth, then remained constant during subsequent growth and decreased in the stationary growth phase. Both spherical and rod-shaped cells excreted adenosine monophosphate but not adenosine triphosphate or adenosine diphosphate during starvation. The intracellular energy charge of spherical cells declined during the initial 10 h and then remained constant for 1 week of starvation at a value of 0.78. The intracellular energy charge of rod-shaped cells declined during the first 24 h of starvation, remained constant for the next 80 h, then decreased to a value of 0.73 after a total of 168 h starvation. Both cell forms remained more than 90% viable during this time. Addition of a carbon and energy source to starving cells resulted in an increase in the ATP concentration and as a result the energy charge increased to the same levels as found during growth.Nonstandard Abbreviations cGMP 3,5 guanosine monophosphate - ppGpp guanosine tetraphosphate - MS mineral salts - HC casein hydrolysate - TEA triethanolamine buffer - Pi inorganic phosphate     

Adenine nucleotide extraction from multicellular organisms and beach sand: ATP recovery,energy charge ratios and determination of carbon/ATP ratios

Investigations were conducted comparing the efficiency of adenine nucleotide extraction from bacteria, unicellular algae, invertebrates (copepods, isopods and polychaetes), and beach sand using boiling buffers and cold acid extraction procedures. Cellular levels of ATP, ADP, and AMP obtained by these procedures were used to calculate the adenylate energy charge ratio ($\text{EC}{}_{\mathrm{<mtext>A</mtext>}}\text{= [}\text{ATP}\text{] +}\text{1}\text{2}\text{[}\text{ADP}\text{]/[}\text{ATP}\text{] + [}\text{ADP}\text{] + [}\text{AMP}\text{]}$). Although both extraction procedures efficiently extract ATP from unicellular micro-organisms, the results with multicells and beach sand indicate that the cold acid procedure preserves a greater percentage of the total adenine nucleotides ([A_T] = [ATP] + [ADP] + [AMP]) in the form of ATP, resulting in higher energy charge ratios. There were relatively large losses of ATP when multicellular organisms were extracted in boiling buffers. These data suggest that ATP hydrolysis may be important in certain fluid-solid mixtures, and also adds experimental support to the thermal gradient hypothesis.The C/ATP ratios calculated from these data indicate that multicellular organisms have C/ATP ratios < 100, as compared with the 250 ratio commonly found in micro-organisms. These results are discussed in terms of the proportion of structural (non-living) carbon vs protoplasm (living) carbon within each of these groups of organisms, as well as the relative intracellular levels of non-adenine nucleotide triphosphates. These differences in the C/ATP ratios must be considered whenever ATP measurements are used for biomass determinations.     

Differential expression of two β-amylase genes of rye during seed development

The expression of two β-amylase loci was analysed in the developing seeds of two inbred lines of rye (Secale cereale L.), one of which was a β-amylase deficient mutant. Enzymatic activity and the contents of enzymatic protein and mRNA specific for each of an endosperm-characteristic and ubiquitous β-amylase were determined throughout the course of caryopsis development. Both loci were expressed in the developing normal line caryopses according to different temporal and quantitative patterns. The ubiquitous enzyme-specific locus β-Amy 2 was expressed earlier; both mRNA and enzymatic protein accumulated to a maximum extent at 10 to 15 days after pollination. In contrast, the highest content of mRNA for endosperm β-amylase (encoded by the β-Amy I locus) was found 20 days after pollination, and the corresponding enzymatic protein accumulated throughout seed development. The expression of the β-Amy I locus was 30- to 40-fold higher than that of the β-Amy 2 locus in terms of maximum specific mRNA accumulation. The expression product of only the β-Amy 2 locus was found in the developing mutant line caryopses. The expression pattern of this locus was similar in the developing normal and mutant line seeds in terms of the temporal accumulation of mRNA and enzymatic protein. However, an approximately 4-fold higher level of ubiquitous β-amylase-specific mRNA was found in the mutant than in the normal line caryopses, and the content of ubiquitous β-amylase protein decreased to near zero at seed maturity in the mutant line, but not in the normal line, caryopses. The enzymatic activities of both β-amylases appeared to be regulated at the level of accumulated enzymatic protein.     

Purification and characterization of β-amylase from leaves of potato (Solanum tuberosum)

Amylolytic activity is widely distributed in plants. In potato leaves ( Solanum tuberosum L.) the abundant amylolytic activity was found to be β-amylase (EC 3.2.1.2, a-1,4-D-glucan maltohydrolase). β-Amylase from potato leaves was purified to homogeneity for study of enzyme characteristics. The purification steps included ammonium sulphate precipitation, anion exchange chromatography, affinity chromatography and gel filtration. The end product of α-1,4-glucan degradation was maltose. The protein is a 111-kDa homo-dimer with a subunit molecular mass of 56 kDa and a pl of 5.6. The pH-optimum is 6.5 using p -nitrophenylmaltopentaoside (PNPG5) as substrate. The optimal temperature for hydrolysis is at 40°C. The enzyme is unstable at temperatures above 40°C. The K_nt-value for PNPG5 is 0.73 m M and the activity is inhibited by cyclodextrins. At a concentration of 1 m M , β-cyclodextrin is a stronger inhibitor than α-cyclodextrin (68 and 20% inhibition, respectively). Branched glucans (e.g. starch and amylopectin) are superior substrates as compared to long, essentially unbranched glucans (e.g. amylose). This study of the catalytic properties of β-amylase from potato leaves indicates the importance of β-amylase as a starch degrading enzyme.